Search results for: Alena Zemanová
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 32

Search results for: Alena Zemanová

32 Time/Temperature-Dependent Finite Element Model of Laminated Glass Beams

Authors: Alena Zemanová, Jan Zeman, Michal Šejnoha

Abstract:

The polymer foil used for manufacturing of laminated glass members behaves in a viscoelastic manner with temperature dependence. This contribution aims at incorporating the time/temperature-dependent behavior of interlayer to our earlier elastic finite element model for laminated glass beams. The model is based on a refined beam theory: each layer behaves according to the finite-strain shear deformable formulation by Reissner and the adjacent layers are connected via the Lagrange multipliers ensuring the inter-layer compatibility of a laminated unit. The time/temperature-dependent behavior of the interlayer is accounted for by the generalized Maxwell model and by the time-temperature superposition principle due to the Williams, Landel, and Ferry. The resulting system is solved by the Newton method with consistent linearization and the viscoelastic response is determined incrementally by the exponential algorithm. By comparing the model predictions against available experimental data, we demonstrate that the proposed formulation is reliable and accurately reproduces the behavior of the laminated glass units.

Keywords: finite element method, finite-strain Reissner model, Lagrange multipliers, generalized Maxwell model, laminated glass, Newton method, Williams-Landel-Ferry equation

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31 Freeze-Thaw Resistance of Concretes with BFSA

Authors: Alena Sicakova

Abstract:

Air-cooled Blast furnace slag aggregate (BFSA) is usually referred to as a material providing for unique properties of concrete. On the other hand, negative influences are also presented in many aspects. The freeze-thaw resistance of concrete is dependent on many factors, including regional specifics and when a concrete mix is specified it is still difficult to tell its exact freeze-thaw resistance due to the different components affecting it. An important consideration in working with BFSA is the granularity and whether slag is sorted or not. The experimental part of the article represents a comparative testing of concrete using both the sorted and unsorted BFSA through the freeze-thaw resistance as an indicator of durability. Unsorted BFSA is able to be successfully used for concretes as they are specified for exposure class XF4 with providing that the type of cement is precisely selected.

Keywords: blast furnace slag aggregate, concrete, freeze-thaw resistance

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30 The Comparative Study of Binary Artifact Repository Managers

Authors: Evgeny Chugunnyy, Alena Gerasimova, Kirill Chernyavskiy, Alexander Krasnov

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One of the primary component of Continuous deployment (CD) is a binary artifact repository — the place where artifacts are stored with metadata in a structured way. The binary artifact repository manager (BARM) is a software, which implements this repository logic and exposes a public application programming interface (API) for managing these artifacts. Almost every programming language ecosystem has its own artifact repository kind. During creating Artipie — BARM constructor and server, we analyzed and implemented a lot of different artifact repositories. In this paper we present criterias for comparing artifact repositories, and analyze the most popular repositories using these metrics. We also describe some of the notable features of different repositories. This paper aimed to help people who are creating, maintaining or optimizing software repository and CI tools.

Keywords: artifact, repository, continuous deployment, build automation, artifacts management

Procedia PDF Downloads 150
29 Enzyme Immobilization: A Strategy to Overcome Enzyme Limitations and Expand Their Applications

Authors: Charline Monnier, Rudolf Andrys, Irene Castellino, Lucie Zemanova

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Due to their inherent sustainability and compatibility with green chemistry principles, enzymes are attracting increasing attention for various applications like bioremediation or biocatalysis. These natural catalysts boast remarkable substrate specificity and operate under mild biological conditions. However, their intrinsic limitations, such as instability at high temperatures or in organic solvents, impede their wider applicability. Enzyme immobilization on supportive matrices emerges as a promising strategy to address these challenges. This approach not only facilitates enzyme reusability but also offers the potential to modulate their stability, activity, and selectivity. The present study investigates the immobilization and application of two distinct groups of hydrolases on supportive matrices: PETases, naturally capable of PolyEthylene Terephthalate (PET) degradation, and cholinesterases (ChEs), key enzymes in neurotransmitter regulation. All tested enzymes will be immobilized on porous and non-porous particles using both covalent and non-covalent methods. Additionally, the stability of PETases and cholinesterases will be explored, followed by exposure to denaturing conditions to assess their resilience under harsh conditions. Furthermore, due to the exceptional catalytic efficiency and selectivity, their biocatalytic efficiency will be tested using xenobiotic substrates, aiming to establish them as replacements for conventional chemical catalysts in environmentally friendly processes. By exploiting the power of enzyme immobilization, this research strives to unlock the full potential of these biocatalysts for sustainable and efficient technological advancements.

Keywords: biocatalysis, bioremediation, enzyme efficiency, enzyme immobilization, green chemistry

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28 Some Aspects of Study the Leaching and Acid Corrosion of Concrete

Authors: Alena Sicakova, Adriana Estokova

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Although properly made concrete is inherently a durable material, there are many physical and chemical forces in the environment which can contribute to its deterioration. This paper deals with two aspects of concrete durability in chemical aggressive environment: degradation effect of particular aggressive exposure and role of particular mineral additives. Results of the study of leaching and acid corrosion processes in samples prepared with specific dosage of microsilica and zeolite are given in the paper. Corrosion progress after 60-day exposition is manifested by increasing rate of both Ca and Si release, what is identified by XRF method. Kind and dosage of additions used in experiment was found to be helpful for stabilization of concrete microstructure. The lowest concentration of mean elements in leachates was observed for mixture V1 (microsilica only) unlike the V2 (microsilica + zeolite). It is surprising in the terms of recommendations of zeolite application for acid exposure. Using microsilica only seems to be more effective.

Keywords: sustainability, durability, concrete, acid corrosion, leaching

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27 Human Rights as Part of the Core Values System of International Organisations: A Comparative Study

Authors: Ayyoub Jamali, Jennie Edlund, Alena Kozlová

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This paper evaluates the monitoring, prevention, and enforcing mechanisms of the core values of international organisations (IOs) in a comparative human rights perspective. The IOs in focus are the European Union, the Council of Europe, the African Union, and the Organization of American States. The paper will take the founding treaties of these IOs and their relevant protocols as a starting point to identify the values and the mechanisms used for their implementation. It will explore the scope of violations, the procedures in place and evaluate what type of response to those breaches seems to work best in terms of achieving its declared objectives. The study will identify and compare the weaknesses and strengths of each mechanism used by the IOs and recognize common challenges and means, thereby drawing inter-organizational comparisons. Consequently, the findings of this paper can be used among the IOs to improve their system and thus enhance their effectiveness.

Keywords: international organizations, core values, human rights, enforcement mechanism, compliance

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26 Microstructural and Magnetic Properties of Ni50Mn39Sn11 and Ni50Mn36Sn14 Heusler Alloys

Authors: Mst Nazmunnahar, Juan del Val, Alena Vimmrova, Blanca Hernando, Julian González

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We report the microstructural and magnetic properties of Ni50Mn39Sn11 and Ni50Mn36Sn14 ribbon Heusler alloys. Experimental results were obtained by differential scanning calorymetry, X-ray diffraction and vibrating sample magnetometry techniques. The Ni-Mn-Sn system undergoes a martensitic structural transformation in a wide temperature range. For example, for Ni50Mn39Sn11 the start and finish temperatures of the martensitic and austenite phase transformation for ribbon alloy were Ms = 336K , Mf = 328K, As = 335K and Af = 343K whereas no structural transformation is observed for Ni50Mn36Sn14 alloys. Magnetic measurements show the typical ferromagnetic behavior with Curie temperature 207K at low applied field of 50 Oe. The complex behavior exhibited by these Heusler alloys should be ascribed to the strong coupling between magnetism and structure, being their magnetic behavior determined by the distance between Mn atoms.

Keywords: as-cast ribbon, Heusler alloys, magnetic properties, structural transformation

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25 Usage of Military Continuity Management System for Flooding Solution

Authors: Jiri Palecek, Radmila Hajkova, Alena Oulehlova, Hana Malachova

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The increase of emergency incidents and crisis situations requires proactive crisis management of authorities and for its solution. Application business continuity management systems help the crisis management authorities quickly and responsibly react to events and to plan more effectively and efficiently powers and resources. The main goal of this article is describing Military Continuity Management System (MCMS) based on the principles of Business Continuity Management System (BCMS) for dealing with floods in the territory of the selected municipalities. There are explained steps of loading, running and evaluating activities in the software application MCMS. Software MCMS provides complete control over the tasks, contribute a comprehensive and responsible approach solutions to solution floods in the municipality.

Keywords: business continuity management, floods plan, flood activity, level of flood activity

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24 Enhancement of Lignin Bio-Degradation through Homogenization with Dimethyl Sulfoxide

Authors: Ivana Brzonova, Asina Fnu, Alena Kubatova, Evguenii Kozliak, Yun Ji

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Bio-decomposition of lignin by Basidiomycetes in the presence of dimethyl sulfoxide (DMSO) was investigated. The addition of 3-5 vol% DMSO to lignin aqueous media significantly increased the lignin solubility based on UV absorbance. After being dissolved in DMSO, the thermal evolution profile also changed significantly, yielding more high-MW organic carbon at the expense of recalcitrant elemental carbon. Medical fungi C. versicolor, G. lucidum and P. pulmonarius, were observed to grow on the lignin in media containing up to 15 vol. % DMSO. Further detailed product characterization by chromatographic methods corroborated these observations, as more low-MW phenolic products were observed with DMSO as a co-solvent. These results may be explained by the high solubility of lignin in DMSO; thus, the addition of DMSO to the medium increases the lignin availability for microorganisms. Some of these low-MW phenolic products host a big potential to be used in medicine. No significant inhibition of enzymatic activity (laccase, MnP, LiP) was observed by the addition of up to 3 vol% DMSO.

Keywords: basidiomycetes, bio-degradation, dimethyl sulfoxide, lignin

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23 Measures Adopted by FIFA and UEFA against Russian Athletes: A Human Rights Perspective

Authors: Ayyoub Jamali, Alena Kozlova

Abstract:

The Russian invasion of Ukraine has tested the mettle of the international community, prompting not only States but also non-state actors to take deterrent action in response. Indeed, international sports organisations, namely FIFA and UEFA, have been rather successful in shifting the power dynamics by introducing a complete ban on the Russian national and club teams. This article aims to inquire into the human rights implications of such actions taken by international sports organisations. First, the article departs from an assessment of the legal status of FIFA and UEFA under international law and reflects on how a legal link could be established vis-à-vis their human rights obligations. Second, it examines the human rights aspects of the impugned measures by FIFA and UEFA on the part of the Russian athletes, further scrutinising them against the international human rights law principle of non-discrimination through a proportionality test. Last, it draws basic pathways for how possible human rights violations committed in the context of measures adopted by such organisations could be remedied, outlining the challenges of arbitration and litigation in Switzerland.

Keywords: FIFA, UEFA, FUR, ban, human rights, Russia, Ukraine, non-state actors

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22 Usage of Military Continuity Management System for Supporting of Emergency Management

Authors: Radmila Hajkova, Jiri Palecek, Hana Malachova, Alena Oulehlova

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Ensuring of continuity of business is the basic strategy of every company. Continuity of organization activities includes comprehensive procedures that help in solving unexpected situations of natural and anthropogenic character (for example flood, blaze, economic situations). Planning of continuity operations is a process that helps identify critical processes and implement plans for the security and recovery of key processes. The aim of this article demonstrates application of system approach to managing business continuity called business continuity management systems in military issues. This article describes the life cycle of business continuity management which is based on the established cycle PDCA (plan-do-check-act). After this is carried out by activities which are making by the University of Defence during activation of forces and means of the Integrated rescue system in case of emergencies - accidents at a nuclear power plant in Czech republic. Activities of various stages of deployment earmarked forces and resources are managed and evaluated by using MCMS application (military continuity management system).

Keywords: business continuity management system, emergency management, military, nuclear safety

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21 Evaluation of European Surveys in the Area of Health and Safety at Work and Identification of New Risks in the Labor Environment

Authors: Alena Dadova, Katarina Holla, Anna Cidlinova, Linda Makovicka Osvaldova, Jiri Vala, Samuel Kockar

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Occupational health and safety (ASH) is an area in which procedures and applications are constantly evolving and changing through legislation and new directives and guidelines. In this way, the relevant organizations strive to ensure continuous progress and the advantage of up-to-date information to ensure safety and prevent occupational accidents. Three ESENER surveys have been carried out in the European Union, led by the Agency for Safety and Health at Work (EU-OSHA). On the basis of surveys, it was determined how European workplaces manage risks and how they manage the field of safety and health protection at work. Thousands of companies and organizations in the European Union were involved in the surveys. Organizations and businesses were presented with a questionnaire that focused on the following topics: the impact of general risks on the field of OSH and the possibility of their management, psychosocial risks and other factors such as stress, harassment and bullying, and employee participation in OSH procedures. The article is dedicated to the fundamental conclusions from these surveys and their subsequent connection with the strategic intent of the Strategic Framework of European Union for the years 2021 - 2027. In the conclusion, emerging risks are identified and EU will soon have to deal with them.

Keywords: ESENER, emerging risks, strategic framework in OSH, EU

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20 The Role of Interpersonal and Institutional Trusts for the Public Support of Welfare State

Authors: Nazim Habibov, Alena Auchynnikava, Lida Fan

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The exploration of the relationship between social trust and the support of the welfare system in transitional countries has attracted growing interests in recent decades. This study estimates the effects of interpersonal and institutional trust on the support of the welfare system in 27 countries in Eastern Europe the former Soviet Union. We estimate the data sets from the Life-in-Transition Survey 2010 and 2016 with binomial regression models. The results indicate that both interpersonal and institutional trust have positive effects on the support for the welfare system in all the three areas under investigation: helping the needy, public healthcare and public education, both in the less developed countries of the former Soviet Union and in the more developed Eastern European countries. Furthermore, the positive effects of interpersonal and institutional trust on support for helping the needy, public healthcare and public education were found to grow over time. In conclusion, this study confirms that interpersonal and institutional trusts have positive effects for the public support of the welfare system in these transitional countries under investigation, regardless of their level of development.

Keywords: central and eastern Europe, former Soviet union, international social welfare policy, comparative social welfare policy

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19 Income-Consumption Relationships in Pakistan (1980-2011): A Cointegration Approach

Authors: Himayatullah Khan, Alena Fedorova

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The present paper analyses the income-consumption relationships in Pakistan using annual time series data from 1980-81 to 2010-1. The paper uses the Augmented Dickey-Fuller test to check the unit root and stationarity in these two time series. The paper finds that the two time series are nonstationary but stationary at their first difference levels. The Augmented Engle-Granger test and the Cointegrating Regression Durbin-Watson test imply that the two time series of consumption and income are cointegrated and that long-run marginal propensity to consume is 0.88 which is given by the estimated (static) equilibrium relation. The paper also used the error correction mechanism to find out to model dynamic relationship. The purpose of the ECM is to indicate the speed of adjustment from the short-run equilibrium to the long-run equilibrium state. The results show that MPC is equal to 0.93 and is highly significant. The coefficient of Engle-Granger residuals is negative but insignificant. Statistically, the equilibrium error term is zero, which suggests that consumption adjusts to changes in GDP in the same period. The short-run changes in GDP have a positive impact on short-run changes in consumption. The paper concludes that we may interpret 0.93 as the short-run MPC. The pair-wise Granger Causality test shows that both GDP and consumption Granger cause each other.

Keywords: cointegrating regression, Augmented Dickey Fuller test, Augmented Engle-Granger test, Granger causality, error correction mechanism

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18 Microbial Degradation of Lignin for Production of Valuable Chemicals

Authors: Fnu Asina, Ivana Brzonova, Keith Voeller, Yun Ji, Alena Kubatova, Evguenii Kozliak

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Lignin, a heterogeneous three-dimensional biopolymer, is one of the building blocks of lignocellulosic biomass. Due to its limited chemical reactivity, lignin is currently processed as a low-value by-product in pulp and paper mills. Among various industrial lignins, Kraft lignin represents a major source of by-products generated during the widely employed pulping process across the pulp and paper industry. Therefore, valorization of Kraft lignin holds great potential as this would provide a readily available source of aromatic compounds for various industrial applications. Microbial degradation is well known for using both highly specific ligninolytic enzymes secreted by microorganisms and mild operating conditions compared with conventional chemical approaches. In this study, the degradation of Indulin AT lignin was assessed by comparing the effects of Basidiomycetous fungi (Coriolus versicolour and Trametes gallica) and Actinobacteria (Mycobacterium sp. and Streptomyces sp.) to two commercial laccases, T. versicolour ( ≥ 10 U/mg) and C. versicolour ( ≥ 0.3 U/mg). After 54 days of cultivation, the extent of microbial degradation was significantly higher than that of commercial laccases, reaching a maximum of 38 wt% degradation for C. versicolour treated samples. Lignin degradation was further confirmed by thermal carbon analysis with a five-step temperature protocol. Compared with commercial laccases, a significant decrease in char formation at 850ºC was observed among all microbial-degraded lignins with a corresponding carbon percentage increase from 200ºC to 500ºC. To complement the carbon analysis result, chemical characterization of the degraded products at different stages of the delignification by microorganisms and commercial laccases was performed by Pyrolysis-GC-MS.

Keywords: lignin, microbial degradation, pyrolysis-GC-MS, thermal carbon analysis

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17 Peril´s Environment of Energetic Infrastructure Complex System, Modelling by the Crisis Situation Algorithms

Authors: Jiří F. Urbánek, Alena Oulehlová, Hana Malachová, Jiří J. Urbánek Jr.

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Crisis situations investigation and modelling are introduced and made within the complex system of energetic critical infrastructure, operating on peril´s environments. Every crisis situations and perils has an origin in the emergency/ crisis event occurrence and they need critical/ crisis interfaces assessment. Here, the emergency events can be expected - then crisis scenarios can be pre-prepared by pertinent organizational crisis management authorities towards their coping; or it may be unexpected - without pre-prepared scenario of event. But the both need operational coping by means of crisis management as well. The operation, forms, characteristics, behaviour and utilization of crisis management have various qualities, depending on real critical infrastructure organization perils, and prevention training processes. An aim is always - better security and continuity of the organization, which successful obtainment needs to find and investigate critical/ crisis zones and functions in critical infrastructure organization models, operating in pertinent perils environment. Our DYVELOP (Dynamic Vector Logistics of Processes) method is disposables for it. Here, it is necessary to derive and create identification algorithm of critical/ crisis interfaces. The locations of critical/ crisis interfaces are the flags of crisis situation in organization of critical infrastructure models. Then, the model of crisis situation will be displayed at real organization of Czech energetic crisis infrastructure subject in real peril environment. These efficient measures are necessary for the infrastructure protection. They will be derived for peril mitigation, crisis situation coping and for environmentally friendly organization survival, continuity and its sustainable development advanced possibilities.

Keywords: algorithms, energetic infrastructure complex system, modelling, peril´s environment

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16 High Expression Levels and Amplification of rRNA Genes in a Mentally Retarded Child with 13p+: A Familial Case Study

Authors: Irina S. Kolesnikova, Alexander A. Dolskiy, Natalya A. Lemskaya, Yulia V. Maksimova, Asia R. Shorina, Alena S. Telepova, Alexander S. Graphodatsky, Dmitry V. Yudkin

Abstract:

A cytogenetic and molecular genetic study of the family with a male child who had mental retardation and autistic features revealed an abnormal chromosome 13 bearing an enlarged p-arm with amplified ribosomal DNA (rDNA) in a boy and his father. Cytogenetic analysis using standard G-banding and FISH with labeled rDNA probes revealed an abnormal chromosome 13 with an enlarged p-arms due to rDNA amplification in a male child, who had clinically confirmed mental retardation and an autistic behavior. This chromosome is evidently inherited from the father, who has morphologically the same chromosome, but is healthy. The karyotype of the mother was normal. Ag-NOR staining showed brightly stained large whole-p-arm nucleolus organizer regions (NORs) in a child and normal-sized NORs in his father with 13p+-NOR-amount mosaicism. qRT-PCR with specific primers showed highly increased levels of 18S, 28S and 5,8 S ribosomal RNA (rRNA) in the patient’s blood samples compared to a normal healthy control donor. Both patient’s father and mother had no elevated levels of rRNAs expression. Thus, in this case, rRNA level seems to correlate with mental retardation in familial individuals with 13p+. Our findings of rRNA overexpression in a patient with mental retardation and his parents may show a possible link between the karyotype (p-arm enlargement due to rDNA amplification), rDNA functionality (rRNA overexpression), functional changes in the brain and mental retardation. The study is supported by Russian Science Foundation Grant 15-15-10001.

Keywords: mental retardation, ribosomal DNA–rDNA, ribosomal RNA–rRNA, nucleolus organizer region–NOR, chromosome 13

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15 Experimental Assessment of the Effectiveness of Judicial Instructions and of Expert Testimony in Improving Jurors’ Evaluation of Eyewitness Evidence

Authors: Alena Skalon, Jennifer L. Beaudry

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Eyewitness misidentifications can sometimes lead to wrongful convictions of innocent people. This occurs in part because jurors tend to believe confident eyewitnesses even when the identification took place under suggestive conditions. Empirical research demonstrated that jurors are often unaware of the factors that can influence the reliability of eyewitness identification. Most common legal safeguards that are designed to educate jurors about eyewitness evidence are judicial instructions and expert testimony. To date, very few studies assessed the effectiveness of judicial instructions and most of them found that judicial instructions make jurors more skeptical of eyewitness evidence or do not have any effect on jurors’ judgments. Similar results were obtained for expert testimony. However, none of the previous studies focused on the ability of legal safeguards to improve jurors’ assessment of evidence obtained from suggestive identification procedures—this is one of the gaps addressed by this paper. Furthermore, only three studies investigated whether legal safeguards improve the ultimate accuracy of jurors’ judgments—that is, whether after listening to judicial instructions or expert testimony jurors can differentiate between accurate and inaccurate eyewitnesses. This presentation includes two studies. Both studies used genuine eyewitnesses (i.e., eyewitnesses who watched the crime) and manipulated the suggestiveness of identification procedures. The first study manipulated the presence of judicial instructions; the second study manipulated the presence of one of two types of expert testimony: a traditional, verbal expert testimony or expert testimony accompanied by visual aids. All participant watched a video-recording of an identification procedure and of an eyewitness testimony. The results indicated that neither judicial instructions nor expert testimony affected jurors’ judgments. However, consistent with the previous findings, when the identification procedure was non-suggestive, jurors believed accurate eyewitnesses more often than inaccurate eyewitnesses. When the procedure was suggestive, jurors believed accurate and inaccurate eyewitnesses at the same rate. The paper will discuss the implications of these studies and directions for future research.

Keywords: expert testimony, eyewitness evidence, judicial instructions, jurors’ decision making, legal safeguards

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14 Numerical Simulation of Precast Concrete Panels for Airfield Pavement

Authors: Josef Novák, Alena Kohoutková, Vladimír Křístek, Jan Vodička

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Numerical analysis software belong to the main tools for simulating the real behavior of various concrete structures and elements. In comparison with experimental tests, they offer an affordable way to study the mechanical behavior of structures under various conditions. The contribution deals with a precast element of an innovative airfield pavement system which is being developed within an ongoing scientific project. The proposed system consists a two-layer surface course of precast concrete panels positioned on a two-layer base of fiber-reinforced concrete with recycled aggregate. As the panels are supposed to be installed directly on the hardened base course, imperfections at the interface between the base course and surface course are expected. Considering such circumstances, three various behavior patterns could be established and considered when designing the precast element. Enormous costs of full-scale experiments force to simulate the behavior of the element in a numerical analysis software using finite element method. The simulation was conducted on a nonlinear model in order to obtain such results which could fully compensate results from the experiments. First, several loading schemes were considered with the aim to observe the critical one which was used for the simulation later on. The main objective of the simulation was to optimize reinforcement of the element subject to quasi-static loading from airplanes. When running the simulation several parameters were considered. Namely, it concerns geometrical imperfections, manufacturing imperfections, stress state in reinforcement, stress state in concrete and crack width. The numerical simulation revealed that the precast element should be heavily reinforced to fulfill all the demands assumed. The main cause of using high amount of reinforcement is the size of the imperfections which could occur at real structure. Improving manufacturing quality, the installation of the precast panels on a fresh base course or using a bedding layer underneath the surface course belong to the main steps how to reduce the size of imperfections and consequently lower the consumption of reinforcement.

Keywords: nonlinear analysis, numerical simulation, precast concrete, pavement

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13 Peculiarities of Microflora of Odontogenic Inflammatory Processes in the Central Kazakhstan Region

Authors: Aliya Tokbergenova, Maida Tusupbekova, Daulet Dzhangaliyev, Alena Lavrinenko

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Background: Odontogenic phlegmons are ranked the first among pyoinflammatory processes in the frequency of hospitalization in maxillofacial surgery in the post-Soviet countries. The main role in etiology is played by obligate anaerobes and aerobes. According to numerous data, the structure of aerobic pathogens is dominated by staphylococci and gram-negative bacteria. Aim: The research aim is to study the microflora of the purulent discharge odontogenic inflammatory processes. Materials and methods: A total of 220 patients have been examined, of which 120 patients aged 25-59 years have been included in the research who did not have comorbidity hospitalized in the maxillofacial hospital in Karaganda (Kazakhstan) from January 2016 to July 2017. The bacteriological research has been carried out on the basis of the multiaccess laboratory of the KSMU, through the Matrix Assisted Laser Desorption/Ionization (MALDI) apparatus. The material sample was pus from the inflammation focus, taken during the operating period. Results: According to the research among 120 patients (100%), 15 patients (12.5%) have had microorganisms not grown. From 105 (87.5%) bacteriological results, it has been revealed the following 1) Streptococcus: 51 (42.5%): Streptococcus beta-haemolytic: 17 (14.2%), Streptococcus pneumoniae: 12 (10%), Streptococcus anginosus: 8 (6.6%), Streptococcus oralis: 8 (6.6%), Streptococcus constellatus: 6 (5.0%); 2) Staphylococci: 27 (22.5%): Staphylococci aureus: 14 (11.7%) and Staphylococci epidermidis: 13 (10.8%); 3) Pseudomonas aeruginosa: 12 (10%); 4) Neisseria: 11 (9.1%): Neisseria mucosa: 5 (4.1%) and Neisseria macacae: 6 (5.0%); 5) Klebsiella pneumoniae: 2 (1.7%); 6) Stenotrophomonas maltophilia: 2 (1.7%). 15 patients (12.5%) experienced complications in the form of 1) The dissemination of the process in 10 patients (8.4%). 2) Osteomyelitis in 3 (2.5%). 3) Mediastinitis in 1 (0.8%). 4) Sinusitis in 1 (0.8%). 15 patients (100%) were carried out repeated bacteriological examination, the following was revealed: 1) Streptococcus: 10 (66.7%): Streptococcus beta-haemolytic: 4 (26.7%), Streptococcus pneumoniae: 2 (13.3%), Streptococcus аnginosus: 2 (13.3%), Streptococcus oralis: 1 (6.7%), Streptococcus constellatus: 1 (6.7%); 2) Staphylococci: 4 (26.7%): Staphylococci aureus: 3 (20%) and Staphylococci epidermidis: 1 (6.7%); 3) Pseudomonas aeruginosa: 1 (6.7%). Conclusions: Thus, according to our research data, streptococci predominate in the odontogenic processes microflora in aerobic flora in the central Kazakhstan region, which refutes the leading role of staphylococci in the development of odontogenic inflammatory processes, thus creating prerequisites for studying new treatment approaches.

Keywords: maxillofacial surgery, microflora, odontogenic phlegmons, pyo-inflammatory

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12 Two-Component Biocompartible Material for Reconstruction of Articular Hyaline Cartilage

Authors: Alena O. Stepanova, Vera S. Chernonosova, Tatyana S. Godovikova, Konstantin A. Bulatov, Andrey Y. Patrushev, Pavel P. Laktionov

Abstract:

Trauma and arthrosis, not to mention cartilage destruction in overweight and elders put hyaline cartilage lesion among the most frequent diseases of locomotor system. These problems combined with low regeneration potential of the cartilage make regeneration of articular cartilage a high-priority task of tissue engineering. Many types of matrices, the procedures of their installation and autologous chondrocyte implantation protocols were offered, but certain aspects including adhesion of the implant with surrounding cartilage/bone, prevention of the ossification and fibrosis were not resolved. Simplification and acceleration of the procedures resulting in restoration of normal cartilage are also required. We have demonstrated that human chondroblasts can be successfully cultivated at the surface of electrospun scaffolds and produce extracellular matrix components in contrast to chondroblasts grown in homogeneous hydrogels. To restore cartilage we offer to use stacks of electrospun scaffolds fixed with photopolymerized solution of prepared from gelatin and chondroitin-4-sulfate both modified by glycidyl methacrylate and non-toxic photoinitator Darocur 2959. Scaffolds were prepared from nylon 6, polylactide-co-glicolide and their mixtures with modified gelatin. Illumination of chondroblasts in photopolymerized solution using 365 nm LED light had no effect on cell viability at compressive strength of the gel less than0,12 MPa. Stacks of electrospun scaffolds provide good compressive strength and have the potential for substitution with cartilage when biodegradable scaffolds are used. Vascularization can be prevented by introduction of biostable scaffolds in the layers contacting the subchondral bone. Studies of two-component materials (2-3 sheets of electrospun scaffold) implanted in the knee-joints of rabbits and fixed by photopolymerization demonstrated good crush resistance, biocompatibility and good adhesion of the implant with surrounding cartilage. Histological examination of the implants 3 month after implantation demonstrates absence of any inflammation and signs of replacement of the biodegradable scaffolds with normal cartilage. The possibility of intraoperative population of the implants with autologous cells is being investigated.

Keywords: chondroblasts, electrospun scaffolds, hyaline cartilage, photopolymerized gel

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11 Towards Modern Approaches of Intelligence Measurement for Clinical and Educational Practices

Authors: Alena Kulikova, Tatjana Kanonire

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Intelligence research is one of the oldest fields of psychology. Many factors have made a research on intelligence, defined as reasoning and problem solving [1, 2], a very acute and urgent problem. Thus, it has been repeatedly shown that intelligence is a predictor of academic, professional, and social achievement in adulthood (for example, [3]); Moreover, intelligence predicts these achievements better than any other trait or ability [4]. The individual level, a comprehensive assessment of intelligence is a necessary criterion for the diagnosis of various mental conditions. For example, it is a necessary condition for psychological, medical and pedagogical commissions when deciding on educational needs and the most appropriate educational programs for school children. Assessment of intelligence is crucial in clinical psychodiagnostic and needs high-quality intelligence measurement tools. Therefore, it is not surprising that the development of intelligence tests is an essential part of psychological science and practice. Many modern intelligence tests have a long history and have been used for decades, for example, the Stanford-Binet test or the Wechsler test. However, the vast majority of these tests are based on the classic linear test structure, in which all respondents receive all tasks (see, for example, a critical review by [5]). This understanding of the testing procedure is a legacy of the pre-computer era, in which blank testing was the only diagnostic procedure available [6] and has some significant limitations that affect the reliability of the data obtained [7] and increased time costs. Another problem with measuring IQ is that classical line-structured tests do not fully allow to measure respondent's intellectual progress [8], which is undoubtedly a critical limitation. Advances in modern psychometrics allow for avoiding the limitations of existing tools. However, as in any rapidly developing industry, at the moment, psychometrics does not offer ready-made and straightforward solutions and requires additional research. In our presentation we would like to discuss the strengths and weaknesses of the current approaches to intelligence measurement and highlight “points of growth” for creating a test in accordance with modern psychometrics. Whether it is possible to create the instrument that will use all achievements of modern psychometric and remain valid and practically oriented. What would be the possible limitations for such an instrument? The theoretical framework and study design to create and validate the original Russian comprehensive computer test for measuring the intellectual development in school-age children will be presented.

Keywords: Intelligence, psychometrics, psychological measurement, computerized adaptive testing, multistage testing

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10 Stress-Strain Relation for Hybrid Fiber Reinforced Concrete at Elevated Temperature

Authors: Josef Novák, Alena Kohoutková

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The performance of concrete structures in fire depends on several factors which include, among others, the change in material properties due to the fire. Today, fiber reinforced concrete (FRC) belongs to materials which have been widely used for various structures and elements. While the knowledge and experience with FRC behavior under ambient temperature is well-known, the effect of elevated temperature on its behavior has to be deeply investigated. This paper deals with an experimental investigation and stress‑strain relations for hybrid fiber reinforced concrete (HFRC) which contains siliceous aggregates, polypropylene and steel fibers. The main objective of the experimental investigation is to enhance a database of mechanical properties of concrete composites with addition of fibers subject to elevated temperature as well as to validate existing stress-strain relations for HFRC. Within the investigation, a unique heat transport test, compressive test and splitting tensile test were performed on 150 mm cubes heated up to 200, 400, and 600 °C with the aim to determine a time period for uniform heat distribution in test specimens and the mechanical properties of the investigated concrete composite, respectively. Both findings obtained from the presented experimental test as well as experimental data collected from scientific papers so far served for validating the computational accuracy of investigated stress-strain relations for HFRC which have been developed during last few years. Owing to the presence of steel and polypropylene fibers, HFRC becomes a unique material whose structural performance differs from conventional plain concrete when exposed to elevated temperature. Polypropylene fibers in HFRC lower the risk of concrete spalling as the fibers burn out shortly with increasing temperature due to low ignition point and as a consequence pore pressure decreases. On the contrary, the increase in the concrete porosity might affect the mechanical properties of the material. To validate this thought requires enhancing the existing result database which is very limited and does not contain enough data. As a result of the poor database, only few stress-strain relations have been developed so far to describe the structural performance of HFRC at elevated temperature. Moreover, many of them are inconsistent and need to be refined. Most of them also do not take into account the effect of both a fiber type and fiber content. Such approach might be vague especially when high amount of polypropylene fibers are used. Therefore, the existing relations should be validated in detail based on other experimental results.

Keywords: elevated temperature, fiber reinforced concrete, mechanical properties, stress strain relation

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9 Social Skills as a Significant Aspect of a Successful Start of Compulsory Education

Authors: Eva Šmelová, Alena Berčíková

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The issue of school maturity and readiness of a child for a successful start of compulsory education is one of the long-term monitored areas, especially in the context of education and psychology. In the context of the curricular reform in the Czech Republic, the issue has recently gained importance. Analyses of research in this area suggest a lack of a broader overview of indicators informing about the current level of children’s school maturity and school readiness. Instead, various studies address partial issues. Between 2009 and 2013 a research study was performed at the Faculty of Education, Palacký University Olomouc (Czech Republic) focusing on children’s maturity and readiness for compulsory education. In this study, social skills were of marginal interest; the main focus was on the mental area. This previous research is smoothly linked with the present study, the objective of which is to identify the level of school maturity and school readiness in selected characteristics of social skills as part of the adaptation process after enrolment in compulsory education. In this context, the following research question has been formulated: During the process of adaptation to the school environment, which social skills are weakened? The method applied was observation, for the purposes of which the authors developed a research tool – record sheet with 11 items – social skills that a child should have by the end of preschool education. The items were assessed by first-grade teachers at the beginning of the school year. The degree of achievement and intensity of the skills were assessed for each child using an assessment scale. In the research, the authors monitored a total of three independent variables (gender, postponement of school attendance, participation in inclusive education). The effect of these independent variables was monitored using 11 dependent variables. These variables are represented by the results achieved in selected social skills. Statistical data processing was assisted by the Computer Centre of Palacký University Olomouc. Statistical calculations were performed using SPSS v. 12.0 for Windows and STATISTICA: StatSoft STATISTICA CR, Cz (software system for data analysis). The research sample comprised 115 children. In their paper, the authors present the results of the research and at the same time point to possible areas of further investigation. They also highlight possible risks associated with weakened social skills.

Keywords: compulsory education, curricular reform, educational diagnostics, pupil, school curriculum, school maturity, school readiness, social skills

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8 Multimodal Analysis of News Magazines' Front-Page Portrayals of the US, Germany, China, and Russia

Authors: Alena Radina

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On the global stage, national image is shaped by historical memory of wars and alliances, government ideology and particularly media stereotypes which represent countries in positive or negative ways. News magazine covers are a key site for national representation. The object of analysis in this paper is the portrayals of the US, Germany, China, and Russia in the front pages and cover stories of “Time”, “Der Spiegel”, “Beijing Review”, and “Expert”. Political comedy helps people learn about current affairs even if politics is not their area of interest, and thus satire indirectly sets the public agenda. Coupled with satirical messages, cover images and the linguistic messages embedded in the covers become persuasive visual and verbal factors, known to drive about 80% of magazine sales. Preliminary analysis identified satirical elements in magazine covers, which are known to influence and frame understandings and attract younger audiences. Multimodal and transnational comparative framing analyses lay the groundwork to investigate why journalists, editors and designers deploy certain frames rather than others. This research investigates to what degree frames used in covers correlate with frames within the cover stories and what these framings can tell us about media professionals’ representations of their own and other nations. The study sample includes 32 covers consisting of two covers representing each of the four chosen countries from the four magazines. The sampling framework considers two time periods to compare countries’ representation with two different presidents, and between men and women when present. The countries selected for analysis represent each category of the international news flows model: the core nations are the US and Germany; China is a semi-peripheral country; and Russia is peripheral. Examining textual and visual design elements on the covers and images in the cover stories reveals not only what editors believe visually attracts the reader’s attention to the magazine but also how the magazines frame and construct national images and national leaders. The cover is the most powerful editorial and design page in a magazine because images incorporate less intrusive framing tools. Thus, covers require less cognitive effort of audiences who may therefore be more likely to accept the visual frame without question. Analysis of design and linguistic elements in magazine covers helps to understand how media outlets shape their audience’s perceptions and how magazines frame global issues. While previous multimodal research of covers has focused mostly on lifestyle magazines or newspapers, this paper examines the power of current affairs magazines’ covers to shape audience perception of national image.

Keywords: framing analysis, magazine covers, multimodality, national image, satire

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7 Detection, Analysis and Determination of the Origin of Copy Number Variants (CNVs) in Intellectual Disability/Developmental Delay (ID/DD) Patients and Autistic Spectrum Disorders (ASD) Patients by Molecular and Cytogenetic Methods

Authors: Pavlina Capkova, Josef Srovnal, Vera Becvarova, Marie Trkova, Zuzana Capkova, Andrea Stefekova, Vaclava Curtisova, Alena Santava, Sarka Vejvalkova, Katerina Adamova, Radek Vodicka

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ASDs are heterogeneous and complex developmental diseases with a significant genetic background. Recurrent CNVs are known to be a frequent cause of ASD. These CNVs can have, however, a variable expressivity which results in a spectrum of phenotypes from asymptomatic to ID/DD/ASD. ASD is associated with ID in ~75% individuals. Various platforms are used to detect pathogenic mutations in the genome of these patients. The performed study is focused on a determination of the frequency of pathogenic mutations in a group of ASD patients and a group of ID/DD patients using various strategies along with a comparison of their detection rate. The possible role of the origin of these mutations in aetiology of ASD was assessed. The study included 35 individuals with ASD and 68 individuals with ID/DD (64 males and 39 females in total), who underwent rigorous genetic, neurological and psychological examinations. Screening for pathogenic mutations involved karyotyping, screening for FMR1 mutations and for metabolic disorders, a targeted MLPA test with probe mixes Telomeres 3 and 5, Microdeletion 1 and 2, Autism 1, MRX and a chromosomal microarray analysis (CMA) (Illumina or Affymetrix). Chromosomal aberrations were revealed in 7 (1 in the ASD group) individuals by karyotyping. FMR1 mutations were discovered in 3 (1 in the ASD group) individuals. The detection rate of pathogenic mutations in ASD patients with a normal karyotype was 15.15% by MLPA and CMA. The frequencies of the pathogenic mutations were 25.0% by MLPA and 35.0% by CMA in ID/DD patients with a normal karyotype. CNVs inherited from asymptomatic parents were more abundant than de novo changes in ASD patients (11.43% vs. 5.71%) in contrast to the ID/DD group where de novo mutations prevailed over inherited ones (26.47% vs. 16.18%). ASD patients shared more frequently their mutations with their fathers than patients from ID/DD group (8.57% vs. 1.47%). Maternally inherited mutations predominated in the ID/DD group in comparison with the ASD group (14.7% vs. 2.86 %). CNVs of an unknown significance were found in 10 patients by CMA and in 3 patients by MLPA. Although the detection rate is the highest when using CMA, recurrent CNVs can be easily detected by MLPA. CMA proved to be more efficient in the ID/DD group where a larger spectrum of rare pathogenic CNVs was revealed. This study determined that maternally inherited highly penetrant mutations and de novo mutations more often resulted in ID/DD without ASD in patients. The paternally inherited mutations could be, however, a source of the greater variability in the genome of the ASD patients and contribute to the polygenic character of the inheritance of ASD. As the number of the subjects in the group is limited, a larger cohort is needed to confirm this conclusion. Inherited CNVs have a role in aetiology of ASD possibly in combination with additional genetic factors - the mutations elsewhere in the genome. The identification of these interactions constitutes a challenge for the future. Supported by MH CZ – DRO (FNOl, 00098892), IGA UP LF_2016_010, TACR TE02000058 and NPU LO1304.

Keywords: autistic spectrum disorders, copy number variant, chromosomal microarray, intellectual disability, karyotyping, MLPA, multiplex ligation-dependent probe amplification

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6 Antimicrobial and Anti-Biofilm Activity of Non-Thermal Plasma

Authors: Jan Masak, Eva Kvasnickova, Vladimir Scholtz, Olga Matatkova, Marketa Valkova, Alena Cejkova

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Microbial colonization of medical instruments, catheters, implants, etc. is a serious problem in the spread of nosocomial infections. Biofilms exhibit enormous resistance to environment. The resistance of biofilm populations to antibiotic or biocides often increases by two to three orders of magnitude in comparison with suspension populations. Subjects of interests are substances or physical processes that primarily cause the destruction of biofilm, while the released cells can be killed by existing antibiotics. In addition, agents that do not have a strong lethal effect do not cause such a significant selection pressure to further enhance resistance. Non-thermal plasma (NTP) is defined as neutral, ionized gas composed of particles (photons, electrons, positive and negative ions, free radicals and excited or non-excited molecules) which are in permanent interaction. In this work, the effect of NTP generated by the cometary corona with a metallic grid on the formation and stability of biofilm and metabolic activity of cells in biofilm was studied. NTP was applied on biofilm populations of Staphylococcus epidermidis DBM 3179, Pseudomonas aeruginosa DBM 3081, DBM 3777, ATCC 15442 and ATCC 10145, Escherichia coli DBM 3125 and Candida albicans DBM 2164 grown on solid media on Petri dishes and on the titanium alloy (Ti6Al4V) surface used for the production joint replacements. Erythromycin (for S. epidermidis), polymyxin B (for E. coli and P. aeruginosa), amphotericin B (for C. albicans) and ceftazidime (for P. aeruginosa) were used to study the combined effect of NTP and antibiotics. Biofilms were quantified by crystal violet assay. Metabolic activity of the cells in biofilm was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric test based on the reduction of MTT into formazan by the dehydrogenase system of living cells. Fluorescence microscopy was applied to visualize the biofilm on the surface of the titanium alloy; SYTO 13 was used as a fluorescence probe to stain cells in the biofilm. It has been shown that biofilm populations of all studied microorganisms are very sensitive to the type of used NTP. The inhibition zone of biofilm recorded after 60 minutes exposure to NTP exceeded 20 cm², except P. aeruginosa DBM 3777 and ATCC 10145, where it was about 9 cm². Also metabolic activity of cells in biofilm differed for individual microbial strains. High sensitivity to NTP was observed in S. epidermidis, in which the metabolic activity of biofilm decreased after 30 minutes of NTP exposure to 15% and after 60 minutes to 1%. Conversely, the metabolic activity of cells of C. albicans decreased to 53% after 30 minutes of NTP exposure. Nevertheless, this result can be considered very good. Suitable combinations of exposure time of NTP and the concentration of antibiotic achieved in most cases a remarkable synergic effect on the reduction of the metabolic activity of the cells of the biofilm. For example, in the case of P. aeruginosa DBM 3777, a combination of 30 minutes of NTP with 1 mg/l of ceftazidime resulted in a decrease metabolic activity below 4%.

Keywords: anti-biofilm activity, antibiotic, non-thermal plasma, opportunistic pathogens

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5 Working Memory and Audio-Motor Synchronization in Children with Different Degrees of Central Nervous System's Lesions

Authors: Anastasia V. Kovaleva, Alena A. Ryabova, Vladimir N. Kasatkin

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Background: The most simple form of entrainment to a sensory (typically auditory) rhythmic stimulus involves perceiving and synchronizing movements with an isochronous beat with one level of periodicity, such as that produced by a metronome. Children with pediatric cancer usually treated with chemo- and radiotherapy. Because of such treatment, psychologists and health professionals declare cognitive and motor abilities decline in cancer patients. The purpose of our study was to measure working memory characteristics with association with audio-motor synchronization tasks, also involved some memory resources, in children with different degrees of central nervous system lesions: posterior fossa tumors, acute lymphoblastic leukemia, and healthy controls. Methods: Our sample consisted of three groups of children: children treated for posterior fossa tumors (PFT-group, n=42, mean age 12.23), children treated for acute lymphoblastic leukemia (ALL-group, n=11, mean age 11.57) and neurologically healthy children (control group, n=36, mean age 11.67). Participants were tested for working memory characteristics with Cambridge Neuropsychological Test Automated Battery (CANTAB). Pattern recognition memory (PRM) and spatial working memory (SWM) tests were applied. Outcome measures of PRM test include the number and percentage of correct trials and latency (speed of participant’s response), and measures of SWM include errors, strategy, and latency. In the synchronization tests, the instruction was to tap out a regular beat (40, 60, 90 and 120 beats per minute) in synchrony with the rhythmic sequences that were played. This meant that for the sequences with an isochronous beat, participants were required to tap into every auditory event. Variations of inter-tap-intervals and deviations of children’s taps from the metronome were assessed. Results: Analysis of variance revealed the significant effect of group (ALL, PFT and control) on such parameters as short-term PRM, SWM strategy and errors. Healthy controls demonstrated more correctly retained elements, better working memory strategy, compared to cancer patients. Interestingly that ALL patients chose the bad strategy, but committed significantly less errors in SWM test then PFT and controls did. As to rhythmic ability, significant associations of working memory were found out only with 40 bpm rhythm: the less variable were inter-tap-intervals of the child, the more elements in memory he/she could retain. The ability to audio-motor synchronization may be related to working memory processes mediated by the prefrontal cortex whereby each sensory event is actively retrieved and monitored during rhythmic sequencing. Conclusion: Our results suggest that working memory, tested with appropriate cognitive methods, is associated with the ability to synchronize movements with rhythmic sounds, especially in sub-second intervals (40 per minute).

Keywords: acute lymphoblastic leukemia (ALL), audio-motor synchronization, posterior fossa tumor, working memory

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4 Scenario-Based Scales and Situational Judgment Tasks to Measure the Social and Emotional Skills

Authors: Alena Kulikova, Leonid Parmaksiz, Ekaterina Orel

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Social and emotional skills are considered by modern researchers as predictors of a person's success both in specific areas of activity and in the life of a person as a whole. The popularity of this scientific direction ensures the emergence of a large number of practices aimed at developing and evaluating socio-emotional skills. Assessment of social and emotional development is carried out at the national level, as well as at the level of individual regions and institutions. Despite the fact that many of the already existing social and emotional skills assessment tools are quite convenient and reliable, there are now more and more new technologies and task formats which improve the basic characteristics of the tools. Thus, the goal of the current study is to develop a tool for assessing social and emotional skills such as emotion recognition, emotion regulation, empathy and a culture of self-care. To develop a tool assessing social and emotional skills, Rasch-Gutman scenario-based approach was used. This approach has shown its reliability and merit for measuring various complex constructs: parental involvement; teacher practices that support cultural diversity and equity; willingness to participate in the life of the community after psychiatric rehabilitation; educational motivation and others. To assess emotion recognition, we used a situational judgment task based on OCC (Ortony, Clore, and Collins) emotions theory. The main advantage of these two approaches compare to classical Likert scales is that it reduces social desirability in answers. A field test to check the psychometric properties of the developed instrument was conducted. The instrument was developed for the presidential autonomous non-profit organization “Russia - Land of Opportunity” for nationwide soft skills assessment among higher education students. The sample for the field test consisted of 500 people, students aged from 18 to 25 (mean = 20; standard deviation 1.8), 71% female. 67% of students are only studying and are not currently working and 500 employed adults aged from 26 to 65 (mean = 42.5; SD 9), 57% female. Analysis of the psychometric characteristics of the scales was carried out using the methods of IRT (Item Response Theory). A one-parameter rating scale model RSM (Rating scale model) and Graded Response model (GRM) of the modern testing theory were applied. GRM is a polyatomic extension of the dichotomous two-parameter model of modern testing theory (2PL) based on the cumulative logit function for modeling the probability of a correct answer. The validity of the developed scales was assessed using correlation analysis and MTMM (multitrait-multimethod matrix). The developed instrument showed good psychometric quality and can be used by HR specialists or educational management. The detailed results of a psychometric study of the quality of the instrument, including the functioning of the tasks of each scale, will be presented. Also, the results of the validity study by MTMM analysis will be discussed.

Keywords: social and emotional skills, psychometrics, MTMM, IRT

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3 A Novel Concept of Optical Immunosensor Based on High-Affinity Recombinant Protein Binders for Tailored Target-Specific Detection

Authors: Alena Semeradtova, Marcel Stofik, Lucie Mareckova, Petr Maly, Ondrej Stanek, Jan Maly

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Recently, novel strategies based on so-called molecular evolution were shown to be effective for the production of various peptide ligand libraries with high affinities to molecular targets of interest comparable or even better than monoclonal antibodies. The major advantage of these peptide scaffolds is mainly their prevailing low molecular weight and simple structure. This study describes a new high-affinity binding molecules based immunesensor using a simple optical system for human serum albumin (HSA) detection as a model molecule. We present a comparison of two variants of recombinant binders based on albumin binding domain of the protein G (ABD) performed on micropatterned glass chip. Binding domains may be tailored to any specific target of interest by molecular evolution. Micropatterened glass chips were prepared using UV-photolithography on chromium sputtered glasses. Glass surface was modified by (3-aminopropyl)trietoxysilane and biotin-PEG-acid using EDC/NHS chemistry. Two variants of high-affinity binding molecules were used to detect target molecule. Firstly, a variant is based on ABD domain fused with TolA chain. This molecule is in vivo biotinylated and each molecule contains one molecule of biotin and one ABD domain. Secondly, the variant is ABD domain based on streptavidin molecule and contains four gaps for biotin and four ABD domains. These high-affinity molecules were immobilized to the chip surface via biotin-streptavidin chemistry. To eliminate nonspecific binding 1% bovine serum albumin (BSA) or 6% fetal bovine serum (FBS) were used in every step. For both variants range of measured concentrations of fluorescently labelled HSA was 0 – 30 µg/ml. As a control, we performed a simultaneous assay without high-affinity binding molecules. Fluorescent signal was measured using inverse fluorescent microscope Olympus IX 70 with COOL LED pE 4000 as a light source, related filters, and camera Retiga 2000R as a detector. The fluorescent signal from non-modified areas was substracted from the signal of the fluorescent areas. Results were presented in graphs showing the dependence of measured grayscale value on the log-scale of HSA concentration. For the TolA variant the limit of detection (LOD) of the optical immunosensor proposed in this study is calculated to be 0,20 µg/ml for HSA detection in 1% BSA and 0,24 µg/ml in 6% FBS. In the case of streptavidin-based molecule, it was 0,04 µg/ml and 0,07 µg/ml respectively. The dynamical range of the immunosensor was possible to estimate just in the case of TolA variant and it was calculated to be 0,49 – 3,75 µg/ml and 0,73-1,88 µg/ml respectively. In the case of the streptavidin-based the variant we didn´t reach the surface saturation even with the 480 ug/ml concentration and the upper value of dynamical range was not estimated. Lower value was calculated to be 0,14 µg/ml and 0,17 µg/ml respectively. Based on the obtained results, it´s clear that both variants are useful for creating the bio-recognizing layer on immunosensors. For this particular system, it is obvious that the variant based on streptavidin molecule is more useful for biosensing on glass planar surfaces. Immunosensors based on this variant would exhibit better limit of detection and wide dynamical range.

Keywords: high affinity binding molecules, human serum albumin, optical immunosensor, protein G, UV-photolitography

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