Search results for: pilot production

Authors: Pierre-Louis Lucas, Corinne Loutelier-Bourhis, Narimane Mati-Baouche, Philippe Chan Tchi-Song, Patrice Lerouge, Elodie Mathieu-Rivet, Muriel Bardor

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N-glycosylation is a post-translational modification taking place in the Endoplasmic Reticulum and the Golgi apparatus where defined glycan features are added on protein in a very specific sequence Asn-X-Thr/Ser/Cys were X can be any amino acid except proline. Because it is well-established that those N-glycans play a critical role in protein biological activity, protein half-life and that a different N-glycan structure may induce an immune response, they are very important in Biopharmaceuticals which are mainly glycoproteins bearing N-glycans. From now, most of the biopharmaceuticals are produced by mammalian cells like Chinese Hamster Ovary cells (CHO) for their N-glycosylation similar to the human, but due to the high production costs, several other species are investigated as the possible alternative system. In this purpose, the green microalgae Chlamydomonas reinhardtii was investigated as the potential production system for Biopharmaceuticals. This choice was influenced by the facts that C. reinhardtii is a well-study microalgae which is growing fast with a lot of molecular biology tools available. This organism is also producing N-glycan on its endogenous proteins. However, the analysis of the N-glycan structure of this microalgae has revealed some differences as compared to the human. Rather than in Human where the glycans are processed by key enzymes called N-acetylglucosaminyltransferase I and II (GnTI and GnTII) adding GlcNAc residue to form a GlcNAc₂Man₃GlcNAc₂ core N-glycan, C. reinhardtii lacks those two enzymes and possess a GnTI independent glycosylation pathway. Moreover, some enzymes like xylosyltransferases and methyltransferases not present in human are supposed to act on the glycans of C. reinhardtii. Furthermore, the recent structural study by mass spectrometry shows that the N-glycosylation precursor supposed to be conserved in almost all eukaryotic cells results in a linear Man₅GlcNAc₂ rather than a branched one in C. reinhardtii. In this work, we will discuss the new released MS information upon C. reinhardtii N-glycan structure and their impact on our attempt to modify the glycan in a Human manner. Two strategies will be discussed. The first one consisted in the study of Xylosyltransferase insertional mutants from the CLIP library in order to remove xyloses from the N-glycans. The second will go further in the humanization by transforming the microalgae with the exogenous gene from Toxoplasma gondii having an activity similar to GnTI and GnTII with the aim to synthesize GlcNAc₂Man₃GlcNAc₂ in C. reinhardtii.

Keywords: Chlamydomonas reinhardtii, N-glycosylation, glycosyltransferase, mass spectrometry, humanization

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Authors: Ljiljana Stojković, Manja Zec, Maja Zivkovic, Maja Bundalo, Marija Glibetić, Dragan Alavantić, Aleksandra Stankovic

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Cardiovascular disease (CVD) is associated with alterations in DNA methylation, the latter modulated by dietary polyphenols. The present pilot study (part of the original clinical study registered as NCT02800967 at www.clinicaltrials.gov) aimed to investigate the impact of 4-week daily consumption of polyphenol-rich Aronia melanocarpa juice on Long Interspersed Nucleotide Element-1 (LINE-1) methylation in peripheral blood leukocytes, in subjects (n=34, age of 41.1±6.6 years) at moderate CVD risk, including an increased body mass index, central obesity, high normal blood pressure and/or dyslipidemia. The goal was also to examine whether factors known to affect DNA methylation, such as folate intake levels, MTHFR C677T gene variant, as well as the anthropometric and metabolic parameters, modulated the LINE-1 methylation levels upon consumption of polyphenol-rich Aronia juice. The experimental analysis of LINE-1 methylation was done by the MethyLight method. MTHFR C677T genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism method. Folate intake was assessed by processing the data from the food frequency questionnaire and repeated 24-hour dietary recalls. Serum lipid profile was determined by using Roche Diagnostics kits. The statistical analyses were performed using the Statistica software package. In women, after vs. before the treatment period, a significant decrease in LINE-1 methylation levels was observed (97.54±1.50% vs. 98.39±0.86%, respectively; P=0.01). The change (after vs. before treatment) in LINE-1 methylation correlated directly with MTHFR 677T allele presence, average daily folate intake and the change in serum low-density lipoprotein cholesterol, while inversely with the change in serum triacylglycerols (R=0.72, R2=0.52, adjusted R2=0.36, P=0.03). The current results imply potential cardioprotective effects of habitual polyphenol-rich Aronia juice consumption achieved through the modifications of DNA methylation pattern in subjects at CVD risk, which should be further confirmed. Hence, the precision nutrition-driven modulations of DNA methylation may become targets for new approaches in the prevention and treatment of CVD.

Keywords: Aronia melanocarpa, cardiovascular risk, LINE-1, methylation, peripheral blood leukocytes, polyphenol

Procedia PDF Downloads 186

Authors: Hoda Rahmani, Gary Weckman

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The body of research examining the integration of drones into various industries is expanding rapidly. Despite progress made in addressing the cybersecurity concerns for commercial drones, knowledge deficits remain in determining potential occupational hazards and risks of drone use to employees’ well-being and health in the workplace. This creates difficulty in identifying key approaches to risk mitigation strategies and thus reflects the need for raising awareness among employers, safety professionals, and policymakers about workplace drone-related accidents. The purpose of this study is to investigate the prevalence of and possible risk factors for drone-related mishaps by comparing the application of drones in construction with manufacturing industries. The chief reason for considering these specific sectors is to ascertain whether there exists any significant difference between indoor and outdoor flights since most construction sites use drones outside and vice versa. Therefore, the current research seeks to examine the causes and patterns of workplace drone-related mishaps and suggest possible ergonomic interventions through data collection. Potential ergonomic practices to mitigate hazards associated with flying drones could include providing operators with professional pieces of training, conducting a risk analysis, and promoting the use of personal protective equipment. For the purpose of data analysis, two data mining techniques, the random forest and association rule mining algorithms, will be performed to find meaningful associations and trends in data as well as influential features that have an impact on the occurrence of drone-related accidents in construction and manufacturing sectors. In addition, Spearman’s correlation and chi-square tests will be used to measure the possible correlation between different variables. Indeed, by recognizing risks and hazards, occupational safety stakeholders will be able to pursue data-driven and evidence-based policy change with the aim of reducing drone mishaps, increasing productivity, creating a safer work environment, and extending human performance in safe and fulfilling ways. This research study was supported by the National Institute for Occupational Safety and Health through the Pilot Research Project Training Program of the University of Cincinnati Education and Research Center Grant #T42OH008432.

Keywords: commercial drones, ergonomic interventions, occupational safety, pattern recognition

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Authors: Zuzana Sedrlova, Eva Slaninova, Ines Fritz, Christina Daffert, Stanislav Obruca

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Cyanobacteria are ecologically extremely important phototrophic gram-negative bacteria capable of oxygenic photosynthesis. They synthesize many interesting metabolites such as glycogen, carotenoids, but the most interesting metabolites are polyhydroxyalkanoates (PHA). The main advantage of cyanobacteria is the fact they do not require costly organic substrate and, oppositely, cyanobacteria can fix CO₂. PHA serves primarily as a carbon and energy source and occurs in the form of intracellular granules in bacterial cells. It is possible, PHA helps cyanobacteria to survive stress conditions since increased PHA synthesis was observed during cultivation in stress conditions. PHA is microbial biopolymers that are biodegradable with similar properties as petrochemical synthetic plastics. Production of PHA by heterotrophic bacteria is expensive; for price reduction waste materials as input, materials are used. Positively, cyanobacteria principally do not require organic carbon substrate since they are capable of CO₂ fixation. In this work, we demonstrated that stress conditions lead to the highest obtained yields of PHA in cyanobacterial cultures. Two cyanobacterial cultures from genera Synechocystis were used in this work. Cultivations were performed either in Erlenmayer flask or in tube multicultivator. Multiple stressors were applied on cyanobacterial cultures, and stressors include PHA precursors. PHA precursors are chemical substances and some of them do not occur naturally in the environment. Cultivation with the same PHA precursors in the same concentration led to a 1,6x higher amount of PHA when a multicultivator was used. The highest amount of PHA reached 25 % of PHA in dry cyanobacterial biomass. Both strains are capable of co-polymer synthesis in the presence of their structural precursor. The composition of co-polymer differs in Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Synechocystis sp. PCC 6803 cultivated with γ-butyrolakton accumulated co-polymer of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) the composition of the copolymer was 56 % of 4HB and 44 % of 3HB. The total amount of PHA, as well as yield of biomass, was lower than in control due to the toxic properties of γ-butyrolakton. Funding: This study was partly funded by the project GA19- 19-29651L of the Czech Science Foundation (GACR) and partly funded by the Austrian Science Fund (FWF), a project I 4082-B25. This work was supported by Brno, Ph.D. Talent – Funded by the Brno City Municipality.

Keywords: co-polymer, cyanobacteria, PHA, synechocystis

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Authors: D. Bjelica, T. Slavinski, V. Vukimrovic, D. Pavlovic, D. Bodroza, V. Dabetic

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Traditional methods and techniques used in higher education may be significantly persuasive on the university students' perception about quality of the teaching process. Students’ satisfaction with the university experience may be affected by chosen educational approaches. Contemporary project management trends recognize agile approaches' beneficial, so modern practice highlights their usage, especially in the IT industry. A key research question concerns the possibility of applying agile methods in youth education. As agile methodology pinpoint iteratively-incremental delivery of results, its employment could be remarkably fruitful in education. This paper demonstrates the agile concept's application in the university students’ education through the continuous delivery of student solutions. Therefore, based on the fundamental values and principles of the agile manifest, paper will analyze students' performance and learned lessons in their encounter with the agile environment. The research is based on qualitative and quantitative analysis that includes sprints, as preparation and realization of student tasks in shorter iterations. Consequently, the performance of student teams will be monitored through iterations, as well as the process of adaptive planning and realization. Grounded theory methodology has been used in this research, as so as descriptive statistics and Man Whitney and Kruskal Wallis test for group comparison. Developed constructs of the model will be showcase through qualitative research, then validated through a pilot survey, and eventually tested as a concept in the final survey. The paper highlights the variability of educational curricula based on university students' feedbacks, which will be collected at the end of every sprint and indicates to university students' satisfaction inconsistency according to approaches applied in education. Values delivered by the lecturers will also be continuously monitored; thus, it will be prioritizing in order to students' requests. Minimal viable product, as the early delivery of results, will be particularly emphasized in the implementation process. The paper offers both theoretical and practical implications. This research contains exceptional lessons that may be applicable by educational institutions in curriculum creation processes, or by lecturers in curriculum design and teaching. On the other hand, they can be beneficial regarding university students' satisfaction increscent in respect of teaching styles, gained knowledge, or even educational content.

Keywords: academic performances, agile, high education, university students' satisfaction

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Authors: Geeta Kumari, Prabu Vairakannu

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Underground coal gasification (UCG) technology is an efficient and an economic in-situ clean coal technology, which converts unmineable coals into calorific valuable gases. This technology avoids ash disposal, coal mining, and storage problems. CO₂ gas can be a potential gasifying medium for UCG. CO₂ is a greenhouse gas and, the liberation of this gas to the atmosphere from thermal power plant industries leads to global warming. Hence, the capture and reutilization of CO₂ gas are crucial for clean energy production. However, the reactivity of high ash Indian coals with CO₂ needs to be assessed. In the present study, two varieties of Indian coals (low ash and high ash) are used for thermogravimetric analyses (TGA). Two low ash north east Indian coals (LAC) and a typical high ash Indian coal (HAC) are procured from the coal mines of India. Low ash coal with 9% ash (LAC-1) and 4% ash (LAC-2) and high ash coal (HAC) with 42% ash are used for the study. TGA studies are carried out to evaluate the activation energy for pyrolysis and gasification of coal under N₂ and CO₂ atmosphere. Coats and Redfern method is used to estimate the activation energy of coal under different temperature regimes. Volumetric model is assumed for the estimation of the activation energy. The activation energy estimated under different temperature range. The inherent properties of coals play a major role in their reactivity. The results show that the activation energy decreases with the decrease in the inherent percentage of coal ash due to the ash layer hindrance. A reverse trend was observed with volatile matter. High volatile matter of coal leads to the estimation of low activation energy. It was observed that the activation energy under CO₂ atmosphere at 400-600°C is less as compared to N₂ inert atmosphere. At this temperature range, it is estimated that 15-23% reduction in the activation energy under CO₂ atmosphere. This shows the reactivity of CO₂ gas with higher hydrocarbons of the coal volatile matters. The reactivity of CO₂ with the volatile matter of coal might occur through dry reforming reaction in which CO₂ reacts with higher hydrocarbon, aromatics of the tar content. The observed trend of Ea in the temperature range of 150-200˚C and 400-600˚C is HAC > LAC-1 >LAC-2 in both N₂ and CO₂ atmosphere. At the temperature range of 850-1000˚C, higher activation energy is estimated when compared to those values in the temperature range of 400-600°C. Above 800°C, char gasification through Boudouard reaction progressed under CO₂ atmosphere. It was observed that 8-20 kJ/mol of activation energy is increased during char gasification above 800°C compared to volatile matter pyrolysis between the temperature ranges of 400-600°C. The overall activation energy of the coals in the temperature range of 30-1000˚C is higher in N₂ atmosphere than CO₂ atmosphere. It can be concluded that higher hydrocarbons such as tar effectively undergoes cracking and reforming reactions in presence of CO₂. Thus, CO₂ gas is beneficial for the production of high calorific value syngas using high ash Indian coals.

Keywords: clean coal technology, CO₂ gasification, activation energy, underground coal gasification

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Authors: Fereshteh Falah, Alireza Vasiee, Farideh Tabatabaei-Yazdi

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In this research, we investigated the usage of dairy sludge in the fermentation process and cyclosporin production. This bioactive compound is a metabolite produced by Tolypocladium inflatum. Results showed that about 200 ppm of cyclosporin can be produced in this fermentation. In order to have a proper and specific function, CyA must be free of any impurities, so we need purification. In this downstream processing, we used chromatographic extraction and evaluation of pharmacological activities of cyA. Results showed that the obtained metabolite has very high activity against Aspergilus niger (25mm clear zone). This cyclosporin was isolated for use as an antibiotic. The current research shows that this drug is very vital and commercially very important.

Keywords: fermentation, cyclosporin A, Tolypocladium inflatum, TLC

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Authors: J. O. Alabi

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Biogas is a clean burning, easily produced natural fuel that is an important source of energy for cooking and heating in rural areas and third world countries. Anaerobic bacteria inside biodigesters break down biomass to produce biogas. (Which is 70% methane)? Currently there is no simple way to compress and store biogas. So, in order to use biogas as a source of energy, a direct feed from biodigeser to the store tap or heater must be made. Any excess biogas is vented into the atmosphere, which is wasteful and car have a negative effect on the environment, we have been tasked with designing a system that will be able to compress biogas using an off-grid power supply, making the biogas portable and makes through the use of large-scale, shared biodigester. Our final design is a system that maximizes simplicity and safety while minimizing cost.

Keywords: biogas, biodigesters, natural fuel, bionanotechnology

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Authors: Bashair Al Riyami

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Background: Acylation stimulating protein (ASP) is a small basic protein that was isolated based on its function as a potent lipogenic factor. The role of ASP in lipid metabolism has been described in numerous studies. Several association studies suggest that ASP may play a prominent role in female fat metabolism and distribution. Progesterone is established as a female lipogenic hormone, however the mechanisms by which progesterone exert its effects are not fully understood. AIM: Since ASP is an established potent lipogenic factor with a known mechanism of action, in this study we aim to investigate acute effects of different hormone treatments on ASP levels in vivo after a fat load. Methods: This is a longitudinal study including 24 female wister rats that were randomly divided into 4 groups including controls (n=6). The rats were ovariectomized, and fourteen days later the fasting rats were injected subcutaneously with a single dose of different hormone treatments (progesterone, estrogen and testosterone). An hour later, olive was administered by oral gavage, and plasma blood samples were collected at several time points after oil administration for ASP and triglyceride measurements. Area under the curve (TG-AUC) was calculated to represent TG clearance Results: RM-ANCOVA and post-analysis showed that only the progesterone treated group had a significant postprandial ASP increase at two hours compared to basal levels and to the controls (439.8± 62.4 vs 253.45± 59.03 ug/ml), P= 0.04. Interestingly, increased postprandial ASP levels coordinated negatively with corresponding TG levels and TG-AUC across the postprandial period most apparent in the progesterone and testosterone treated groups that behaved in an opposite manner. ASP levels were 3-fold higher in the progesterone compared to the testosterone treated group, whereas TG-AUC was significantly lower in the progesterone treated group compared to the testosterone treated group. Conclusion: These findings suggest that progesterone treatment enhances ASP production and TG clearance in a simultaneous manner. The strong association of postprandial ASP levels and TG clearance in the progesterone treated group support the notion of a stimulatory role for progesterone on ASP mediated TG clearance. This is the first functional study to demonstrate a cause-effect relationship between hormone treatment and ASP levels in vivo. These findings are promising and may contribute to further understanding the mechanism of progesterone function as a female lipogenic hormone through enhancing ASP production and plasma levels.

Keywords: ASP, lipids, sex hormones, wister rats

Procedia PDF Downloads 331

Authors: Amanda Watson

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The goal of this project is to create the conditions wherein every teacher, especially subjectarea experts, sees themselves as a teacher of language and vocabulary. Assessment and observational data suggest that students are not getting the support they need in vocabulary and reading comprehension, and secondary teachers do not currently have the confidence or expertise to provide this support. This study seeks to examine the impact of 10-20 minutes of daily, targeted instruction around orthography and vocabulary on student competence with the navigation of complex vocabulary and comprehension of subject-specific concepts and texts. The first phase of the pilot included 6 participating classroom teachers of grades 9 and 10 English (95 students in total) who administered an initial reading comprehension assessment. The results of this assessment indicated that the vast majority of students were reading below grade level. Teachers were then provided with a slide deck of complete lessons on orthography, vocabulary (etymology, roots and affixes) and reading comprehension strategies. For five weeks, teachers delivered lessons with their students, implementing the recommended evidence-based teaching strategies. Students and teachers completed surveys to provide feedback on the value and impact of the method. The results confirmed that this was new learning for the students and that the teaching strategies improved engagement. The lessons succeeded in providing equitable access to challenge by simultaneously offering theoretical learning to proficient readers, and exposure and practice to weaker readers. A second reading comprehension was administered after 5 weeks of daily instruction. Average scores increased by 41%, and almost every student experienced progress. The first phase was not long enough to measure the impact of the method on vocabulary acquisition or reading comprehension of subject-specific texts, however. The project will use the results of the first phase to design the second phase, and new teaching and learning strategies will be added. The goals of the second phases are to increase motivation, and to grow the daily practice beyond English class and into science and / or math. This team will continue to document a continuation of the daily lessons, Commented [E1]: Please do not use rhetorical questions in the abstract. measure the impact of the strategies, and address questions about the correlation between daily practice and improvements in the skills students need for vocabulary acquisition and disciplinary reading comprehension.

Keywords: adolescent, comprehension, orthography, reading, vocabulary, etymology, word study, disciplinary, teaching strategies

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Authors: Michael Tupý, Jaroslav Císař, Pavel Mokrejš, Dagmar Měřínská, Alice Tesaříková-Svobodová

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The worldwide problem is that the recycled PVB is wildly stored in landfills. However, PVB have very similar chemical properties such as PVC. Moreover, both of them are used in plasticized form. Thus, the thermal properties of plasticized PVC obtained from primary production and the PVB was obtained by recycling of windshields are compared. It is carried out in order to find degradable conditions and decide if blend of PVB/PVC can be processable together. Tested PVC contained 38 % of plasticizer diisononyl phthalate (DINP) and PVB was plasticized with 28 % of triethylene glycol, bis(2-ethylhexanoate) (3GO). Thermal and thermo-oxidative decomposition of both vinyl polymers are compared such as DSC and OOT analysis. The tensile strength analysis is added.

Keywords: polyvinyl chloride, polyvinyl butyral, recycling, reprocessing, thermal analysis, decomposition

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Authors: V. Ziegler, F. Schneider, M. Pesch

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With the EDM264, GRIMM offers a solution for mobile short- and long-term measurements in outdoor areas and at production sites. For research as well as permanent areal observations on a near reference quality base. The model EDM264 features a powerful and robust measuring cell based on optical particle counting (OPC) principle with all the advantages that users of GRIMM's portable aerosol spectrometers are used to. The system is embedded in a compact weather-protection housing with all-weather sampling, heated inlet system, data logger, and meteorological sensor. With TSP, PM10, PM4, PM2.5, PM1, and PMcoarse, the EDM264 provides all fine dust fractions real-time, valid for outdoor applications and calculated with the proven GRIMM enviro-algorithm, as well as six additional dust mass fractions pm10, pm2.5, pm1, inhalable, thoracic and respirable for IAQ and workplace measurements. This highly versatile instrument performs real-time monitoring of particle number, particle size and provides information on particle surface distribution as well as dust mass distribution. GRIMM's EDM264 has 31 equidistant size channels, which are PSL traceable. A high-end data logger enables data acquisition and wireless communication via LTE, WLAN, or wired via Ethernet. Backup copies of the measurement data are stored in the device directly. The rinsing air function, which protects the laser and detector in the optical cell, further increases the reliability and long term stability of the EDM264 under different environmental and climatic conditions. The entire sample volume flow of 1.2 L/min is analyzed by 100% in the optical cell, which assures excellent counting efficiency at low and high concentrations and complies with the ISO 21501-1standard for OPCs. With all these features, the EDM264 is a world-leading dust monitor for precise monitoring of particulate matter and particle number concentration. This highly reliable instrument is an indispensable tool for many users who need to measure aerosol levels and air quality outdoors, on construction sites, or at production facilities.

Keywords: aerosol research, aerial observation, fence line monitoring, wild fire detection

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Authors: Samuel Attias, Elad Schiff, Zahi Arnon, Eran Ben-Arye, Yael Keshet, Ibrahim Matter, Boker Lital Keinan

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Background: Despite the extensive use of manual therapies, reflexology in particular, no validated tools have been developed to evaluate patients' beliefs, attitudes and expectations regarding reflexology. Such tools however are essential to improve the results of the reflexology treatment, by better adjusting it to the patients' attitudes and expectations. The tool also enables assessing correlations with clinical results of interventional studies using reflexology. Methods: The IBEAR (Inpatient Beliefs, Expectations and Attitudes toward Reflexology) tool contains 25 questions (8 demographic and 17 specifically addressing reflexology), and was constructed in several stages: brainstorming by a multidisciplinary team of experts; evaluation of each of the proposed questions by the experts' team; and assessment of the experts' degree of agreement per each question, based on a Likert 1-7 scale (1 – don't agree at all; 7 – agree completely). Cronbach's Alpha was computed to evaluate the questionnaire's reliability while the Factor analysis test was used for further validation (228 patients). The questionnaire was tested and re-tested (48h) on a group of 199 patients to assure clarity and reliability, using the Pearson coefficient and the Kappa test. It was modified based on these results into its final form. Results: After its construction, the IBEAR questionnaire passed the expert group's preliminary consensus, evaluation of the questions' clarity (from 5.1 to 7.0), inner validation (from 5.5 to 7) and structural validation (from 5.5 to 6.75). Factor analysis pointed to two content worlds in a division into 4 questions discussing attitudes and expectations versus 5 questions on belief and attitudes. Of the 221 questionnaires collected, a Cronbach's Alpha coefficient was calculated on nine questions relating to beliefs, expectations, and attitudes regarding reflexology. This measure stood at 0.716 (satisfactory reliability). At the Test-Retest stage, 199 research participants filled in the questionnaire a second time. The Pearson coefficient for all questions ranged between 0.73 and 0.94 (good to excellent reliability). As for dichotomic answers, Kappa scores ranged between 0.66 and 1.0 (mediocre to high). One of the questions was removed from the IBEAR following questionnaire validation. Conclusions: The present study provides evidence that the proposed IBEAR-16 questionnaire is a valid and reliable tool for the characterization of potential reflexology patients and may be effectively used in settings which include the evaluation of inpatients' beliefs, expectations, and attitudes toward reflexology.

Keywords: reflexology, attitude, expectation, belief, CAM, inpatient

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Authors: Fiorenzo Lenzi, Doris Abt, Besnik Bytyqi

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Recent developments in composite materials for the interior cabin market provide more sustainable solutions for industrial applications. One contribution comes from epoxy-based prepregs recently developed to substitute phenolic prepregs in order to reduce the environmental impact of their production process and to eliminate health and safety issues related to their handling. Another example is the use of Mica-based products for improving the fire protection of interior cabin parts. Minerals, such as Mica, can be used as reinforcement in composites to reduce the heat release rate or, more traditionally, to improve the burn-through performance of fuselage and cargo lining components.

Keywords: prepreg, epoxy, Mica, battery protection

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Authors: Zarine Avetisyan

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The process of speech production and transmission naturally implies the occurrence of certain defective assumptions and erroneous formulations which may be both spontaneous, caused by haste, carelessness, etc., or deliberate. Whether deliberate or not, fallacies always act by way of “faux pas”. In the latter case, we deal with fake or deceptive arguments which are the focus of the given paper. The paper departs from the assumption that fallacies are arguments that prove nothing. Additionally and more importantly, political discourse becomes the main domain for scholarly “cultivation” while pinning down fallacies. The fallacy of telling the truth but deliberately omitting important key details in order to falsify the larger picture called “the half truth” captures special attention in the given paper.

Keywords: break in the information chain, fallacy, half truth, political discourse

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Authors: Annette Cosgrove

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The objectives of this research study is to examine the impact of professional development in Technology Enhanced Learning (TEL) and the digitisation of learning in teaching communities across multiple higher education sites in the ATU (Atlantic Technological University *) ( 2020-2025), including the proposal of an evidence based digital teaching model for use in a future pandemic. The research strategy undertaken for this PhD Study is a multi-site study using mixed methods. Qualitative & quantitative methods are being used in the study to collect data. A pilot study was carried out initially , feedback collected and the research instrument was edited to reflect this feedback, before being administered. The purpose of the staff questionnaire is to evaluate the impact of professional development in the area of TEL, and to capture the practitioners views on the perceived impact on their teaching practice in the higher education sector across ATU (West of Ireland – 5 Higher education locations ). The phenomenon being explored is ‘ the impact of professional development in the area of technology enhanced learning and on teaching practice in a higher education institution.’ The research methodology chosen for this study is an Action based Research Study. The researcher has chosen this approach as it is a prime strategy for developing educational theory and enhancing educational practice . This study includes quantitative and qualitative methods to elicit data which will quantify the impact that continuous professional development in the area of digital teaching practice and technologies has on the practitioner’s teaching practice in higher education. The research instruments / data collection tools for this study include a lecturer survey with a targeted TEL Practice group ( Pre and post covid experience) and semi-structured interviews with lecturers.. This research is currently being conducted across the ATU multisite campus and targeting Higher education lecturers that have completed formal CPD in the area of digital teaching. ATU, a west of Ireland university is the focus of the study , The research questionnaire has been deployed, with 75 respondents to date across the ATU - the primary questionnaire and semi- formal interviews are ongoing currently – the purpose being to evaluate the impact of formal professional development in the area of TEL and its perceived impact on the practitioners teaching practice in the area of digital teaching and learning . This paper will present initial findings, reflections and data from this ongoing research study.

Keywords: TEL, DTL, digital teaching, digital assessment

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Authors: Ajit Rai, Hamza Deroui, Blandine Vacher, Khwansiri Ninpan, Arthur Aumont, Francesco Vitillo, Robert Plana

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Large-scale critical industrial scheduling problems are based on Resource-Constrained Project Scheduling Problems (RCPSP), that necessitate integration with other optimization problems (e.g., vehicle routing, supply chain, or unique industrial ones), thus requiring practical solutions (i.e., modular, computationally efficient with feasible solutions). To the best of our knowledge, the current industrial state of the art is not addressing this holistic problem. We propose an original modular solution that answers the issues exhibited by the delivery of complex projects. With three interlinked entities (project, task, resources) having their constraints, it uses a greedy heuristic with a dynamic cost function for each task with a situational assessment at each time step. It handles large-scale data and can be easily integrated with other optimization problems, already existing industrial tools and unique constraints as required by the use case. The solution has been tested and validated by domain experts on three use cases: outage management in Nuclear Power Plants (NPPs), planning of future NPP maintenance operation, and application in the defense industry on supply chain and factory relocation. In the first use case, the solution, in addition to the resources’ availability and tasks’ logical relationships, also integrates several project-specific constraints for outage management, like, handling of resource incompatibility, updating of tasks priorities, pausing tasks in a specific circumstance, and adjusting dynamic unit of resources. With more than 20,000 tasks and multiple constraints, the solution provides a feasible schedule within 10-15 minutes on a standard computer device. This time-effective simulation corresponds with the nature of the problem and requirements of several scenarios (30-40 simulations) before finalizing the schedules. The second use case is a factory relocation project where production lines must be moved to a new site while ensuring the continuity of their production. This generates the challenge of merging job shop scheduling and the RCPSP with location constraints. Our solution allows the automation of the production tasks while considering the rate expectation. The simulation algorithm manages the use and movement of resources and products to respect a given relocation scenario. The last use case establishes a future maintenance operation in an NPP. The project contains complex and hard constraints, like on Finish-Start precedence relationship (i.e., successor tasks have to start immediately after predecessors while respecting all constraints), shareable coactivity for managing workspaces, and requirements of a specific state of "cyclic" resources (they can have multiple states possible with only one at a time) to perform tasks (can require unique combinations of several cyclic resources). Our solution satisfies the requirement of minimization of the state changes of cyclic resources coupled with the makespan minimization. It offers a solution of 80 cyclic resources with 50 incompatibilities between levels in less than a minute. Conclusively, we propose a fast and feasible modular approach to various industrial scheduling problems that were validated by domain experts and compatible with existing industrial tools. This approach can be further enhanced by the use of machine learning techniques on historically repeated tasks to gain further insights for delay risk mitigation measures.

Keywords: deterministic scheduling, optimization coupling, modular scheduling, RCPSP

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Authors: Sontana Mimapan, Phattarawadee Wattanasuntorn, Phanom Saijit

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Aflatoxin contamination in maize, one of several agriculture crops grown for livestock feeding, is still a problem throughout the world mainly under hot and humid weather conditions like Thailand. In this study Aspergillus flavus (A. Flavus), the key fungus for aflatoxin production especially aflatoxin B1 (AFB1), isolated from naturally infected maize were identified and characterized according to colony morphology and PCR using ITS, Beta-tubulin and calmodulin genes. The strains were analysed for the presence of four aflatoxigenic biosynthesis genes in relation to their capability to produce AFB1, Ver1, Omt1, Nor1, and aflR. Aflatoxin production was then confirmed using immunoaffinity column technique. A loop-mediated isothermal amplification (LAMP) was applied as an innovative technique for rapid detection of target nucleic acid. The reaction condition was optimized at 65C for 60 min. and calcein flurescent reagent was added before amplification. The LAMP results showed clear differences between positive and negative reactions in end point analysis under daylight and UV light by the naked eye. In daylight, the samples with AFB1 producing A. Flavus genes developed a yellow to green color, but those without the genes retained the orange color. When excited with UV light, the positive samples become visible by bright green fluorescence. LAMP reactions were positive after addition of purified target DNA until dilutions of 10⁻⁶. The reaction products were then confirmed and visualized with 1% agarose gel electrophoresis. In this regards, 50 maize samples were collected from dairy farms and tested for the presence of four aflatoxigenic biosynthesis genes using LAMP technique. The results were positive in 18 samples (36%) but negative in 32 samples (64%). All of the samples were rechecked by PCR and the results were the same as LAMP, indicating 100% specificity. Additionally, when compared with the immunoaffinity column-based aflatoxin analysis, there was a significant correlation between LAMP results and aflatoxin analysis (r= 0.83, P < 0.05) which suggested that positive maize samples were likely to be a high- risk feed. In conclusion, the LAMP developed in this study can provide a simple and rapid approach for detecting AFB1 producing A. Flavus genes from maize and appeared to be a promising tool for the prediction of potential aflatoxigenic risk in livestock feedings.

Keywords: Aflatoxin B1, Aspergillus flavus genes, maize, loop-mediated isothermal amplification

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Authors: Mehrnaz Mostafavi

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Perovskite nanocrystals and quantum dots have emerged as leaders in the field of photovoltaic technologies, demonstrating exceptional light-harvesting abilities and stability. This study investigates the substantial progress and potential of these nano-sized materials in transforming solar energy conversion. The research delves into the foundational characteristics and production methods of perovskite nanocrystals and quantum dots, elucidating their distinct optical and electronic properties that render them well-suited for photovoltaic applications. Specifically, it examines their outstanding light absorption capabilities, enabling more effective utilization of a wider solar spectrum compared to traditional silicon-based solar cells. Furthermore, this paper explores the improved durability achieved in perovskite nanocrystals and quantum dots, overcoming previous challenges related to degradation and inconsistent performance. Recent advancements in material engineering and techniques for surface passivation have significantly contributed to enhancing the long-term stability of these nanomaterials, making them more commercially feasible for solar cell usage. The study also delves into the advancements in device designs that incorporate perovskite nanocrystals and quantum dots. Innovative strategies, such as tandem solar cells and hybrid structures integrating these nanomaterials with conventional photovoltaic technologies, are discussed. These approaches highlight synergistic effects that boost efficiency and performance. Additionally, this paper addresses ongoing challenges and research endeavors aimed at further improving the efficiency, stability, and scalability of perovskite nanocrystals and quantum dots in photovoltaics. Efforts to mitigate concerns related to material degradation, toxicity, and large-scale production are actively pursued, paving the way for broader commercial application. In conclusion, this paper emphasizes the significant role played by perovskite nanocrystals and quantum dots in advancing photovoltaic technologies. Their exceptional light-harvesting capabilities, combined with increased stability, promise a bright future for next-generation solar cells, ushering in an era of highly efficient and cost-effective solar energy conversion systems.

Keywords: perovskite nanocrystals, quantum dots, photovoltaic technologies, light-harvesting, solar energy conversion, stability, device designs

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Authors: Shoji Katagiri, Hugang Han

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This paper clarifies the role of ICT capital in Economic Growth. Albeit ICT remarkably contributes to economic growth, there are few studies on ICT capital in ICT sector from theoretical point of view. In this paper, production function of ICT which is used as input of intermediate good in final good and ICT sectors is incorporated into our model. In this setting, we analyze the role of ICT on balance growth path and show the possibility of general equilibrium solutions for this model. Through the simulation of the equilibrium solutions, we find that when ICT impacts on economy and economic growth increases, it is necessary that increases of efficiency at ICT sector and of accumulation of non-ICT and ICT capitals occur simultaneously.

Keywords: endogenous economic growth, ICT, intensity, capital accumulation

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Authors: Luz Estrada

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The currents of evolutionary materialistic thought have argued that knowledge about an object is not obtained through the abstractive method. That is, the object cannot come to be understood if founded upon itself, nor does it take place by the encounter between form and matter. According to this affirmation, the research presented here identified as a problematic situation the absence of comprehension of the formal creation as a generative operation. This has been referred to as a recurrent lack in the production of objects and corresponds to the need to conceive the configurative process from the reality of its genesis. In this case, it is of interest to explore ways of creation that consider the object as if it were a living organism, as well as responding to the object’s experience as embodied in the designer since it unfolds its genesis simultaneously to the ways of existence of those who are involved in the generative experience.

Keywords: architecture, theoretical graphics, evolutionary genetics, formal perception

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Authors: Shashank Gupta, Shiva Garg

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The purpose of the present paper is to study the changes in the strength characteristics of autoclaved aerated concrete (AAC) and also the density when different expansion agents are used. The expansion agent so used releases air in the concrete thereby making it lighter by reducing its density. It also increases the workability of the concrete. The various air entraining agents used for this study are hydrogen peroxide, oleic acid, and olive oil. The addition of these agents causes the concrete to rise like cake but it reduces the strength of concrete due to the formation of air voids. The amount of agents chosen for concrete production are 0.5%, 1%, 1.5% by weight of cement.

Keywords: AAC, olive oil, hydrogen peroxide, oleic acid, steam curing

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

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Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

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Authors: Azeem Tariq, Stephane De Tourdonnet, Lars Stoumann Jensen, Reiner Wassmann, Bjoern Ole Sander, Quynh Duong Vu, Trinh Van Mai, Andreas De Neergaard

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Rice growing area is increasing to meet the food demand of increasing population. Mostly, rice is growing on lowland, small landholder fields in most part of the world, which is one of the major sources of greenhouse gases (GHG) emissions from agriculture fields. The strategies such as, altering water and residues (carbon) management practices are assumed to be essential to mitigate the GHG emissions from flooded rice system. The actual implementation and potential of these measures on small farmer fields is still challenging. A field study was conducted on red river delta in Northern Vietnam to identify the potential challenges and barriers to the small rice farmers for implementation of climate smart rice practices. The objective of this study was to develop and access the feasibility of climate smart rice prototypes under actual farmer conditions. Field and scientific oriented framework was used to meet our objective. The methodological framework composed of six steps: i) identification of stakeholders and possible options, ii) assessment of barrios, drawbacks/advantages of new technologies, iii) prototype design, iv) assessment of mitigation potential of each prototype, v) scenario building and vi) scenario assessment. A farm survey was conducted to identify the existing farm practices and major constraints of small rice farmers. We proposed the two water (pre transplant+midseason drainage and early+midseason drainage) and one straw (full residue incorporation) management option keeping in views the farmers constraints and barriers for implementation. To test new typologies with existing prototypes (midseason drainage, partial residue incorporation) at farmer local conditions, a participatory field experiment was conducted for two consecutive rice seasons at farmer fields. Following the results of each season a workshop was conducted with stakeholders (farmers, village leaders, cooperatives, irrigation staff, extensionists, agricultural officers) at local and district level to get feedbacks on new tested prototypes and to develop possible scenarios for climate smart rice production practices. The farm analysis survey showed that non-availability of cheap labor and lacks of alternatives for straw management influence the small farmers to burn the residues in the fields except to use for composting or other purposes. Our field results revealed that application of early season drainage significantly mitigates (40-60%) the methane emissions from residue incorporation. Early season drainage was more efficient and easy to control under cooperate manage system than individually managed water system, and it leads to both economic (9-11% high rice yield, low cost of production, reduced nutrient loses) and environmental (mitigate methane emissions) benefits. The participatory field study allows the assessment of adaptation potential and possible benefits of climate smart practices on small farmer fields. If farmers have no other residue management option, full residue incorporation with early plus midseason drainage is adaptable and beneficial (both environmentally and economically) management option for small rice farmers.

Keywords: adaptation, climate smart agriculture, constrainsts, smallholders

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Authors: Nenita Pambid Domingo

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The komedya otherwise known as moro-moro or pretending to be Moors, is a traditional Filipino play in the vernacular adapted from the Spanish comedia de capa y espada. It was used by Spanish colonizers in the Philippines, circa 1766 to evangelize and strengthen the faith of Indios or Filipino natives to Christianity. Unlike the Moros y Cristianos festival held all over Spain celebrating the Reconquista from the 8th to the 15th century, the Philippine Moro-Moro or Komedya is a romance between a Muslim and a Christian and the battles between Christians and Moros, where the Moros are always defeated and the Muslim prince is converted to the Christian faith and marries the Christian princess at the end of the play. For over 200 years, the komedya has been part of the Filipinos’ life and has been dubbed by some Philippine scholars as the Philippine’s national theater. Until now postings of performances in different parts of the Philippines in different Philippine languages are uploaded at youtube. In the US, “San Dionisio sa America (SDA),” an organization of natives from Barrio San Dionisio, Parañaque, Philippines has been performing the komedya for the past 16 years during their town’s fiesta, in honor of the barrio's patron saints St Denis of Paris, France and Saint Joseph whom the devotees fondly call "Tata Dune" and "Tata Hosep". The komedya performed in the US is infused with modern elements in the production and content, but retain the basic form in verse and the stylized war dance, marches, and singsong delivery of lines. Most of the Celebras or town fiestas and komedya performances are held at The Barnsdall Art Park and Gallery Theatre in Hollywood, Los Angeles. The presentation will focus on the linguistic and content analysis of the Tagalog verses in the 2010 komedya entitled Mga Prinsesa ng Cordova (The Princesses of Cordova) publicized as a modern komedya. The presentation will also touch on the healing function of the language and performance that is part of the town’s religious festivities. It will also look into the aesthetics of the production, audience reception, participation of the sponsors, producers called Hermana/Hermano Mayor, the performers who are a mix of Filipinos from the Philippines and Filipino-Americans who are starting to lose the Tagalog language and the non-Filipino participants, as well as the general audience who are from Parañaque and those not from Parañaque, who come to witness the event and enjoy the festivities.

Keywords: devotion, diaspora nationalism, komedya, st. denis of Paris, France, traditional Philippine theater

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Authors: Rohit Vashistha, Amit Kumar Gupta, G. P. Ravishankar, Mahesh P. Padwale

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Accurate thrust measurement is required for aircraft during takeoff and after ski-jump. In a developmental aircraft, takeoff from ship is extremely critical and thrust produced by the engine should be known to the pilot before takeoff so that if thrust produced is not sufficient then take-off can be aborted and accident can be avoided. After ski-jump, thrust produced by engine is required because the horizontal speed of aircraft is less than the normal takeoff speed. Engine should be able to produce enough thrust to provide nominal horizontal takeoff speed to the airframe within prescribed time limit. The contemporary low bypass gas turbine engines generally have three mounts where the two side mounts transfer the engine thrust to the airframe. The third mount only takes the weight component. It does not take any thrust component. In the present method of thrust estimation, the strain gauging of the two side mounts is carried out. The strain produced at various power settings is used to estimate the thrust produced by the engine. The quarter Wheatstone bridge is used to acquire the strain data. The engine mount assembly is subjected to Universal Test Machine for determination of equivalent elasticity of assembly. This elasticity value is used in the analytical approach for estimation of engine thrust. The estimated thrust is compared with the test bed load cell thrust data. The experimental strain data is also compared with strain data obtained from FEM analysis. Experimental setup: The strain gauge is mounted on the tapered portion of the engine mount sleeve. Two strain gauges are mounted on diametrically opposite locations. Both of the strain gauges on the sleeve were in the horizontal plane. In this way, these strain gauges were not taking any strain due to the weight of the engine (except negligible strain due to material's poison's ratio) or the hoop's stress. Only the third mount strain gauge will show strain when engine is not running i.e. strain due to weight of engine. When engine starts running, all the load will be taken by the side mounts. The strain gauge on the forward side of the sleeve was showing a compressive strain and the strain gauge on the rear side of the sleeve shows a tensile strain. Results and conclusion: the analytical calculation shows that the hoop stresses dominate the bending stress. The estimated thrust by strain gauge shows good accuracy at higher power setting as compared to lower power setting. The accuracy of estimated thrust at max power setting is 99.7% whereas at lower power setting is 78%.

Keywords: engine mounts, finite elements analysis, strain gauge, stress

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Authors: Kira Rysakova, Vitaly Novikov

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An improved method of obtaining enzymatic collagen hydrolysate from the tissues of marine hydrobionts is proposed, which allows to obtain hydrolysate without pre-isolation of pure collagen. The method can be used to isolate enzymatic collagen hydrolysate from the waste of industrial processing of Red King crab and non-traditional objects - marine holothurias. Comparative analysis of collagen hydrolysates has shown the possibility of their use in a number of nutrient media, but this requires additional optimization of their composition and biological tests on wide sets of test strains of microorganisms.

Keywords: collagen hydrolysate, marine hydrobionts, red king crab, marine holothurias, enzymes, exclusive HPLC

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Authors: Ruina Xu, Peixue Jiang, Feng Luo

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The novel concept of enhanced geothermal system with CO2 instead of water as working fluid (CO2-EGS) has attracted wide attention due to additional benefit of CO2 geological storage during the power generation process. In this research, numerical investigation on a doublet CO2-EGS system is performed, focusing on the influence of the injection/production well perforation location in the targeted geothermal reservoir. Three different reservoir inlet and outlet boundary conditions are used in simulations since the well constrains are different in reality. The results show that CO2-EGS system performance of power generation and power cost vary greatly among cases of different wells perforation locations, and the optimum options under different boundary conditions are also different.

Keywords: Enhanced Geothermal System, supercritical CO2, heat transfer, CO2-EGS

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Authors: Elena Listo Ubeda, Miguel Marchamalo Sacristan

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The demand for water doubles every twenty years, at a rate which is twice as fast as the world´s population growth. Despite it´s great importance, water is one of the most degraded natural resources in the world, mainly because of the reduction of natural vegetation coverage, population growth, contamination and changes in the soil use which reduces its capacity to collect water. This situation is especially serious in Central America, as reflected in the Human Development reports. The objective of this project is to assist in the improvement of water production and quality in Central America. In order to do these two watersheds in Costa Rica were selected as experiments: that of the Virilla-Durazno River, located in the extreme north east of the central valley which has an Atlantic influence; and that of the Jabillo River, which flows directly into the Pacific. The Virilla river watershed is located over andisols, and that of the Jabillo River is over alfisols, and both are of great importance for water supply to the Greater Metropolitan Area and the future tourist resorts respectively, as well as for the production of agriculture, livestock and hydroelectricity. The hydrological reaction in different soil-cover complexes, varying from the secondary forest to natural vegetation and degraded pasture, was analyzed according to the evaluation of the properties of the soil, infiltration, soil compaction, as well as the effects of the soil cover complex on erosion, calculated by the C factor of the Revised Universal Soil Loss Equation (RUSLE). A water balance was defined for each watershed, in which the volume of water that enters and leaves were estimated, as well as the evapotranspiration, runoff, and infiltration. Two future scenarios, representing the implementation of reforestation and deforestation plans, were proposed, and were analyzed for the effects of the soil cover complex on the water balance in each case. The results obtained show an increase of the ground water recharge in the humid forest areas, and an extension of the study of the dry areas is proposed since the ground water recharge here is diminishing. These results are of great significance for the planning, design of Payment Schemes for Environmental Services and the improvement of the existing water supply systems. In Central America spatial planning is a priority, as are the watersheds, in order to assess the water resource socially and economically, and securing its availability for the future.

Keywords: Costa Rica, infiltration, soil, water

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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