Search results for: phenol formaldehyde resin

Authors: Andrzej Szewczak

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Study of the effect of high temperatures on polymer coatings represents an important field of research of their properties. Polymers, as materials with numerous features (chemical resistance, ease of processing and recycling, corrosion resistance, low density and weight) are currently the most widely used modern building materials, among others in the resin concrete, plastic parts, and hydrophobic coatings. Unfortunately, the polymers have also disadvantages, one of which decides about their usage - low resistance to high temperatures and brittleness. This applies in particular thin and flexible polymeric coatings applied to other materials, such a steel and concrete, which degrade under varying thermal conditions. Research about improvement of this state includes methods of modification of the polymer composition, structure, conditioning conditions, and the polymerization reaction. At present, ways are sought to reflect the actual environmental conditions, in which the coating will be operating after it has been applied to other material. These studies are difficult because of the need for adopting a proper model of the polymer operation and the determination of phenomena occurring at the time of temperature fluctuations. For this reason, alternative methods are being developed, taking into account the rapid modeling and the simulation of the actual operating conditions of polymeric coating’s materials in real conditions. The nature of a duration is typical for the temperature influence in the environment. Studies typically involve the measurement of variation one or more physical and mechanical properties of such coating in time. Based on these results it is possible to determine the effects of temperature loading and develop methods affecting in the improvement of coatings’ properties. This paper contains a description of the stability studies of silicone coatings deposited on the surface of a ceramic brick. The brick’s surface was hydrophobized by two types of inorganic polymers: nano-polymer preparation based on dialkyl siloxanes (Series 1 - 5) and an aqueous solution of the silicon (series 6 - 10). In order to enhance the stability of the film formed on the brick’s surface and immunize it to variable temperature and humidity loading, the nano silica was added to the polymer. The right combination of the polymer liquid phase and the solid phase of nano silica was obtained by disintegration of the mixture by the sonification. The changes of viscosity and surface tension of polymers were defined, which are the basic rheological parameters affecting the state and the durability of the polymer coating. The coatings created on the brick’s surfaces were then subjected to a temperature loading of 100° C and moisture by total immersion in water, in order to determine any water absorption changes caused by damages and the degradation of the polymer film. The effect of moisture and temperature was determined by measurement (at specified number of cycles) of changes in the surface hardness (using a Vickers’ method) and the absorption of individual samples. As a result, on the basis of the obtained results, the degradation process of polymer coatings related to their durability changes in time was determined.

Keywords: silicones, siloxanes, surface hardness, temperature, water absorption

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Authors: Yogesh Dalvi, Ruby Varghese, Nibu Varghese, C. K. Krishnan Nair

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Introduction: The Genus Phellinus, locally known as Phansomba is a well-known traditional folk medicine. Especially, in Western Ghats of India, many tribes use several species of Phellinus for various ailments related to teeth, throat, tongue, stomach and even wound healing. It is one of the few mushrooms which play a pivotal role in Ayurvedic Dravyaguna. Aim: The present study focuses on to investigate phytochemical analysis, antioxidant, and antitumor (in vitro and in vivo) potential of Phellinus robinae from South India, Kerala Material and Methods: The present study explores the following: 1. Phellinus samples were collected from Ranni, Pathanamthitta district of Kerala state, India from Artocarpus heterophyllus Lam. and species were identified using rDNA region. 2. The fruiting body was shadow dried, powdered and extracted with 50% alcohol using water bath at 60°C which was further condensed by rotary evaporator and lyophilized at minus 40°C temperature. 3. Secondary metabolites were analyzed by using various phytochemical screening assay (Hager’s Test, Wagner’s Test, Sodium hydroxide Test, Lead acetate Test, Ferric chloride Test, Folin-ciocalteu Test, Foaming Test, Benedict’s test, Fehling’s Test and Lowry’s Test). 4. Antioxidant and free radical scavenging activity were analyzed by DPPH, FRAP and Iron chelating assay. 5. The antitumor potential of Water alcohol extract of Phellinus (PAWE) is evaluated through In vitro condition by Trypan blue dye exclusion method in DLA cell line and In vivo by murine model. Result and Discussion: Preliminary phytochemical screening by various biochemical tests revealed presence of a variety of active secondary molecules like alkaloids, flavanoids, saponins, carbohydrate, protein and phenol. In DPPH and FRAP assay PAWE showed significantly higher antioxidant activity as compared to standard Ascorbic acid. While, in Iron chelating assay, PAWE exhibits similar antioxidant activity that of Butylated Hydroxytoluene (BHT) as standard. Further, in the in vitro study, PAWE showed significant inhibition on DLA cell proliferation in dose dependent manner and showed no toxicity on mice splenocytes, when compared to standard chemotherapy drug doxorubicin. In vivo study, oral administration of PAWE showed dose dependent tumor regression in mice and also raised the immunogenicity by restoring levels of antioxidant enzymes in liver and kidney tissue. In both in vitro and in vivo gene expression studies PAWE up-regulates pro-apoptotic genes (Bax, Caspases 3, 8 and 9) and down- regulates anti-apoptotic genes (Bcl2). PAWE also down regulates inflammatory gene (Cox-2) and angiogenic gene (VEGF). Conclusion: Preliminary phytochemical screening revealed that PAWE contains various secondary metabolites which contribute to its antioxidant and free radical scavenging property as evaluated by DPPH, FRAP and Iron chelating assay. PAWE exhibits anti-proliferative activity by the induction of apoptosis through a signaling cascade of death receptor-mediated extrinsic (Caspase8 and Tnf-α), as well as mitochondria-mediated intrinsic (caspase9) and caspase pathways (Caspase3, 8 and 9) and also by regressing angiogenic factor (VEGF) without any inflammation or adverse side effects. Hence, PAWE serve as a potential antioxidant and antitumor agent.

Keywords: antioxidant, antitumor, Dalton lymphoma ascites (DLA), fungi, Phellinus robinae

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Authors: Yusuf Hesham Mohamed

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Introduction: Three-dimensional printing technology includes multiple procedures that fabricate three-dimensional objects through consecutively layering two-dimensional cross-sections on top of each other. 3D bioprinting is a promising field of 3D printing, which fabricates tissues and organs by accurately controlling the proper arrangement of diverse biological components. 3D bioprinting uses software and prints biological materials and their supporting components layer-by-layer on a substrate or in a tissue culture plate to produce complex live tissues and organs. 3D food printing is an emerging field of 3D bioprinting in which the 3D printed products are food products that are cheap, require less effort to produce, and have more desirable traits. The Aim of the Study is the development of an affordable 3D bioprinter by altering a locally made CNC instrument with an open-source platform to suit the 3D bio-printer purposes. Later, we went through applying the prototype in several applications regarding food technology and drug testing, including the organ-On-Chip. Materials and Methods: An off-the-shelf 3D printer was modified by designing and fabricating the syringe unit, which was designed on the basis of the Milli-fluidics system. Sodium alginate and gelatin hydrogels were prepared, followed by leaf cell suspension preparation from narrow sections of Fragaria’s viable leaves. The desired 3D structure was modeled, and 3D printing preparations took place. Cell-free and cell-laden hydrogels were printed at room temperature under sterile conditions. Post printing curing process was performed. The printed structure was further studied. Results: Positive results have been achieved using the altered 3D bioprinter where a 3D hydrogel construct of two layers made of the combination of sodium alginate to gelatin (15%: 0.5%) has been printed. DLP 3D printer was used to design the syringe component with a transparent PLA-Pro resin for the creation of a microfluidics system having two channels altered to the double extruder. The hydrogel extruder’s design was based on peristaltic pumps, which utilized a stepper motor. The design and fabrication were made using DIY-3D printed parts. Hard plastic PLA was the material utilized for printing. SEM was used to carry out the porous 3D construct imaging. Multiple physical and chemical tests were performed in order to ensure that the cell line was suitable for hosting. Fragaria plant was developed by suspending Fragaria’s cells from its leaves using the 3D bioprinter. Conclusion: 3D bioprinting is considered to be an emerging scientific field that can facilitate and improve many scientific tests and studies. Thus, having a 3D bioprinter in labs is considered to be an essential requirement. 3D bioprinters are very expensive; however, the fabrication of a 3D printer into a 3D bioprinter can lower the cost of the bioprinter. The 3D bioprinter implemented made use of peristaltic pumps instead of syringe-based pumps in order to extend the ability to print multiple types of materials and cells.

Keywords: scaffold, eco on chip, 3D bioprinter, DLP printer

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Authors: O. Squillace, R. L. Thompson

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Migration of small molecules to, and across the surface of polymer matrices is a little-studied problem with important industrial applications. Tackifiers in adhesives, flavors in foods and binding agents in paints all present situations where the function of a product depends on the ability of small molecules to migrate through a polymer matrix to achieve the desired properties such as softness, dispersion of fillers, and to deliver an effect that is felt (or tasted) on a surface. It’s been shown that the chemical and molecular structure, surface free energies, phase behavior, close environment and compatibility of the system, influence the migrants’ motion. When differences in behavior, such as occurrence of segregation to the surface or not, are observed it is then of crucial importance to identify and get a better understanding of the driving forces involved in the process of molecular migration. In this aim, experience is meant to be allied with theory in order to deliver a validated theoretical and computational toolkit to describe and predict these phenomena. The systems that have been chosen for this study aim to address the effect of polarity mismatch between the migrants and the polymer matrix and that of a second migrant over the first one. As a non-polar resin polymer, polyvinyl acetate is used as the material to which more or less polar migrants (sorbitol, carvone, octanoic acid (OA), triacetin) are to be added. Through contact angle measurement a surface excess is seen for sorbitol (polar) mixed with PVAc as the surface energy is lowered compare to the one of pure PVAc. This effect is increased upon the addition of carvon or triacetin (non-polars). Surface micro-structures are also evidenced by atomic force microscopy (AFM). Ion beam analysis (Nuclear Reaction Analysis), supplemented by neutron reflectometry can accurately characterize the self-organization of surfactants, oligomers, aromatic molecules in polymer films in order to relate the macroscopic behavior to the length scales that are amenable to simulation. The nuclear reaction analysis (NRA) data for deuterated OA 20% shows the evidence of a surface excess which is enhanced after annealing. The addition of 10% triacetin, as a second migrant, results in the formation of an underlying layer enriched in triacetin below the surface excess of OA. The results show that molecules in polarity mismatch with the matrix tend to segregate to the surface, and this is favored by the addition of a second migrant of the same polarity than the matrix. As studies have been restricted to materials that are model supported films under static conditions in a first step, it is also wished to address the more challenging conditions of materials under controlled stress or strain. To achieve this, a simple rig and PDMS cell have been designed to stretch the material to a defined strain and to probe these mechanical effects by ion beam analysis and atomic force microscopy. This will make a significant step towards exploring the influence of extensional strain on surface segregation, flavor release in cross-linked rubbers.

Keywords: polymers, surface segregation, thin films, molecular migration

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Authors: N. Naz, A. D. Domenico, M. N. Huda

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Soft Robotics is a rapidly growing multidisciplinary field where robots are fabricated using highly deformable materials motivated by bioinspired designs. The high dexterity and adaptability to the external environments during contact make soft robots ideal for applications such as gripping delicate objects, locomotion, and biomedical devices. The actuation system of soft robots mainly includes fluidic, tendon-driven, and smart material actuation. Among them, Soft Pneumatic Actuator, also known as SPA, remains the most popular choice due to its flexibility, safety, easy implementation, and cost-effectiveness. However, at present, most of the fabrication of SPA is still based on traditional molding and casting techniques where the mold is 3d printed into which silicone rubber is cast and consolidated. This conventional method is time-consuming and involves intensive manual labour with the limitation of repeatability and accuracy in design. Recent advancements in direct 3d printing of different soft materials can significantly reduce the repetitive manual task with an ability to fabricate complex geometries and multicomponent designs in a single manufacturing step. The aim of this research work is to design and analyse the Soft Pneumatic Actuator (SPA) utilizing both conventional casting and modern direct 3d printing technologies. The mold of the SPA for traditional casting is 3d printed using fused deposition modeling (FDM) with the polylactic acid (PLA) thermoplastic wire. Hyperelastic soft materials such as Ecoflex-0030/0050 are cast into the mold and consolidated using a lab oven. The bending behaviour is observed experimentally with different pressures of air compressor to ensure uniform bending without any failure. For direct 3D-printing of SPA fused deposition modeling (FDM) with thermoplastic polyurethane (TPU) and stereolithography (SLA) with an elastic resin are used. The actuator is modeled using the finite element method (FEM) to analyse the nonlinear bending behaviour, stress concentration and strain distribution of different hyperelastic materials after pressurization. FEM analysis is carried out using Ansys Workbench software with a Yeon-2nd order hyperelastic material model. FEM includes long-shape deformation, contact between surfaces, and gravity influences. For mesh generation, quadratic tetrahedron, hybrid, and constant pressure mesh are used. SPA is connected to a baseplate that is in connection with the air compressor. A fixed boundary is applied on the baseplate, and static pressure is applied orthogonally to all surfaces of the internal chambers and channels with a closed continuum model. The simulated results from FEM are compared with the experimental results. The experiments are performed in a laboratory set-up where the developed SPA is connected to a compressed air source with a pressure gauge. A comparison study based on performance analysis is done between FDM and SLA printed SPA with the molded counterparts. Furthermore, the molded and 3d printed SPA has been used to develop a three-finger soft pneumatic gripper and has been tested for handling delicate objects.

Keywords: finite element method, fused deposition modeling, hyperelastic, soft pneumatic actuator

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Authors: Chang-Hui Shen, Michelle Esposito, Andrew J. Shen, Michael Adejokun, Diana Laterman

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The packaging of DNA into nucleosomes is critical to genomic compaction, yet it can leave gene promoters inaccessible to activator proteins or transcription machinery and thus prevents transcriptional initiation. Both chromatin remodelers and histone acetylases (HATs) are the two main transcription co-activators that can reconfigure chromatin structure for transcriptional activation. Ino80p is the core component of the INO80 remodeling complex. Recently, it was shown that Ino80p dissociates from the yeast INO1 promoter after induction. However, when certain HATs were deleted or mutated, Ino80p accumulated at the promoters during gene activation. This suggests a link between HATs’ presence and Ino80p’s dissociation. However, it has yet to be demonstrated that Ino80p can be acetylated. To determine if Ino80p can be acetylated, wild-type Saccharomyces cerevisiae cells carrying Ino80p engineered with a double FLAG tag (MATa INO80-FLAG his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) were grown to mid log phase, as were non-tagged wild type (WT) (MATa his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) and ino80∆ (MATa ino80∆::TRP1 his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) cells as controls. Cells were harvested, and the cell lysates were subjected to immunoprecipitation (IP) with α-FLAG resin to isolate Ino80p. These eluted IP samples were subjected to SDS-PAGE and Western blot analysis. Subsequently, the blots were probed with the α-FLAG and α-acetyl lysine antibodies, respectively. For the blot probed with α-FLAG, one prominent band was shown in the INO80-FLAG cells, but no band was detected in the IP samples from the WT and ino80∆ cells. For the blot probed with the α-acetyl lysine antibody, we detected acetylated Ino80p in the INO80-FLAG strain while no bands were observed in the control strains. As such, our results showed that Ino80p can be acetylated. This acetylation can explain the co-activator’s recruitment patterns observed in current gene activation models. In yeast INO1, it has been shown that Ino80p is recruited to the promoter during repression, and then dissociates from the promoter once de-repression begins. Histone acetylases, on the other hand, have the opposite pattern of recruitment, as they have an increased presence at the promoter as INO1 de-repression commences. This Ino80p recruitment pattern significantly changes when HAT mutant strains are studied. It was observed that instead of dissociating, Ino80p accumulates at the promoter in the absence of functional HATs, such as Gcn5p or Esa1p, under de-repressing processes. As such, Ino80p acetylation may be required for its proper dissociation from the promoters. The remodelers’ dissociation mechanism may also have a wide range of implications with respect to transcriptional initiation, elongation, or even repression as it allows for increased spatial access to the promoter for the various transcription factors and regulators that need to bind in that region. Our findings here suggest a previously uncharacterized interaction between Ino80p and other co-activators recruited to promoters. As such, further analysis of Ino80p acetylation not only will provide insight into the role of epigenetic modifications in transcriptional activation, but also gives insight into the interactions occurring between co-activators at gene promoters during gene regulation.

Keywords: acetylation, chromatin remodeler, epigenetic modification, Ino80p

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Authors: David F. Nettleton, Elodie Bugnicourt, Christian Wasiak, Alejandro Rosales

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In this presentation, preliminary results are given for the modeling and calibration of two different industrial winding MIMO (Multiple Input Multiple Output) processes using machine learning techniques. In contrast to previous approaches which have typically used ‘black-box’ linear statistical methods together with a definition of the mechanical behavior of the process, we use non-linear machine learning algorithms together with a ‘white-box’ rule induction technique to create a supervised model of the fitting error between the expected and real force measures. The final objective is to build a precise model of the winding process in order to control de-tension of the material being wound in the first case, and the friction of the material passing through the die, in the second case. Case 1, Tension Control of a Winding Process. A plastic web is unwound from a first reel, goes over a traction reel and is rewound on a third reel. The objectives are: (i) to train a model to predict the web tension and (ii) calibration to find the input values which result in a given tension. Case 2, Friction Force Control of a Micro-Pullwinding Process. A core+resin passes through a first die, then two winding units wind an outer layer around the core, and a final pass through a second die. The objectives are: (i) to train a model to predict the friction on die2; (ii) calibration to find the input values which result in a given friction on die2. Different machine learning approaches are tested to build models, Kernel Ridge Regression, Support Vector Regression (with a Radial Basis Function Kernel) and MPART (Rule Induction with continuous value as output). As a previous step, the MPART rule induction algorithm was used to build an explicative model of the error (the difference between expected and real friction on die2). The modeling of the error behavior using explicative rules is used to help improve the overall process model. Once the models are built, the inputs are calibrated by generating Gaussian random numbers for each input (taking into account its mean and standard deviation) and comparing the output to a target (desired) output until a closest fit is found. The results of empirical testing show that a high precision is obtained for the trained models and for the calibration process. The learning step is the slowest part of the process (max. 5 minutes for this data), but this can be done offline just once. The calibration step is much faster and in under one minute obtained a precision error of less than 1x10-3 for both outputs. To summarize, in the present work two processes have been modeled and calibrated. A fast processing time and high precision has been achieved, which can be further improved by using heuristics to guide the Gaussian calibration. Error behavior has been modeled to help improve the overall process understanding. This has relevance for the quick optimal set up of many different industrial processes which use a pull-winding type process to manufacture fibre reinforced plastic parts. Acknowledgements to the Openmind project which is funded by Horizon 2020 European Union funding for Research & Innovation, Grant Agreement number 680820

Keywords: data model, machine learning, industrial winding, calibration

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Authors: Milica B. Veljković, Milica B. Simović, Marija M. Ćorović, Ana D. Milivojević, Anja I. Petrov, Katarina M. Banjanac, Dejan I. Bezbradica

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In recent decades, the scientific community has recognized the growing importance of prebiotics, and therefore, numerous studies are focused on their economic production due to their low presence in natural resources. It has been confirmed that prebiotics is a source of energy for probiotics in the gastrointestinal tract (GIT) and enable their proliferation, consequently leading to the normal functioning of the intestinal microbiota. Also, products of their fermentation are short-chain fatty acids (SCFA), which play a key role in maintaining and improving the health not only of the GIT but also of the whole organism. Among several confirmed prebiotics, fructooligosaccharides (FOS) are considered interesting candidates for use in a wide range of products in the food industry. They are characterized as low-calorie and non-cariogenic substances that represent an adequate sugar substitute and can be considered suitable for use in products intended for diabetics. The subject of this research will be the production of FOS by transforming sucrose using a fructosyltransferase (FTase) present in commercial preparation Pectinex® Ultra SP-L, with special emphasis on the development of adequate FTase immobilization method that would enable selective isolation of the enzyme responsible for the synthesis of FOS from the complex enzymatic mixture. This would lead to considerable enzyme purification and allow its direct incorporation into different sucrose-based products without the fear that the action of the other hydrolytic enzymes may adversely affect the products' functional characteristics. Accordingly, the possibility of selective immobilization of the enzyme using support with primary amino groups, Purolite® A109, which was previously activated and modified using glutaraldehyde (GA), was investigated. In the initial phase of the research, the effects of individual immobilization parameters such as pH, enzyme concentration, and immobilization time were investigated to optimize the process using support chemically activated with 15% and 0.5% GA to form dimers and monomers, respectively. It was determined that highly active immobilized preparations (371.8 IU/g of support - dimer and 213.8 IU/g of support – monomer) were achieved under acidic conditions (pH 4) provided that an enzyme concentration was 50 mg/g of support after 7 h and 3 h, respectively. Bearing in mind the obtained results of the expressed activity, it is noticeable that the formation of dimers showed higher reactivity compared to the form of monomers. Also, in the case of support modification using 15% GA, the value of the ratio of FTase and pectinase (as dominant enzyme mixture component) activity immobilization yields was 16.45, indicating the high feasibility of selective immobilization of FTase on modified polystyrene resin. After obtaining immobilized preparations of satisfactory features, they were tested in a reaction of FOS synthesis under determined optimal conditions. The maximum FOS yields of approximately 50% of total carbohydrates in the reaction mixture were recorded after 21 h. Finally, it can be concluded that the examined immobilization method yielded highly active, stable and, more importantly, refined enzyme preparation that can be further utilized on a larger scale for the development of continual processes for FOS synthesis, as well as for modification of different sucrose-based mediums.

Keywords: chemical modification, fructooligosaccharides, glutaraldehyde, immobilization of fructosyltransferase

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Authors: Diego Giuseppe Romano, Gianvito Apuleo, Jiri Duda

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Mobility is one of the most important societal needs for amusement, business activities and health. Thus, transport needs are continuously increasing, with the consequent traffic congestion and pollution increase. Aeronautic effort aims at smarter infrastructures use and in introducing greener concepts. A possible solution to address the abovementioned topics is the development of Small Air Transport (SAT) system, able to guarantee operability from today underused airfields in an affordable and green way, helping meanwhile travel time reduction, too. In the framework of Horizon2020, EU (European Union) has funded the Clean Sky 2 SAT TA (Transverse Activity) initiative to address market innovations able to reduce SAT operational cost and environmental impact, ensuring good levels of operational safety. Nowadays, most of the key technologies to improve passenger comfort and to reduce community noise, DOC (Direct Operating Costs) and pilot workload for SAT have reached an intermediate level of maturity TRL (Technology Readiness Level) 3/4. Thus, the key technologies must be developed, validated and integrated on dedicated ground and flying aircraft demonstrators to reach higher TRL levels (5/6). Particularly, SAT TA focuses on the integration at aircraft level of the following technologies [1]: 1)    Low-cost composite wing box and engine nacelle using OoA (Out of Autoclave) technology, LRI (Liquid Resin Infusion) and advance automation process. 2) Innovative high lift devices, allowing aircraft operations from short airfields (< 800 m). 3) Affordable small aircraft manufacturing of metallic fuselage using FSW (Friction Stir Welding) and LMD (Laser Metal Deposition). 4)       Affordable fly-by-wire architecture for small aircraft (CS23 certification rules). 5) More electric systems replacing pneumatic and hydraulic systems (high voltage EPGDS -Electrical Power Generation and Distribution System-, hybrid de-ice system, landing gear and brakes). 6) Advanced avionics for small aircraft, reducing pilot workload. 7) Advanced cabin comfort with new interiors materials and more comfortable seats. 8) New generation of turboprop engine with reduced fuel consumption, emissions, noise and maintenance costs for 19 seats aircraft. (9) Alternative diesel engine for 9 seats commuter aircraft. To address abovementioned market innovations, two different platforms have been designed: Reference and Green aircraft. Reference aircraft is a virtual aircraft designed considering 2014 technologies with an existing engine assuring requested take-off power; Green aircraft is designed integrating the technologies addressed in Clean Sky 2. Preliminary integration of the proposed technologies shows an encouraging reduction of emissions and operational costs of small: about 20% CO₂ reduction, about 24% NO_x reduction, about 10 db (A) noise reduction at measurement point and about 25% DOC reduction. Detailed description of the performed studies, analyses and validations for each technology as well as the expected benefit at aircraft level are reported in the present paper.

Keywords: affordable, European, green, mobility, technologies development, travel time reduction

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Authors: Jessica P. Fiorotti, Maria C. Freitas, Caio J. B. Coutinho-Rodrigues, Mariana G. Camargo, Emily S. Mesquita, Amanda R. C. Corval, Ricardo O. B. Bitencourt, Allan F. Marciano, Diva D. Spadacci-Morena, Patricia S. Golo, Isabele C. Angelo, Vania R. E. P. Bittencourt

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The bovine tick, Rhipicephalus microplus, is an arthropod of great importance in veterinary medicine leading to anemia, weight loss, animals' leather depreciation and also acting as a vector of many pathogens. In this way, the parasitism causes a loss of 3.24 billion dollars per year in Brazil. Knowingly, entomopathogenic fungi act as natural controller of some arthropods, acting mainly by active penetration through the cuticle. However, it can also act on the hemolymph and through the production of mycotoxins. Hemocytes are responsible for the cellular immune response and participate in the processes of phagocytosis, nodulation and encapsulation and may undergo changes when challenged by pathogens. The aim of the present study was to evaluate changes in R. microplus hemocytes after inoculation of Metarhizium robertsii using transmission electron microscopy. The isolate ARSEF 2575 and 200 engorged R. microplus females were used. The groups were divided into control, in which the females were inoculated with 5 μL of sterile distilled water solution and 0.1% Tween 80, and a group inoculated with 5 μL of fungal suspension at the concentration of 10⁷ conidia mL⁻¹. The experiment was performed in duplicate and each group contained 50 females. Twenty-four hours after fungal inoculation, hemolymph was collected through the cuticle dorsal surface perforation of the tick females. After collection, the hemolymph samples were centrifuged at 500 x g for 3 minutes at 4 °C, the plasma was discarded and the hemocyte pellet was resuspended in 50 μl PBS. The suspension material was fixed in 2% glutaraldehyde in Millonig buffer for three hours. After fixation, the material was centrifuged at 500 x g for 3 minutes, the supernatant was discarded and the cells were resuspended in a wash solution. Subsequently, the cells were post-fixed with 1% osmium tetroxide in phosphate buffer for one hour at room temperature and dehydrated in increasing concentrations of ethanol, and then embedded in Epon resin. The ultrathin sections were examined under the LEO EM 906E transmission electron microscopy at 80kV. The ultrastructural results revealed that.in control group, the cells were considered intact, in which the granulocytes were observed with granules of different electrodensities, intact mitochondria and cytoplasm without vacuolization. In addition, granulocytes showed plasma membrane projections similar to pseudopodia. Plasmatocytes presented as irregularly shaped cells, with the eccentric nucleus, agranular cytoplasm and some cells presented pseudopodia. Nevertheless, in the group exposed to the fungus, most of the cells presented in degeneration. The granulocytes found had fewer granules in the cytoplasm and more vacuoles. Plasmatocytes, after treatment, presented many vacuoles also in the cytoplasm and the lysosomes presented great amount of electrodense material in their interior. Thus, the results suggest that the fungus has a depressant action in the immune system of the tick, not only by the cell degranulation, but also suggesting that this leads to morphological changes in the hemocytes and may even trigger processes such as phagocytosis.

Keywords: bovine tick, cellular defense, entomopathogenic fungi, immune response

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Authors: Kristina Karapetyan, Flora Tkhruni, Tsovinar Balabekyan, Arevik Israyelyan, Tatyana Khachatryan

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The ability of the lactic acid bacteria (LAB) to prevent and cure a variety of diseases, their protective role against infections and colonization of pathogenic microorganisms in the digestive tract, has lead to the coining of the term probiotics or pro-life. LAB inhibiting the growth of pathogenic and food spoilage microorganisms, maintaining the nutritive quality and improving the shelf life of foods. They have also been used as flavor and texture producers. Enterococcus strains have been used for treatment of diseases such as diarrhea or antibiotic associated diarrhea, inflammatory pathologies that affect colon such as irritable bowel syndrome, or immune regulation, diarrhea caused by antibiotic treatments. The obtaining and investigation of biological properties of proteinoceous antibiotics, on the basis of probiotic LAB shown, that bacteriocins, metabiotics, and peptides of LAB represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics. The genotyping by 16S rRNA sequencing for LAB were used. Cell free culture broth (CFC) broth was purified by the Gel filtration method on the Sephadex Superfine G 25 resin. Antimicrobial activity was determined by spot-on-lawn method and expressed in arbitrary units (AU/ml). The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republics of Armenia and Nagorno Karabakh were examined. It was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. The influences of partially purified antimicrobial preparations (AMP), obtained from the different strains of Enterococcus genus on the growth of MDR pathogenic bacteria were investigated. It was shown, that bacteriocin containing partially purified preparations, obtained from different strains of Enterococcus faecium and durans species, possess bactericidal or bacteriostatic activity against antibiotic resistant intestinal, spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella. Endemic strains of LAB, isolated from Matsoni made from donkey, buffalo and goat milk, shown broad spectrum of activity against food spoiling microorganisms, moulds and fungi, such as Salmonella sp., Esherichia coli, Aspergillus and Penicillium species. Highest activity against MDR pathogens shown bacteria, isolated from goat milk products. High stability of the investigated strains of the genus Enerococcus, isolated from samples of matsun from different regions of Nagorno-Karabakh (NKR) to the antibiotics was shown. The obtained data show high stability of the investigated different strains of the genus Enerococcus. The high genetic diversity in Enterococcus group suggests adaptations for specific mutations in different environments. Thus, endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms isolated from different sources and belong to different taxonomic group. Prospect of the use of certain antimicrobial preparations against pathogenic strains is obvious. These AMP can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.

Keywords: antimicrobial biopreparation, endemic lactic acid bacteria, intra-species diversity, multidrug resistance of pathogens

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