Search results for: Raya Kathreen T. Fuentes

Authors: Raya Sulaiman

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Critical discourse analysis investigates how discourse is used to abuse power relationships. Political debates constitute discourses which mirror aspects of ideologies. The Arab world has been one of the most unsettled zones in the world and has dominated global politics due to the Arab revolutions which started in 2010. This study aimed at uncovering the ideological intentions in the formulation and circulation of hegemonic political ideology in the TV political debates of the 2011 to 2012 Yemen revolution, how ideology was used as a tool of hegemony. The study specifically examined the ideologies associated with the use of protesters as a social actor. Data of the study consisted of four debates (17350 words) from four live debate programs: The Opposite Direction, In Depth, Behind the News and the Revolution Talk that were staged at Al Jazeera TV channel between 2011 and 2012. Data was readily transcribed by Al Jazeera online. Al Jazeera was selected for the study because it is the most popular TV network in the Arab world and has a strong presence, especially during the Arab revolutions. Al Jazeera has also been accused of inciting protests across the Arab region. Two debate sites were identified in the data: government and anti-government. The government side represented the president Ali Abdullah Saleh and his regime while the anti-government side represented the gathering squares who demanded the president to ‘step down’. The study analysed verbal discourse aspects of the debates using critical discourse analysis: aspects from the Social Actor Network model of van Leeuwen. This framework provides a step-by-step analysis model, and analyses discourse from specific grammatical processes into broader semantic issues. It also provides representative findings since it considers discourse as representative and reconstructed in social practice. Study findings indicated that Al Jazeera and the anti-government had similarities in terms of the ideological intentions related to the protesters. Al Jazeera victimized and incited the protesters which were similar to the anti-government. Al Jazeera used assimilation, nominalization, and active role allocation as the linguistic aspects in order to reach its ideological intentions related to the protesters. Government speakers did not share the same ideological intentions with Al Jazeera. Study findings indicated that Al Jazeera had excluded the government from its debates causing a violation to its slogan, the opinion, and the other opinion. This study implies the powerful role of discourse in shaping ideological media intentions and influencing the media audience.

Keywords: Al Jazeera network, critical discourse analysis, ideology, Yemeni revolution

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Authors: Alicia Yanadira Nava Fuentes, Ilse Cervantes Camacho, Amadeo José Argüelles Cruz, Ana María Balboa Verduzco

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Augmented reality promises being present in future driving, with its immersive technology let to show directions and maps to identify important places indicating with graphic elements when the car driver requires the information. On the other side, driving is considered a multitasking activity and, for some people, a complex activity where different situations commonly occur that require the immediate attention of the car driver to make decisions that contribute to avoid accidents; therefore, the main aim of the project is the instrumentation of a platform with biometric sensors that allows evaluating the performance in driving vehicles with the influence of augmented reality devices to detect the level of attention in drivers, since it is important to know the effect that it produces. In this study, the physiological sensors EPOC X (EEG), ECG06 PRO and EMG Myoware are joined in the driving test platform with a Logitech G29 steering wheel and the simulation software City Car Driving in which the level of traffic can be controlled, as well as the number of pedestrians that exist within the simulation obtaining a driver interaction in real mode and through a MSP430 microcontroller achieves the acquisition of data for storage. The sensors bring a continuous analog signal in time that needs signal conditioning, at this point, a signal amplifier is incorporated due to the acquired signals having a sensitive range of 1.25 mm/mV, also filtering that consists in eliminating the frequency bands of the signal in order to be interpretative and without noise to convert it from an analog signal into a digital signal to analyze the physiological signals of the drivers, these values are stored in a database. Based on this compilation, we work on the extraction of signal features and implement K-NN (k-nearest neighbor) classification methods and decision trees (unsupervised learning) that enable the study of data for the identification of patterns and determine by classification methods different effects of augmented reality on drivers. The expected results of this project include are a test platform instrumented with biometric sensors for data acquisition during driving and a database with the required variables to determine the effect caused by augmented reality on people in simulated driving.

Keywords: augmented reality, driving, physiological signals, test platform

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Authors: Leonardo Parra, Daniel Martínez, Jorge Pizarro, Fernando Ortega, Manuel Chacón-Fuentes, Andrés Quiroz

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Control of Dalaca pallens or blackworms, one of the most important hypogeous pest in grassland in southern Chile, is based on the use of broad-spectrum insecticides such as organophosphates and pyrethroids. However, the rapid development of insecticide resistance in field populations of this insect and public concern over the environmental impact of these insecticides has resulted in the search for other control methods. Specifically, the use of endophyte fungi for controlling pest has emerged as an interesting and promising strategy. Endophytes from ryegrass (Lolium perenne), establish a biotrophic relationship with the host, defined as mutualistic symbiosis. The plant-fungi association produces alkaloids where peramine is the main toxic substance against Listronotus bonariensis, the most important epigean pest of ryegrass. Nevertheless, the effect of peramina on others pest insects, such as D. pallens, to our knowledge has not been studied, and also its possible metabolization in the body of the larvae. Therefore, we addressed the following research question: Do larvae of D. pallens store peramine after consumption of ryegrass endophyte infected (E+)? For this, specimens of blackworms were fed with ryegrass plant of seven experimental lines and one commercial cultivar endophyte free (E-) sown at the Instituto de Investigaciones Agropecuarias Carillanca (Vilcún, Chile). Once the feeding period was over, ten larvae of each treatment were examined. Individuals were dissected, and their gut was removed to exclude any influence of remaining material. The rest of the larva's body was dried at 60°C by 24-48 h and ground into a fine powder using a mortar. 25 mg of dry powder was transferred to a microcentrifuge tube and extracted in 1 mL of a mixture of methanol:water:formic acid. Then, the samples were centrifuged at 16,000 rpm for 3 min, and the supernatant was colected and injected in the liquid chromatography of high resolution (HPLC). The results confirmed the presence of peramine in the larva's body of D. pallens. The insects that fed the experimental lines LQE-2 and LQE-6 were those where peramine was present in high proportion (0.205 and 0.199 ppm, respectively); while LQE-7 and LQE-3 obtained the lowest concentrations of the alkaloid (0.047 and 0.053 ppm, respectively). Peramine was not detected in the insects when the control cultivar Jumbo (E-) was tested. These results evidenced the storage and metabolism of peramine during consumption of the larvae. However, the effect of this alkaloid present in 'future ryegrass cultivars' (LQE-2 and LQE-6) on the performance and survival of blackworms must be studied and confirmed experimentally.

Keywords: blackworms, HPLC, alkaloid, pest

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Authors: Radek Novotny, Michal Novak, Daniela Marusakova, Monika Sipova, Hugo Fuentes, Peter Borst

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This research has been conducted within the European HORIZON 2020 project ECC-SMART. The main objective is to assess whether it is feasible to design and develop a small modular reactor (SMR) that would be cooled by supercritical water (SCW). One of the main objectives for material research concerns the corrosion of the candidate cladding materials. The experimental part has been conducted in support of the qualification procedure of the future SCW-SMR constructional materials. The last objective was to identify the gaps in current norms and guidelines. Apart from corrosion, resistance testing of candidate materials stresses corrosion cracking susceptibility tests have been performed in supercritical water. This paper describes part of these tests, in particular, those slow strain rate tensile loading applied for tangential ring shape specimens of two candidate materials, Alloy 800H and 310S stainless steel. These ring tensile tests are one the methods used for tensile testing of nuclear cladding. Here full circular heads with dimensions roughly equal to the inner diameter of the sample and the gage sections are placed in the parallel direction to the applied load. Slow strain rate tensile tests have been conducted in 380 or 500oC supercritical water applying two different elongation rates, 1x10-6 and 1x10-7 s-1. The effect of temperature and dissolved oxygen content on the SCC susceptibility of Alloy 800H and 310S stainless steel was investigated when two different temperatures and concentrations of dissolved oxygen were applied in supercritical water. The post-fracture analysis includes fractographic analysis of the fracture surfaces using SEM as well as cross-sectional analysis on the occurrence of secondary cracks. Assessment of the effect of environment and dissolved oxygen content was by comparing to the results of the reference tests performed in air and N2 gas overpressure. The effect of high temperature on creep and its role in the initiation of SCC was assessed as well. It has been concluded that the applied test method could be very useful for the investigation of stress corrosion cracking susceptibility of candidate cladding materials in supercritical water.

Keywords: stress corrosion cracking, ring tensile tests, super-critical water, alloy 800H, 310S stainless steel

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Authors: L. C. Sánchez, L. C. Corrales, A. G. Lancheros, E. Castañeda, Y. Ariza, L. S. Fuentes, L. Sierra, J. L. Cuervo

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The importance of environment friendly alternatives in agricultural processes is the reason why the research group Ceparium, the Colegio Mayor de Cundinamarca University, Colombia, investigated the genus Bacillus and its applicability for improving crops of economic importance in Colombia. In this investigation, we presented a study in which the genus Bacillus plays a leading role as beneficial microorganism. The objective was to identify the biochemical potential of three indigenous species of Bacillus, which were able to carry out actions for biological control against pathogens and pests or promoted growth to improve productivity of crops in Colombia. The procedures were performed in three phases: first, the production of biomass of an indigenous strain and a reference strain starting from culture media for production of spores and toxins were made. Spore count was done in a Neubauer chamber, concentrations of spores of Bacillus sphaericus were prepared and a bioassay was done at the Laboratory of Entomology at the University Jorge Tadeo Lozano of Plutella xylostella larvae, insect pest of crucifers in several Colombian regions. The second phase included the extraction in the liquid state fermentation, a secondary metabolite that has antibiosis action against fungi, call iturin B, and was obtained from strains of Bacillus subtilis. The molecule was identified using High Resolution Chromatography (HPLC) and its biocontrol effect on Fusarium sp fungus causes vascular wilt in economically important plant varieties, was confirmed using testing of antagonism in Petri dish. In the third phase, an initial procedure in that let recover and identify microorganisms of the genus Bacillus from the rhizosphere in two aromatic herbs, Rosmarinus officinalis and Thymus vulgaris L. was used. Subsequently, testing of antagonism against Fusarium sp were made and an assay was done under greenhouse conditions to observe biocontrol and growth promoting action by comparing growth in length and dry weight. In the first experiment, native Bacillus sphaericus was lethal to 92% Plutella xylostella larvae in 10 DDA. In the second experiment, iturin B was identified and biological control of Fusarium sp was demonstrated. In the third study, all strains demonstrated biological control and the B14 strain identified as Bacillus megaterium increased root length and productivity of the two plants in terms of weight. It was concluded that the native microorganisms of the genus Bacillus has a great biochemical potential that provides a beneficial interactions with plants, improve their growth and development and therefore a greater impact on production.

Keywords: genus bacillus, biological control, PGPRs, biochemical potential

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Authors: Cristina Diaz, Lucia Perez-Gandarillas, Gonzalo Garcia-Fuentes, Simone Visigalli, Roberto Canziani, Giuseppe Di Florio, Paolo Gronchi

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During their lifetime, industrial machines are often subjected to chemical, mechanical and thermal extreme conditions. In some cases, the loss of efficiency comes from the degradation of the surface as a result of its exposition to abrasive environments that can cause wear. This is a common problem to be solved in industries of diverse nature such as food, paper or concrete industries, among others. For this reason, a good selection of the material is of high importance. In the machine design context, stainless steels such as AISI 304 and 316 are widely used. However, the severity of the external conditions can require additional protection for the steel and sometimes coating solutions are demanded in order to extend the lifespan of these materials. Therefore, the development of effective coatings with high wear resistance is of utmost technological relevance. In this research, bilayer coatings made of Titanium-Tantalum, Titanium-Niobium, Titanium-Hafnium, and Titanium-Zirconium have been developed using magnetron sputtering configuration by PVD (Physical Vapor Deposition) technology. Their tribological behavior has been measured and evaluated under different environmental conditions. Two kinds of steels were used as substrates: AISI 304, AISI 316. For the comparison with these materials, titanium alloy substrate was also employed. Regarding the characterization, wear rate and friction coefficient were evaluated by a tribo-tester, using a pin-on-ball configuration with different lubricants such as tomato sauce, wine, olive oil, wet compost, a mix of sand and concrete with water and NaCl to approximate the results to real extreme conditions. In addition, topographical images of the wear tracks were obtained in order to get more insight of the wear behavior and scanning electron microscope (SEM) images were taken to evaluate the adhesion and quality of the coating. The characterization was completed with the measurement of nanoindentation hardness and elastic modulus. Concerning the results, thicknesses of the samples varied from 100 nm (Ti-Zr layer) to 1.4 µm (Ti-Hf layer) and SEM images confirmed that the addition of the Ti layer improved the adhesion of the coatings. Moreover, results have pointed out that these coatings have increased the wear resistance in comparison with the original substrates under environments of different severity. Furthermore, nanoindentation hardness results showed an improvement of the elastic strain to failure and a high modulus of elasticity (approximately 200 GPa). As a conclusion, Ti-Ta, Ti-Zr, Ti-Nb, and Ti-Hf are very promising and effective coatings in terms of tribological behavior, improving considerably the wear resistance and friction coefficient of typically used machine materials.

Keywords: coating, stainless steel, tribology, wear

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Authors: Manuel Chacon-Fuentes, Leonardo Bardehle, Marcelo Lizama, Claudio Reyes, Andres Quiroz

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The insect-plant interaction is a complex process in which the plant is able to release chemical signaling that modifies the behavior of insects. Insect herbivory can trigger mechanisms that allow the increase in the production of secondary metabolites that allow coping against the herbivores. Monoterpenes are a kind of secondary metabolites involved in direct defense acting as repellents of herbivorous or even in indirect defense acting as attractants for insect predators. In addition, an increase of the monoterpenes concentration is an effect commonly associated with the herbivory. Hence, plants subjected to damage by herbivory increase the monoterpenes production in comparison to plants without herbivory. In this framework, co-evolutionary aspects play a fundamental role in the adaptation of the herbivorous to their host and in the counter-adaptive strategies of the plants to avoid the herbivorous. In this context, Ugni molinae 'murtilla' is a native shrub from Chile characterized by its antioxidant activity mainly related to the phenolic compounds presents in its fruits. The larval stage of the red grape caterpillar Chilesia rudis Butler (Lepidoptera: Arctiidae) has been reported as an important defoliator of U. molinae. This insect is native from Chile and probably has been involved in a co-evolutionary process with murtilla. Therefore, we hypothesized that herbivory by the red grape caterpillar increases the emission of monoterpenes in Ugni molinae. Ecotypes 19-1 and 22-1 of murtilla were established and maintained at 25° C in the Laboratorio de Química Ecológica at Universidad de La Frontera. Red grape caterpillars of ⁓40 mm were collected near to Temuco (Chile) from grasses, and they were deprived of food for 24 h before performing the assays. Ten caterpillars were placed on the foliage of the ecotypes 19-1 and 22-1 and allowed to feed during 48 h. After this time, caterpillars were removed from the ecotypes and monoterpenes were collected. A glass chamber was used to enclose the ecotypes and a Porapak-Q column was used to trap the monoterpenes. After 24 h of capturing, columns were desorbed with hexane. Then, samples were injected in a gas chromatograph coupled to mass spectrometer and monoterpenes were determined according to the NIST library. All the experiments were performed in triplicate. Results showed that α-pinene, β-phellandrene, limonene, and 1,8 cineole were the main monoterpenes released by murtilla ecotypes. For the ecotype 19-1, the abundance of α-pinene was significantly higher in plants subjected to herbivory (100%) in relation to control plants (54.58%). Moreover, β-phellandrene and 1,8 cineole were observed only in control plants. For ecotype 22-1, there was no significant difference in monoterpenes abundance. In conclusion, the results suggest a trade-off of β-phellandrene and 1,8 cineole in response to herbivory damage by red grape caterpillar generating an increase in α-pinene abundance.

Keywords: Chilesia rudis, gas chromatography, monoterpenes, Ugni molinae

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Authors: Leonardo Parra, Manuel Chacón-Fuentes, Andrés Quiroz

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One of the most important factors in beef and dairy production in the world as well as also in Chile, is related to the correct choice of cultivars or mixtures of forage grasses and legumes to ensure high yields and quality of grassland. However, a great problem is the persistence of the grasses as a result of the action of different hypogeous as epigean pests. The complex insect pests associated with grassland include white grubs (Hylamorpha elegans, Phytoloema herrmanni), blackworm (Dalaca pallens) and Argentine stem weevil (Listronotus bonariensis). In Chile, the principal strategy utilized for controlling this pest is chemical control, through the use of synthetic insecticides, however, underground feeding habits of larval and flight activity of adults makes this uneconomic method. Furthermore, due to problems including environmental degradation, development of resistance and chemical residues, there is a worldwide interest in the use of alternative environmentally friendly pest control methods. In this sense, in recent years there has been an increasing interest in determining the role of endophyte fungi in controlling epigean and hypogeous pest. Endophytes from ryegrass (Lolium perenne), establish a biotrophic relationship with the host, defined as mutualistic symbiosis. The plant-fungi association produces a “cocktail of alkaloids” where peramine is the main toxic substance present in endophyte of ryegrass and responsible for damage reduction of L. bonariensis. In the last decade, few studies have been developed on the effectiveness of new ryegrass cultivars carriers of endophyte in controlling insect pests. Therefore, the aim of this research is to provide knowledge concerning to evaluate the alkaloid content, such as peramine and Lolitrem B, present in new experimental lines of ryegrass and feasible to be used in grasslands of southern Chile. For this, during 2016, ryegrass plants of six experimental lines and two commercial cultivars sown at the Instituto de Investigaciones Agropecuarias Carrillanca (Vilcún, Chile) were collected and subjected to a process of chemical extraction to identify and quantify the presence of peramine and lolitrem B by the technique of liquid chromatography of high resolution (HPLC). The results indicated that the experimental lines EL-1 and EL-3 had high content of peramine (0.25 and 0.43 ppm, respectively) than with lolitrem B (0.061 and 0.19 ppm, respectively). Furthermore, the higher contents of lolitrem B were detected in the EL-4 and commercial cultivar Alto (positive control) with 0.08 and 0.17 ppm, respectively. Peramine and lolitrem B were not detected in the cultivar Jumbo (negative control). These results suggest that EL-3 would have potential as future cultivate because it has high content of peramine, alkaloid responsible for controlling insect pest. However, their current role on the complex insects attacking ryegrass grasslands should be evaluated. The information obtained in this research could be used to improve control strategies against hypogeous and epigean pests of grassland in southern Chile and also to reduce the use of synthetic pesticides.

Keywords: HPLC, Lolitrem B, peramine, pest

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Authors: Andrés Quiroz, Emilio Hormazabal, Ana Mutis, Fernando Ortega, Manuel Chacón-Fuentes, Leonardo Parra

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Red clover (Trifolium pratense L.) is an important forage species in temperate regions of the world. The main limitation of this species worldwide is a lack of persistence related to the high mortality of plants due to a complex of biotic and abiotic factors, determining a life span of two or three seasons. Because of the importance of red clover in Chile, a red clover breeding program was started at INIA Carillanca Research Center in 1989, with the main objective of improving the survival of plants, forage yield, and persistence. The main selection criteria for selecting new varieties have been based on agronomical parameters and biotic factors. The main biotic factor associated with red clover mortality in Chile is Hylastinus obscurus (Coleoptera: Curculionidae). Both larval and adults feed on the roots, causing weakening and subsequent death of clover plants. Pesticides have not been successful for controlling infestations of this root borer. Therefore, alternative strategies for controlling this pest are a high priority for red clover producers. Currently, the role of semiochemical in the interaction between H. obscurus and red clover plants has been widely studied for our group. Specifically, from the red clover foliage has been identified limonene is eliciting repellency from the root borer. Limonene is generated in the plant from two independent biosynthetic pathways, the mevalonic acid, and deoxyxylulose pathway. Mevalonate pathway enzymes are localized in the cytosol, whereas the deoxyxylulose phosphate pathway enzymes are found in plastids. In summary, limonene can be determinated by enzymatic bioassay using GPP as substrate and by limonene synthase expression. Therefore, the main objective of this work was to study genetic variation of limonene in material provided by INIA´s Red Clover breeding program. Protein extraction was carried out homogenizing 250 mg of leave tissue and suspended in 6 mL of extraction buffer (PEG 1500, PVP-30, 20 mM MgCl2 and antioxidants) and stirred on ice for 20 min. After centrifugation, aliquots of 2.5 mL were desalted on PD-10 columns, resulting in a final volume of 3.5 mL. Protein determination was performed according to Bradford with BSA as a standard. Monoterpene synthase assays were performed with 50 µL of protein extracts transferred into gas-tight 2 mL crimp seal vials after addition of 4 µL MgCl₂ and 41 µL assay buffer. The assay was started by adding 5 µL of a GPP solution. The mixture was incubated for 30 min at 40 °C. Biosynthesized limonene was quantified in a GC equipped with a chiral column and using synthetic R and S-limonene standards. The enzymatic the production of R and S-limonene from different Superqueli-Carillanca genotypes is shown in this work. Preliminary results showed significant differences in limonene content among the genotypes analyzed. These results constitute an important base for selecting genotypes with a high content of this repellent monoterpene towards H. obscurus.

Keywords: head space, limonene enzymatic determination, red clover, Hylastinus obscurus

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Authors: Cristina Diaz, Lucia Perez, Simone Visigalli, Giuseppe Di Florio, Gonzalo Fuentes, Roberto Canziani, Paolo Gronchi

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During the last 50 years, the selection of materials is one of the main decisions in machine design for different industrial applications. It is due to numerous physical, chemical, mechanical and technological factors to consider in it. Corrosion effects are related with all of these factors and impact in the life cycle, machine incidences and the costs for the life of the machine. Corrosion affects the deterioration or destruction of metals due to the reaction with the environment, generally wet. In food industry, dewatering industry, concrete industry, paper industry, etc. corrosion is an unsolved problem and it might introduce some alterations of some characteristics in the final product. Nowadays, depending on the selected metal, its surface and its environment of work, corrosion prevention might be a change of metal, use a coating, cathodic protection, use of corrosion inhibitors, etc. In the vast majority of the situations, use of a corrosion resistant material or in its defect, a corrosion protection coating is the solution. Stainless steels are widely used in machine design, because of their strength, easily cleaned capacity, corrosion resistance and appearance. Typical used are AISI 304 and AISI 316. However, their benefits don’t fit every application, and some coatings are required against corrosion such as some paintings, galvanizing, chrome plating, SiO₂, TiO₂ or ZrO₂ coatings, etc. In this work, some coatings based in a bilayer made of Titanium-Tantalum, Titanium-Niobium, Titanium-Hafnium or Titanium-Zirconium, have been developed used magnetron sputtering configuration by PVD (Physical Vapor Deposition) technology, for trying to reduce corrosion effects on AISI 304, AISI 316 and comparing it with Titanium alloy substrates. Ti alloy display exceptional corrosion resistance to chlorides, sour and oxidising acidic media and seawater. In this study, Ti alloy (99%) has been included for comparison with coated AISI 304 and AISI 316 stainless steel. Corrosion tests were conducted by a Gamry Instrument under ASTM G5-94 standard, using different electrolytes such as tomato salsa, wine, olive oil, wet compost, a mix of sand and concrete with water and NaCl for testing corrosion in different industrial environments. In general, in all tested environments, the results showed an improvement of corrosion resistance of all coated AISI 304 and AISI 316 stainless steel substrates when they were compared to uncoated stainless steel substrates. After that, comparing these results with corrosion studies on uncoated Ti alloy substrate, it was observed that in some cases, coated stainless steel substrates, reached similar current density that uncoated Ti alloy. Moreover, Titanium-Zirconium and Titanium-Tantalum coatings showed for all substrates in study including coated Ti alloy substrates, a reduction in current density more than two order in magnitude. As conclusion, Ti-Ta, Ti-Zr, Ti-Nb and Ti-Hf coatings have been developed for improving corrosion resistance of AISI 304 and AISI 316 materials. After corrosion tests in several industry environments, substrates have shown improvements on corrosion resistance. Similar processes have been carried out in Ti alloy (99%) substrates. Coated AISI 304 and AISI 316 stainless steel, might reach similar corrosion protection on the surface than uncoated Ti alloy (99%). Moreover, coated Ti Alloy (99%) might increase its corrosion resistance using these coatings.

Keywords: coatings, corrosion, PVD, stainless steel

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Authors: Lizeth Fuentes-Mera, Vanessa Perez-Silos, Nidia K. Moncada-Saucedo, Alejandro Garcia-Ruiz, Alberto Camacho, Jorge Lara-Arias, Ivan Marino-Martinez, Victor Romero-Diaz, Adolfo Soto-Dominguez, Humberto Rodriguez-Rocha, Hang Lin, Victor Pena-Martinez

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Biphasic scaffolds in cartilage tissue engineering have been designed to influence not only the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone to promote the implant integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a biphasic scaffold based on the assembly of a cartilage phase constituted by fibroin biofunctionalized with bovine cartilage matrix; cellularized with differentiated pre-chondrocytes from adipose tissue stem cells (autologous) and well attached to a bone phase (bone bovine decellularized) to mimic the structure of the nature of native tissue and to promote the cartilage regeneration in a model of joint damage in pigs. Biphasic scaffolds were assembled by fibroin crystallization with methanol. The histological and ultrastructural architectures were evaluated by optical and scanning electron microscopy respectively. Mechanical tests were conducted to evaluate Young's modulus of the implant. For the biological evaluation, pre-chondrocytes were loaded onto the scaffolds and cellular adhesion, proliferation, and gene expression analysis of cartilage extracellular matrix components was performed. The scaffolds that were cellularized and matured for 10 days were implanted into critical 3 mm in diameter and 9-mm in depth osteochondral defects in a porcine model (n=4). Three treatments were applied per knee: Group 1: monophasic cellular scaffold (MS) (single chondral phase), group 2: biphasic scaffold, cellularized only in the chondral phase (BS1), group 3: BS cellularized in both bone and chondral phases (BS2). Simultaneously, a control without treatment was evaluated. After 4 weeks of surgery, integration and regeneration tissues were analyzed by x-rays, histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular biphasic composites exhibited Young's modulus of 805.01 kPa similar to native cartilage (400-800 kPa). In vitro biological studies revealed the chondroinductive ability of the biphasic implant, evidenced by an increase in sulfated glycosaminoglycan (GAGs) and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, In group 1, the defects were not reconstructed. In group 2 and 3 a good integration of the implant with the surrounding tissue was observed. Defects in group 2 were fulfilled by hyaline cartilage and normal bone. Group 3 defects showed fibrous repair tissue. In conclusion; our findings demonstrated the efficacy of biphasic and bioactive scaffold based on silk fibroin, which entwined chondroinductive features and biomechanical capability with appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Keywords: biphasic scaffold, extracellular cartilage matrix, silk fibroin, osteochondral tissue engineering

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