Search results for: Luciana Di Thommazo-Luporini

Authors: Luciana Abednego, Cecilia Nugraheni

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There are many approaches proposed for solving Sudoku puzzles. One of them is by modelling the puzzles as block world problems. There have been three model for Sudoku solvers based on this approach. Each model expresses Sudoku solver as a parameterized multi agent systems. In this work, we propose a new model which is an improvement over the existing models. This paper presents the development of a Sudoku solver that implements all the proposed models. Some experiments have been conducted to determine the performance of each model.

Keywords: Sudoku puzzle, Sudoku solver, block world problem, parameterized multi agent systems

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Authors: Marcela dos P. G. Baltazar, Louise H. Gracioso, Luciana J. Gimenes, Bruno Karolski, Ingrid Avanzi, Elen A. Perpetuo

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In this work, bacterial strains were isolated from environmental samples from a copper mine and three of them presented potential for bioremediation of copper. All the strains were identified by mass spectrometry (MALDI-TOF-Biotyper) and grown in three diferent media supplemented with 100 ppm of copper chloride in flasks of 500mL and it was incubated at 28 °C and 180 rpm. Periodically, samples were taken and monitored for cellular growth and copper biosorption by spectrophotometer UV-Vis (600 nm) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), respectively. At the end of exponential phase of cellular growth, the biomass was utilized to construct a correlation curve between absorbance and dry mass of the cells. Among the three isolates with potential for biorremediation, 1 strain exhibit capacity the most for bioremediation of effluents contaminated by copper being identified as Rhodococcus erythropolis.

Keywords: bioprocess, bioremediation, biosorption, copper

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Authors: Mafalda Fabiene Ferreira Pantoja, Luciana Lins Do Amaral Sobreira

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The present work seeks to exemplify the precepts of the sustainable tectonics, through a built work. This is the architectural group ‘Chácara dos Professores’ in Brasília, Federal District, Brazil. Such an architectural ensemble allows elucidating vernacular constructive techniques that can still be used in contemporary times, on the way to more sustainable constructions. It seeks to show the principles of sustainable tectonics as a design tool, seeking an architecture that can give answers to contemporary issues and that concern caring for the planet. Firstly, a brief investigation will be made about sustainable tectonics, its application in architecture, and later, to correlate such precepts as a tool for reflections and sustainable projects in the contemporary world. For this, will be exemplified, by means of a visit in loco, the construction methods used in the ‘Chácara dos Professores’ project and its results in the constructed work. The goal is to draw a theoretical reflection on the precepts of tectonics, and to show if such precepts are able to enhance contemporary architecture and help it move towards sustainability, seeking projects increasingly consistent with the reality of today.

Keywords: contemporary architecture, constructive techniques, sustainability, tectonics

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Authors: Josiane R. Pires, Marco A. S. González, Bruna L. Brenner, Luciana S. Roos

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The concern with sustainability brought the need for optimization of the buildings to reduce consumption of natural resources. Almost 1/3 of energy demanded by Brazilian housings is used to provide thermal solutions. AEC sector may contribute applying bioclimatic strategies on building design. The aim of this research is to investigate the viability of applying some alternative solutions in residential buildings. The research was developed with computational simulation on single family social housing, examining envelope type, absorptance, and insolation. The analysis of the thermal performance applied both Brazilian standard NBR 15575 and degree-hour method, in the scenery of Porto Alegre, a southern Brazilian city. We used BIM modeling through Revit/Autodesk and used Energy Plus to thermal simulation. The payback of the investment was calculated comparing energy savings and building costs, in a period of 50 years. The results shown that with the increment of envelope’s insulation there is thermal comfort improvement and energy economy, with a pay-back period of 24 to 36 years, in some cases.

Keywords: civil construction, design, thermal performance, energy, economic analysis

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Authors: Luciana Marrone

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Natural dyes are a great resource for today's artists and designers providing endless possibilities for design and sustainability. This research and development project focuses on the idea of making these dyeing or painting methodologies reach the widest possible range of students. The main objective is to inform and train, free of charge, teachers and students from different academic institutions, at different levels, kindergarten, primary, secondary, tertiary and university. In this research and dissemination project, in the first instance, institutions from Argentina, Chile, Uruguay, Mexico, Spain, Italy, Colombia, Paraguay, Venezuela, Brazil and Australia joined the project, reaching the grassroots of education from the very beginning. Natural dyes will become part of everyday life for more people, achieving their own colors for art, textiles or any other application. The knowledge of the techniques and resources of the student a fundamental tool, sustainable and opens endless possibilities even in places or homes with few economic resources, thus achieving that natural dyes are not only part of the world of designers but also that they are incorporated from the basics and can thus become a resource applicable in different areas even in places with few economic or development possibilities.

Keywords: art, education, natural dyes, sustainability, textile design.

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Authors: Marcus C. Garcia, Giselle C. Guimarães, Luciana H. Yamane, Renato R. Siman

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The environmental indicators and the classification of beach waste are essential tools to diagnose the current situation and to indicate ways to improve the quality of this environment. The purpose of this paper was to perform a quali-quantitative analysis of the beach waste on the Curva da Jurema Beach (Espírito Santo - Brazil). Three transects were used with equidistant positioning over the total length of the beach for the solid waste collection. Solid wastes were later classified according to their use and primary raw material from the low and high summer season. During the low season, average values of 7.10 items.m^-1, 18.22 g.m^-1 and 0.91 g.m^-2 were found for the whole beach, and transect 3 contributed the most waste, with the total sum of items equal to 999 (49%), a total mass of 5.62 kg and a total volume of 21.31 L. During the high summer season, average values of 8.22 items.m^-1, 54.40 g.m^-1 and 2.72 g.m^-2 were found, with transect 2 contributing the most to the total sum with 1,212 items (53%), a total mass of 10.76 kg and a total volume of 51.99 L. Of the total collected, plastic materials represented 51.4% of the total number of items, 35.9% of the total mass and 68% of the total volume. The implementation of reactive and proactive measures is necessary so that the management of the solid wastes on Curva da Jurema Beach is in accordance with principles of sustainability.

Keywords: beach solid waste, environmental indicators, quali-quantitative analysis, waste management

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Authors: Angga Hidayah Ramadhan, Luciana Andrawina, M. Azani Hasibuan

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Data will be as a core of the decision if it has a good treatment or process, which is process that data into information, and information into knowledge to make a wisdom or decision. Today, many companies have not realize it include XYZ University Admission Directorate as executor of National Admission called Seleksi Masuk Bersama (SMB) that during the time, the workers only uses their feeling to make a decision. Whereas if it done, then that company can analyze the data to make a right decision to get a pin sales from student candidate or registrant that follow SMB as many as possible. Therefore, needs Knowledge Management System (KMS) with Geographic Information System (GIS) use 5C4C that can process that company data becomes more useful and can help make decisions. This information system can process data into information based on the pin sold data with 5C (Contextualized, Categorize, Calculation, Correction, Condensed) and convert information into knowledge with 4C (Comparing, Consequence, Connection, Conversation) that has been several steps until these data can be useful to make easier to take a decision or wisdom, resolve problems, communicate, and quicker to learn to the employees have not experience and also for ease of viewing/visualization based on spatial data that equipped with GIS functionality that can be used to indicate events in each province with indicator that facilitate in this system. The system also have a function to save the tacit on the system then to be proceed into explicit in expert system based on the problems that will be found from the consequences of information. With the system each team can make a decision with same ways, structured, and the important is based on the actual event/data.

Keywords: 5C4C, data, information, knowledge

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Authors: Luciana Steffen

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People with disabilities are often neglected in the exercise of their sexuality, facing several prejudices and discrimination in this area. For women with disabilities, the negligence is even major. Studies that relate sexual and reproductive rights with the experience of women with disabilities are rare, and in the field of Theology, practically nonexistent in Brazil. The aim of this work is to reflect on the relationship between women with disabilities, sexual and reproductive rights and Theology, according to a feminist perspective. The work is a literature review and involves the areas of Gender Studies, Disability Studies, Feminist Studies and Theology. In the article it will be addressed the relations between disability, sexual and reproductive rights, feminism, as well as the relations with the area of Theology, reflecting on these themes toward a fairer and more inclusive understanding of feminism, sexuality and women with disabilities. To reflect on sexual and reproductive rights of women with disabilities, it is important to reflect on religious concepts about the body, sexuality, reproduction and gender roles, because they are all connected. So, a critical analysis of traditional theological values taking into consideration the dimensions of sexuality and women with disability is important for a more liberating and inclusive understand about sexual and reproductive rights of women with disabilities. Theology should help the other areas in the understanding that all people have the right to live their lives with completeness, dignity and respect, so women with disabilities must have the opportunity of making their own choices on the fields of sexuality and reproduction.

Keywords: gender, disability, sexual and reproductive rights, theology

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Authors: Luciana Brandao Ferreira, Janaina de Moura Engracia Giraldi, Fabiana Gondim Mariutti, Marina Toledo de Arruda Lourencao

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Developing countries, such as BRICS (Brazil, Russia, India, China and South Africa) are hosting sports mega-events to promote socio-economic development and image enhancement. Thus, this paper aims to verify the image of Rio de Janeiro, in Brazil, as the host city of 2016 Olympic Games, considering the main cognitive and affective image dimensions. The research design uses exploratory factorial analysis to find the most important factors highlighted in the city image dimensions. The data were collected by structured questionnaires with an international respondents sample (n=274) with high international travel experience. The results show that Rio’s image as a sport mega-event host city has two main factors in each dimension: Cognitive ('General Infrastructure'; 'Services and Attractions') and Affective ('Positive Feelings'; 'Negative Feelings'). The most important factor related to cognitive dimension was 'Services and Attractions' which is more related to tourism activities. In the affective dimension 'Positive Feelings' was the most important factor, which means a good result considering that is a city in an emerging country with many unmet social demands.

Keywords: Rio de Janeiro, 2016 olympic games, host city image, cognitive image dimension, affective image dimension

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Authors: Andressa S. T. Gomes, Luiza A. Souza, Luciana H. Yamane, Renato R. Siman

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The segregation of waste of electrical and electronic equipment (WEEE) in the generating source, its characterization (quali-quantitative) and identification of origin, besides being integral parts of classification reports, are crucial steps to the success of its integrated management. The aim of this paper was to count WEEE generation at the Federal University of Espírito Santo (UFES), Brazil, as well as to define sources, temporary storage sites, main transportations routes and destinations, the most generated WEEE and its recycling potential. Quantification of WEEE generated at the University in the years between 2010 and 2015 was performed using data analysis provided by UFES’s sector of assets management. EEE and WEEE flow in the campuses information were obtained through questionnaires applied to the University workers. It was recorded 6028 WEEEs units of data processing equipment disposed by the university between 2010 and 2015. Among these waste, the most generated were CRT screens, desktops, keyboards and printers. Furthermore, it was observed that these WEEEs are temporarily stored in inappropriate places at the University campuses. In general, these WEEE units are donated to NGOs of the city, or sold through auctions (2010 and 2013). As for recycling potential, from the primary processing and further sale of printed circuit boards (PCB) from the computers, the amount collected could reach U$ 27,839.23. The results highlight the importance of a WEEE management policy at the University.

Keywords: solid waste, waste of electrical and electronic equipment, waste management, institutional solid waste generation

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Authors: Luciana C. Ramos, Leandro J. Sousa, Antônio Ferreira da Silva, Valéria Gomes Oliveira Falcão, Suzana T. Cunha Lima

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The commercial production of biodiesel using microalgae demands a high-energy input for harvesting biomass, making production economically unfeasible. Methods currently used involve mechanical, chemical, and biological procedures. In this work, a flocculation system is presented as a cost and energy effective process to increase biomass production of Phaeodactylum tricornutum. This diatom is the only species of the genus that present fast growth and lipid accumulation ability that are of great interest for biofuel production. The algae, selected from the Bank of Microalgae, Institute of Biology, Federal University of Bahia (Brazil), have been bred in tubular reactor with photoperiod of 12 h (clear/dark), providing luminance of about 35 μmol photons m^-2s^-1, and temperature of 22 °C. The medium used for growing cells was the Conway medium, with addition of silica. The seaweed growth curve was accompanied by cell count in Neubauer camera and by optical density in spectrophotometer, at 680 nm. The precipitation occurred at the end of the stationary phase of growth, 21 days after inoculation, using two methods: centrifugation at 5000 rpm for 5 min, and electro-flocculation at 19 EPD and 95 W. After precipitation, cells were frozen at -20 °C and, subsequently, lyophilized. Biomass obtained by electro-flocculation was approximately four times greater than the one achieved by centrifugation. The benefits of this method are that no addition of chemical flocculants is necessary and similar cultivation conditions can be used for the biodiesel production and pharmacological purposes. The results may contribute to improve biodiesel production costs using marine microalgae.

Keywords: biomass, diatom, flocculation, microalgae

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Authors: Ana C. V. Freitas, Luciana B. M. Pires, Joao P. Martins

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In recent years, fire dynamics in deforestation areas of tropical forests have received considerable attention because of their relationship to climate change. Climate models project great increases in the frequency and area of drought in the Amazon region, which may increase the occurrence of fires. This study analyzes the historical record number of fire outbreaks in 2017 using satellite-derived data sets of active fire detections, burned area, precipitation, and data of the Fire Program from the Center for Weather Forecasting and Climate Studies (CPTEC/INPE). A downward trend in the number of fire outbreaks occurred in the first half of 2017, in relation to the previous year. This decrease can be related to the fact that 2017 was not an El Niño year and, therefore, the observed rainfall and temperature in the Amazon region was close to normal conditions. Meanwhile, the worst period in history for fire outbreaks began with the subsequent arrival of the dry season. September of 2017 exceeded all monthly records for number of fire outbreaks per month in the entire series. This increase was mainly concentrated in Bolivia and in the states of Amazonas, northeastern Pará, northern Rondônia and Acre, regions with high densities of rural settlements, which strongly suggests that human action is the predominant factor, aggravated by the lack of precipitation during the dry season allowing the fires to spread and reach larger areas. Thus, deforestation in the Amazon is primarily a human-driven process: climate trends may be providing additional influences.

Keywords: Amazon forest, climate change, deforestation, human-driven process, fire outbreaks

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Authors: Ingrid R. Avanzi, Marcela dos P. G. Baltazar, Louise H. Gracioso, Luciana J. Gimenes, Bruno Karolski, Elen A. Perpetuo, Claudio Auguto Oller do Nascimento

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In this study was developed a natural process using a biological system for the uptake of Copper and possible removal of copper from wastewater by dead biomass of the strain Burkholderia cenocepacia. Dead and live biomass of Burkholderia cenocepacia was used to analyze the equilibrium and kinetics of copper biosorption by this strain in function of the pH. Living biomass exhibited the highest biosorption capacity of copper, 50 mg g−1, which was achieved within 5 hours of contact, at pH 7.0, temperature of 30°C, and agitation speed of 150 rpm. The dead biomass of Burkholderia cenocepacia may be considered an efficiently bioprocess, being fast and low-cost to production of copper and also a probably nano-adsorbent of this metal ion in wastewater in bioremediation process. In this study was developed a natural process using a biological system for the uptake of Copper and possible removal of copper from wastewater by dead biomass of the strain Burkholderia cenocepacia. Dead and live biomass of Burkholderia cenocepacia was used to analyze the equilibrium and kinetics of copper biosorption by this strain in function of the pH. Living biomass exhibited the highest biosorption capacity of copper, 50 mg g−1, which was achieved within 5 hours of contact, at pH 7.0, temperature of 30°C, and agitation speed of 150 rpm. The dead biomass of Burkholderia cenocepacia may be considered an efficiently bioprocess, being fast and low-cost to production of copper and also a probably nano-adsorbent of this metal ion in wastewater in bioremediation process.

Keywords: biosorption, dead biomass, biotechnology, copper recovery

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Authors: Luciana P. Mazur, Tatiana A. Pozdniakova, Rui A. R. Boaventura, Vitor J. P. Vilar

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The electroplating industry requires a lot of process water, which generates a large volume of wastewater loaded with heavy metals. Two different wastewaters were collected in a company’s wastewater treatment plant, one after the use of zinc in the metal plating process and the other after the use of chromium. The main characteristics of the Zn(II) and Cr(VI) wastewaters are: pH = 6.7/5.9; chemical oxygen demand = 55/<5 mg/L; sodium, potassium, magnesium and calcium ions concentrations of 326/28, 4/28, 11/7 and 46/37 mg/L, respectively; zinc(II) = 11 mg/L and Cr(VI) = 39 mg/L. Batch studies showed that L. hyperborea can be established as a natural cation exchanger for heavy metals uptake mainly due to the presence of negatively charged functional groups in the surface of the biomass. Beyond that, L. hyperborea can be used as a natural electron donor for hexavalent chromium reduction to trivalent chromium at acidic medium through the oxidation of the biomass, and Cr(III) can be further bound to the negatively charged functional groups. The uptake capacity of Cr(III) by the oxidized biomass after Cr(VI) reduction was higher than by the algae in its original form. This can be attributed to the oxidation of the biomass during Cr(VI) reduction, turning other active sites available for Cr(III) binding. The brown macroalgae Laminaria hyperborea was packed in a fixed-bed column in order to evaluate the feasibility of the system for the continuous treatment of the two galvanization wastewaters. The column, with an internal diameter of 4.8 cm, was packed with 59 g of algae up to a bed height of 27 cm. The operation strategy adopted for the treatment of the two wastewaters consisted in: i) treatment of the Zn(II) wastewater in the first sorption cycle; ii) desorption of pre-loaded Zn(II) using an 1.0 M HCl solution; iii) treatment of the Cr(VI) wastewater, taking advantage of the acidic conditions of the column after the desorption cycle, for the reduction of the Cr(VI) to Cr(III), in the presence of the electrons resulting from the biomass oxidation. This cycle ends when all the oxidizing groups are used.

Keywords: biosorption, brown marine macroalgae, zinc, chromium

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Authors: Tatiana A. Pozdniakova, Maria A. P. Cechinel, Luciana P. Mazur, Rui A. R. Boaventura, Vitor J. P. Vilar.

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Two brown macroalgae, Laminaria hyperborea and Pelvetia canaliculata, were employed as natural cation exchangers in a fixed-bed column for Zn(II) removal from a galvanization wastewater. The column (4.8 cm internal diameter) was packed with 30-59 g of previously hydrated algae up to a bed height of 17-27 cm. The wastewater or eluent was percolated using a peristaltic pump at a flow rate of 10 mL/min. The effluent used in each experiment presented similar characteristics: pH of 6.7, 55 mg/L of chemical oxygen demand and about 300, 44, 186 and 244 mg/L of sodium, calcium, chloride and sulphate ions, respectively. The main difference was nitrate concentration: 20 mg/L for the effluent used with L. hyperborean and 341 mg/L for the effluent used with P. canaliculata. The inlet zinc concentration also differed slightly: 11.2 mg/L for L. hyperborean and 8.9 mg/L for P. canaliculata experiments. The breakthrough time was approximately 22.5 hours for both macroalgae, corresponding to a service capacity of 43 bed volumes. This indicates that 30 g of biomass is able to treat 13.5 L of the galvanization wastewater. The uptake capacities at the saturation point were similar to that obtained in batch studies (unpublished data) for both algae. After column exhaustion, desorption with 0.1 M HNO3 was performed. Desorption using 9 and 8 bed volumes of eluent achieved an efficiency of 100 and 91%, respectively for L. hyperborean and P. canaliculata. After elution with nitric acid, the column was regenerated using different strategies: i) convert all the binding sites in the sodium form, by passing a solution of 0.5 M NaCl, until achieve a final pH of 6.0; ii) passing only tap water in order to increase the solution pH inside the column until pH 3.0, and in this case the second sorption cycle was performed using protonated algae. In the first approach, in order to remove the excess of salt inside the column, distilled water was passed through the column, leading to the algae structure destruction and the column collapsed. Using the second approach, the algae remained intact during three consecutive sorption/desorption cycles without loss of performance.

Keywords: biosorption, zinc, galvanization wastewater, packed-bed column

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Authors: Soultana Konstantinidou, Tiziana Schmidt, Elena Landi, Alessandro De Carli, Giovanni Maltinti, Darius Witt, Alicja Dziadosz, Agnieszka Lindstaedt, Michele Lai, Mauro Pistello, Valentina Cappello, Luciana Dente, Chiara Gabellini, Piotr Barski, Vittoria Raffa

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CRISPR/Cas9 technology has gained the interest of researchers in the field of biotechnology for genome editing. Since its discovery as a microbial adaptive immune defense, this system has been widely adopted and is acknowledged for having a variety of applications. However, critical barriers related to safety and delivery are persisting. Here, we propose a new concept of genome engineering, which is based on a nano-formulation of Cas9. The Cas9 enzyme was conjugated to a gold nanoparticle (AuNP-Cas9). The AuNP-Cas9 maintained its cleavage efficiency in vitro, to the same extent as the ribonucleoprotein, including non-conjugated Cas9 enzyme, and showed high gene editing efficiency in vivo in zebrafish embryos. Since CRISPR/Cas9 technology is extensively used in cancer research, melanoma was selected as a validation target. Cell studies were performed in A375 human melanoma cells. Particles per se had no impact on cell metabolism and proliferation. Intriguingly, the AuNP-Cas9 internalized spontaneously in cells and localized as a single particle in the cytoplasm and organelles. More importantly, the AuNP-Cas9 showed a high nuclear localization signal. The AuNP-Cas9, overcoming the delivery difficulties of Cas9, could be used in cellular biology and localization studies. Taking advantage of the plasmonic properties of gold nanoparticles, this technology could potentially be a bio-tool for combining gene editing and photothermal therapy in cancer cells. Further work will be focused on intracellular interactions of the nano-formulation and characterization of the optical properties.

Keywords: CRISPR/Cas9, gene editing, gold nanoparticles, nanotechnology

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Authors: Luciana P. Mazur, Tatiana A. Pozdniakova, Rui A. R. Boaventura, Vitor J. P. Vilar

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The principal mechanism of metal ions sequestration by brown algae involves the formation of complexes between the metal ion and functional groups present on the cell wall of the biological material. To understand the role of functional groups on copper(II) uptake by Ascophyllum nodosum, some functional groups were chemically modified. The esterification of carboxylic groups was carried out by suspending the biomass in a methanol/HCl solution under stirring for 48 h and the blocking of the sulfonic groups was performed by repeating the same procedure for 4 cycles of 48 h. The methylation of amines was conducted by suspending the biomass in a formaldehyde/formic acid solution under shaking for 6 h and the chemical modification of sulfhydryl groups on the biomass surface was achieved using dithiodipyridine for 1 h. Equilibrium sorption studies for Cu2+ using the raw and esterified algae were performed at pH 2.0 and 4.0. The experiments were performed using an initial copper concentration of 300 mg/L and algae dose of 1.0 g/L. After reaching the equilibrium, the metal in solution was quantified by atomic absorption spectrometry. The biological material was analyzed by Fourier Transform Infrared Spectroscopy and Potentiometric Titration techniques for functional groups identification and quantification, respectively. The results using unmodified algae showed that the maximum copper uptake capacity at pH 4.0 and 2.0 was 1.17 and 0.52 mmol/g, respectively. At acidic pH values most carboxyl groups are protonated and copper sorption suffered a significant reduction of 56%. Blocking the carboxylic, sulfonic, amines and sulfhydryl functional groups, copper uptake decreased by 24/26%, 69/81%, 1/23% and 40/27% at pH 2.0/4.0, respectively, when compared to the unmodified biomass. It was possible to conclude that the carboxylic and sulfonic groups are the main functional groups responsible for copper binding (>80%). This result is supported by the fact that the adsorption capacity is directly related to the presence of carboxylic groups of the alginate polymer, and the second most abundant acidic functional group in brown algae is the sulfonic acid of fucoidan that contributes, to a lower extent, to heavy metal binding, particularly at low pH.

Keywords: biosorption, brown marine macroalgae, copper, ion-exchange

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Authors: Luciana Salomon

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The Andes hold one of the highest plant species diversity in the world. How such diversity originated is one of the most intriguing questions in studies addressing the pattern of plant diversity worldwide. Recently, the explosive adaptive radiations found in high Andean groups have been pointed as major triggers of this spectacular diversity. The Andes are one of the most species-rich area for the largest genus from the Asteraceae family, Senecio. There, the genus presents an incredible variation in growth form and ecological niche space. If this diversity of Andean Senecio can be explained by a monophyletic origin and subsequent radiation has not been tested up to now. Previous studies trying to disentangle the evolutionary history of some Andean Senecio struggled with the relatively low resolution and support of the phylogenies, which is indicative of recently radiated groups. Using Hyb-Seq, a powerful approach is available to address phylogenetic questions in groups whose evolutionary histories are recent and rapid. This approach was used for Senecio to build a phylogenetic backbone on which to study the mechanisms shaping its hyper-diversity in the Andes, focusing on Senecio ser. Culcitium, an exclusively Andean and well circumscribed group presenting large morphological variation and which is widely distributed across the Andes. Hyb-Seq data for about 130 accessions of Seneciowas generated. Using standard data analysis work flows and a newly developed tool to utilize paralogs for phylogenetic reconstruction, robustness of the species treewas investigated. Fully resolved and moderately supported species trees were obtained, showing Senecio ser. Culcitium as monophyletic. Within this group, some species formed well-supported clades congruent with morphology, while some species would not have exclusive ancestry, in concordance with previous studies showing a geographic differentiation. Additionally, paralogs were detected for a high number of loci, indicating duplication events and hybridization, known to be common in Senecio ser. Culcitium might have lead to hybrid speciation. The rapid diversification of the group seems to have followed a south-north distribution throughout the Andes, having accelerated in the conquest of new habitats more recently available: i.e., Montane forest, Paramo, and Superparamo.

Keywords: evolutionary radiations, andes, paralogy, hybridization, senecio

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Authors: Luciana Aparecida Bastos, Tatiana Diair L. F. Rosa, Jesus Creapldi

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The idea of Latin American integration occurred from the ideals of Simón Bolívar that, in 1824, called the Ibero-American nations to Amphictyonic Congress of Panama, on June 22, 1826, where he would defend the importance of Latin American unity. However, this congress was frustrating and the idea of Bolívar went no further. It was only after the European Union to start the process, driven by the end of World War II that the subject returned to emerge in Latin America. Thus, in 1960, supported by the European integration process, started in 1957 with the excellent result of the ECSC - European Coal and Steel Community, a result of the Customs Union of the BENELUX (integration between Belgium, the Netherlands and Luxembourg) in 1948, was created in Latin America, LAFTA - Latin American Free Trade Association, in 1960. In 1980, LAFTA was replaced by LAAI- Latin American Association, both with the same goal: to integrate Latin America, it´s economy and its trade. Most researchers in this period agree that the regional market would be expanded through the integration. The creation of one or more economic blocs in the region would provide the union of Latin American countries through a fusion of common interests and by their geographical proximity, which would try to develop common projects to promote mutual growth and economic development, tariff reductions, promotion of increased trade between, among many other goals set together. Thus, taking into account Mercosur, the main Latin-American block, created in 1994, the aim of this paper is to make a brief analysis of the trade balance performance of Brazil (larger economy of the block) in Mercosur in the periods: 1989-1994 and 1994-2012. The choice of this period was because the objective is to compare the period before and after the integration of Brazil in Mercosur. The methodologies used were the literature review and descriptive statistics. The results showed that after the integration of Brazil in Mercosur, the exports and imports grew within the bloc and the country turned out to become the leading importer of other economies of Mercosur after integration, that is, Brazil, after integration to Mercosur, was largely responsible for promoting the expansion of regional trade through the import of products from other members of the block.

Keywords: Brazil, mercosur, integration, trade balance, comparison

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Authors: Natan C. G. Silva, Carlos A. C. Girao Neto, Marcele M. S. Vasconcelos, Luciana R. B. Goncalves, Maria Valderez P. Rocha

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Galactooligosaccharides (GOS) are sugars with prebiotic function that can be synthesized chemically or enzymatically, and this last one can be promoted by the action of β-galactosidases. In addition to favoring the transgalactosylation reaction to form GOS, these enzymes can also catalyze the hydrolysis of lactose. A highly studied type of GOS is lactulose because it presents therapeutic properties and is a health promoter. Among the different raw materials that can be used to produce lactulose, whey stands out as the main by-product of cheese manufacturing, and its discarded is harmful to the environment due to the residual lactose present. Therefore, its use is a promising alternative to solve this environmental problem. Thus, lactose from whey is hydrolyzed into glucose and galactose by β-galactosidases. However, in order to favor the transgalactosylation reaction, the medium must contain fructose, due this sugar reacts with galactose to produce lactulose. Then, the glucose-isomerase enzyme can be used for this purpose, since it promotes the isomerization of glucose into fructose. In this scenario, the aim of the present work was first to develop β-galactosidase biocatalysts of Kluyveromyces lactis and to apply it in the integrated reactions of hydrolysis, isomerization (with the glucose-isomerase from Streptomyces murinus) and transgalactosylation reaction, using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% glutaraldehyde was evaluated using different enzymatic loads (2, 5, 7, 10, and 12 mg/g). Subsequently, the hydrolysis and transgalactosylation reactions were studied and conducted at 50°C, 120 RPM for 20 minutes. In parallel, the isomerization of glucose into fructose was evaluated under conditions of 70°C, 750 RPM for 90 min. After, the integration of the three processes for the production of lactulose was investigated. Among the evaluated loads, 7 mg/g was chosen because the best activity of the derivative (44.3 U/g) was obtained, being this parameter determinant for the reaction stages. The other parameters of immobilization yield (87.58%) and recovered activity (46.47%) were also satisfactory compared to the other conditions. Regarding the integrated process, 94.96% of lactose was converted, achieving 37.56 g/L and 37.97 g/L of glucose and galactose, respectively. In the isomerization step, conversion of 38.40% of glucose was observed, obtaining a concentration of 12.47 g/L fructose. In the transgalactosylation reaction was produced 13.15 g/L lactulose after 5 min. However, in the integrated process, there was no formation of lactulose, but it was produced other GOS at the same time. The high galactose concentration in the medium probably favored the reaction of synthesis of these other GOS. Therefore, the integrated process proved feasible for possible production of prebiotics. In addition, this process can be economically viable due to the use of an industrial residue as a substrate, but it is necessary a more detailed investigation of the transgalactosilation reaction.

Keywords: beta-galactosidase, glucose-isomerase, galactooligosaccharides, lactulose, whey

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Authors: Louise H. Gracioso, Marcela P.G. Baltazar, Ingrid R. Avanzi, Bruno Karolski, Luciana J. Gimenes, Claudio O. Nascimento, Elen A. Perpetuo

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Introduction: Higher copper concentrations promote a selection pressure on organisms such as plants, fungi and bacteria, which allows surviving only the resistant organisms to the contaminated site. This selective pressure keeps only the organisms most resistant to a specific condition and subsequently increases their bioremediation potential. Despite the bacteria importance for biosphere maintenance, it is estimated that only a small fraction living microbial species has been described and characterized. Due to the molecular biology development, tools based on analysis 16S ribosomal RNA or another specific gene are making a new scenario for the characterization studies and identification of microorganisms in the environment. News identification of microorganisms methods have also emerged like Biotyper (MALDI / TOF), this method mass spectrometry is subject to the recognition of spectroscopic patterns of conserved and features proteins for different microbial species. In view of this, this study aimed to isolate bacteria resistant to copper present in a Copper Processing Area (Sossego Mine, Canaan, PA) and identifies them in two different methods: Recent (spectrometry mass) and conventional. This work aimed to use them for a future bioremediation of this Mining. Material and Methods: Samples were collected at fifteen different sites of five periods of times. Microorganisms were isolated from mining wastes by culture enrichment technique; this procedure was repeated 4 times. The isolates were inoculated into MJS medium containing different concentrations of chloride copper (1mM, 2.5mM, 5mM, 7.5mM and 10 mM) and incubated in plates for 72 h at 28 ºC. These isolates were subjected to mass spectrometry identification methods (Biotyper – MALDI/TOF) and 16S gene sequencing. Results: A total of 105 strains were isolated in this area, bacterial identification by mass spectrometry method (MALDI/TOF) achieved 74% agreement with the conventional identification method (16S), 31% have been unsuccessful in MALDI-TOF and 2% did not obtain identification sequence the 16S. These results show that Biotyper can be a very useful tool in the identification of bacteria isolated from environmental samples, since it has a better value for money (cheap and simple sample preparation and MALDI plates are reusable). Furthermore, this technique is more rentable because it saves time and has a high performance (the mass spectra are compared to the database and it takes less than 2 minutes per sample).

Keywords: copper mining area, bioremediation, microorganisms, identification, MALDI/TOF, RNA 16S

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Authors: Priscila A. Bernardes, Marcos E. Buzanskas, Luciana C. A. Regitano, Ricardo V. Ventura, Danisio P. Munari

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The best linear unbiased predictor (BLUP) is a method commonly used in genetic evaluations of breeding programs. However, this approach can lead to higher inbreeding coefficients in the population due to the intensive use of few bulls with higher genetic potential, usually presenting some degree of relatedness. High levels of inbreeding are associated to low genetic viability, fertility, and performance for some economically important traits and therefore, should be constantly monitored. Unreliable pedigree data can also lead to misleading results. Genomic information (i.e., single nucleotide polymorphism – SNP) is a useful tool to estimate the inbreeding coefficient. Runs of homozygosity have been used to evaluate homozygous segments inherited due to direct or collateral inbreeding and allows inferring population selection history. This study aimed to evaluate runs of homozygosity (ROH) and inbreeding in a population of Nelore beef cattle. A total of 814 animals were genotyped with the Illumina BovineHD BeadChip and the quality control was carried out excluding SNPs located in non-autosomal regions, with unknown position, with a p-value in the Hardy-Weinberg equilibrium lower than 10⁻⁵, call rate lower than 0.98 and samples with the call rate lower than 0.90. After the quality control, 809 animals and 509,107 SNPs remained for analyses. For the ROH analysis, PLINK software was used considering segments with at least 50 SNPs with a minimum length of 1Mb in each animal. The inbreeding coefficient was calculated using the ratio between the sum of all ROH sizes and the size of the whole genome (2,548,724kb). A total of 25.711 ROH were observed, presenting mean, median, minimum, and maximum length of 3.34Mb, 2Mb, 1Mb, and 80.8Mb, respectively. The number of SNPs present in ROH segments varied from 50 to 14.954. The longest ROH length was observed in one animal, which presented a length of 634Mb (24.88% of the genome). Four bulls were among the 10 animals with the longest extension of ROH, presenting 11% of ROH with length higher than 10Mb. Segments longer than 10Mb indicate recent inbreeding. Therefore, the results indicate an intensive use of few sires in the studied data. The distribution of ROH along the chromosomes showed that chromosomes 5 and 6 presented a large number of segments when compared to other chromosomes. The mean, median, minimum, and maximum inbreeding coefficients were 5.84%, 5.40%, 0.00%, and 24.88%, respectively. Although the mean inbreeding was considered low, the ROH indicates a recent and intensive use of few sires, which should be avoided for the genetic progress of breed.

Keywords: autozygosity, Bos taurus indicus, genomic information, single nucleotide polymorphism

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Authors: Priscila A. Bernardes, Marcos E. Buzanskas, Luciana C. A. Regitano, Ricardo V. Ventura, Danisio P. Munari

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Genotype imputation has been used to reduce genomic selections costs. In order to increase haplotype detection accuracy in methods that considers the linkage disequilibrium, another approach could be used, such as combined genotype data from different panels. Therefore, this study aimed to evaluate the linkage disequilibrium and haplotype blocks in two high-density panels before and after the imputation to a combined panel in Nelore beef cattle. A total of 814 animals were genotyped with the Illumina BovineHD BeadChip (IHD), wherein 93 animals (23 bulls and 70 progenies) were also genotyped with the Affymetrix Axion Genome-Wide BOS 1 Array Plate (AHD). After the quality control, 809 IHD animals (509,107 SNPs) and 93 AHD (427,875 SNPs) remained for analyses. The combined genotype panel (CP) was constructed by merging both panels after quality control, resulting in 880,336 SNPs. Imputation analysis was conducted using software FImpute v.2.2b. The reference (CP) and target (IHD) populations consisted of 23 bulls and 786 animals, respectively. The linkage disequilibrium and haplotype blocks studies were carried out for IHD, AHD, and imputed CP. Two linkage disequilibrium measures were considered; the correlation coefficient between alleles from two loci (r²) and the |D’|. Both measures were calculated using the software PLINK. The haplotypes' blocks were estimated using the software Haploview. The r² measurement presented different decay when compared to |D’|, wherein AHD and IHD had almost the same decay. For r², even with possible overestimation by the sample size for AHD (93 animals), the IHD presented higher values when compared to AHD for shorter distances, but with the increase of distance, both panels presented similar values. The r² measurement is influenced by the minor allele frequency of the pair of SNPs, which can cause the observed difference comparing the r² decay and |D’| decay. As a sum of the combinations between Illumina and Affymetrix panels, the CP presented a decay equivalent to a mean of these combinations. The estimated haplotype blocks detected for IHD, AHD, and CP were 84,529, 63,967, and 140,336, respectively. The IHD were composed by haplotype blocks with mean of 137.70 ± 219.05kb, the AHD with mean of 102.10kb ± 155.47, and the CP with mean of 107.10kb ± 169.14. The majority of the haplotype blocks of these three panels were composed by less than 10 SNPs, with only 3,882 (IHD), 193 (AHD) and 8,462 (CP) haplotype blocks composed by 10 SNPs or more. There was an increase in the number of chromosomes covered with long haplotypes when CP was used as well as an increase in haplotype coverage for short chromosomes (23-29), which can contribute for studies that explore haplotype blocks. In general, using CP could be an alternative to increase density and number of haplotype blocks, increasing the probability to obtain a marker close to a quantitative trait loci of interest.

Keywords: Bos taurus indicus, decay, genotype imputation, single nucleotide polymorphism

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Authors: Paula K. Novelli, Margarida M. Barros, Luciana F. Flueri

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Utilization of agricultural by-products, wheat ban and soybean bran, as substrate for solid state fermentation (SSF) was studied, aiming the achievement of different enzymes from Aspergillus sp. with distinct biological characteristics and its application and improvement on animal nutrition. Aspergillus niger and Aspergillus oryzea were studied as they showed very high yield of phytase and protease production, respectively. Phytase activity was measure using p-nitrophenilphosphate as substrate and a standard curve of p-nitrophenol, as the enzymatic activity unit was the quantity of enzyme necessary to release one μmol of p-nitrophenol. Protease activity was measure using azocasein as substrate. Activity for phytase and protease substantially increased when the different biochemical characteristics were considered in the study. Optimum pH and stability of the phytase produced by A. niger with wheat bran as substrate was between 4.0 - 5.0 and optimum temperature of activity was 37oC. Phytase fermented in soybean bran showed constant values at all pHs studied, for optimal and stability, but low production. Phytase with both substrates showed stable activity for temperatures higher than 80oC. Protease from A. niger showed very distinct behavior of optimum pH, acid for wheat bran and basic for soybean bran, respectively and optimal values of temperature and stability at 50oC. Phytase produced by A. oryzae in wheat bran had optimum pH and temperature of 9 and 37oC, respectively, but it was very unstable. On the other hand, proteases were stable at high temperatures, all pH’s studied and showed very high yield when fermented in wheat bran, however when it was fermented in soybean bran the production was very low. Subsequently the upscale production of phytase from A. niger and proteases from A. oryzae were applied as an enzyme additive in fish fed for digestibility studies. Phytases and proteases were produced with stable enzyme activity of 7,000 U.g-1 and 2,500 U.g-1, respectively. When those enzymes were applied in a plant protein based fish diet for digestibility studies, they increased protein, mineral, energy and lipids availability, showing that these new enzymes can improve animal production and performance. In conclusion, the substrate, as well as, the microorganism species can affect the biochemical character of the enzyme produced. Moreover, the production of these enzymes by SSF can be up to 90% cheaper than commercial ones produced with the same fungi species but submerged fermentation. Add to that these cheap enzymes can be easily applied as animal diet additives to improve production and performance.

Keywords: agricultural by-products, animal nutrition, enzymes production, solid state fermentation

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Authors: Lívia Pinheiro Carvalho, Luciana Di Thommazo-Luporini, Rafael Luís Luporini, José Carlos Bonjorno Junior, Renata Pedrolongo Basso Vanelli, Manoel Carneiro de Oliveira Junior, Rodolfo de Paula Vieira, Renata Trimer, Renata G. Mendes, Mylène Aubertin-Leheudre, Audrey Borghi-Silva

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Rationale: Cardiorespiratory fitness (CRF) and functional capacity in young obese and normal-weight people are associated with metabolic and cardiovascular diseases and mortality. However, it remains unclear whether their metabolically healthy (MH) or at risk (AR) phenotype influences cardiorespiratory fitness in this vulnerable population such as obese adults but also in normal-weight people. HOMA insulin resistance index (HI) and leptin-adiponectin ratio (LA) are strong markers for characterizing those phenotypes that we hypothesized to be associated with physical fitness. We also hypothesized that an easy and feasible exercise test could identify a subpopulation at risk to develop metabolic and related disorders. Methods: Thirty-nine sedentary men and women (20-45y; 18.530 kg.m-2) underwent a clinical evaluation, including the six-minute step test (ST), a well-validated and reliable test for young people. Body composition assessment was done by a tetrapolar bioimpedance in a fasting state and in the folicular phase for women. A maximal cardiopulmonary exercise testing, as well as the ST, evaluated the oxygen uptake at the peak of the test (VO2peak) by an ergospirometer Oxycon Mobile. Lipids, glucose, insulin were analysed and the ELISA method quantified the serum leptin and adiponectin from blood samples. Volunteers were divided in two groups: AR or MH according to a HI cutoff of 1.95, which was previously determined in the literature. T-test for comparison between groups, Pearson´s test to correlate main variables and ROC analysis for discriminating AR from up-and-down cycles in ST (SC) were applied (p<0.05). Results: Higher LA, fat mass (FM) and lower HDL, SC, leg lean mass (LM) and VO2peak were found in AR than in MH. Significant correlations were found between VO2peak and SC (r= 0.80) as well as between LA and FM (r=0.87), VO2peak (r=-0.73), and SC (r=-0.65). Area under de curve showed moderate accuracy (0.75) of SC <173 to discriminate AR phenotype. Conclusion: Our study found that at risk obese and normal-weight subjects showed an unhealthy metabolism as well as a poor CRF and functional daily activity capacity. Additionally, a simple and less costly functional test associated with above-mentioned aspects is able to identify ‘at risk’ subjects for primary intervention with important clinical and health implications.

Keywords: aerobic capacity, exercise, fitness, metabolism, obesity, 6MST

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Authors: Patrícia Severino, Luciana Nalone, Daniele Martins, Marco Chaud, Classius Ferreira, Cristiane Bani, Ricardo Albuquerque

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Hydrogels produced with polymers have been used in the development of dressings for wound treatment and tissue revitalization. Our study on polymer nanocomposites containing silver nanoparticles shows antimicrobial activity and applications in wound healing. The effects are linked with the slow oxidation and Ag⁺ liberation to the biological environment. Furthermore, bacterial cell membrane penetration and metabolic disruption through cell cycle disarrangement also contribute to microbial cell death. The silver antimicrobial activity has been known for many years, and previous reports show that low silver concentrations are safe for human use. This work aims to develop a hydrogel using natural polymers (sodium alginate and gelatin) combined with silver nanoparticles for wound healing and with antimicrobial properties in cutaneous lesions. The hydrogel development utilized different sodium alginate and gelatin proportions (20:80, 50:50 and 80:20). The silver nanoparticles incorporation was evaluated at the concentrations of 1.0, 2.0 and 4.0 mM. The physico-chemical properties of the formulation were evaluated using ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermogravimetric (TG) analysis. The morphological characterization was made using transmission electron microscopy (TEM). Human fibroblast (L2929) viability assay was performed with a minimum inhibitory concentration (MIC) assessment as well as an in vivo cicatrizant test. The results suggested that sodium alginate and gelatin in the (80:20) proportion with 4 mM of AgNO₃ in the (UV-Vis) exhibited a better hydrogel formulation. The nanoparticle absorption spectra of this analysis showed a maximum band around 430 - 450 nm, which suggests a spheroidal form. The TG curve exhibited two weight loss events. DSC indicated one endothermic peak at 230-250 °C, due to sample fusion. The polymers acted as stabilizers of a nanoparticle, defining their size and shape. Human fibroblast viability assay L929 gave 105 % cell viability with a negative control, while gelatin presented 96% viability, alginate: gelatin (80:20) 96.66 %, and alginate 100.33 % viability. The sodium alginate:gelatin (80:20) exhibited significant antimicrobial activity, with minimal bacterial growth at a ratio of 1.06 mg.mL⁻¹ in Pseudomonas aeruginosa and 0.53 mg.mL⁻¹ in Staphylococcus aureus. The in vivo results showed a significant reduction in wound surface area. On the seventh day, the hydrogel-nanoparticle formulation reduced the total area of injury by 81.14 %, while control reached a 45.66 % reduction. The results suggest that silver-hydrogel nanoformulation exhibits potential for wound dressing therapeutics.

Keywords: nanocomposite, wound healing, hydrogel, silver nanoparticle

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Authors: Luciana S. Almeida

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Nanotechnology has revolutionized the world of science and technology bringing great expectations due to its great potential of application in the most varied industrial sectors. The same characteristics that make nanoparticles interesting from the point of view of the technological application, these may be undesirable when released into the environment. The small size of nanoparticles facilitates their diffusion and transport in the atmosphere, water, and soil and facilitates the entry and accumulation of nanoparticles in living cells. The main objective of this study is to evaluate the environmental regulatory process of nanomaterials in the Brazilian scenario. Three specific objectives were outlined. The first is to carry out a global scientometric study, in a research platform, with the purpose of identifying the main lines of study of nanomaterials in the environmental area. The second is to verify how environmental agencies in other countries have been working on this issue by means of a bibliographic review. And the third is to carry out an assessment of the Brazilian Nanotechnology Draft Law 6741/2013 with the state environmental agencies. This last one has the aim of identifying the knowledge of the subject by the environmental agencies and necessary resources available in the country for the implementation of the Policy. A questionnaire will be used as a tool for this evaluation to identify the operational elements and build indicators through the Environment of Evaluation Application, a computational application developed for the development of questionnaires. At the end will be verified the need to propose changes in the Draft Law of the National Nanotechnology Policy. Initial studies, in relation to the first specific objective, have already identified that Brazil stands out in the production of scientific publications in the area of nanotechnology, although the minority is in studies focused on environmental impact studies. Regarding the general panorama of other countries, some findings have also been raised. The United States has included the nanoform of the substances in an existing program in the EPA (Environmental Protection Agency), the TSCA (Toxic Substances Control Act). The European Union issued a draft of a document amending Regulation 1907/2006 of the European Parliament and Council to cover the nanoform of substances. Both programs are based on the study and identification of environmental risks associated with nanomaterials taking into consideration the product life cycle. In relation to Brazil, regarding the third specific objective, it is notable that the country does not have any regulations applicable to nanostructures, although there is a Draft Law in progress. In this document, it is possible to identify some requirements related to the environment, such as environmental inspection and licensing; industrial waste management; notification of accidents and application of sanctions. However, it is not known if these requirements are sufficient for the prevention of environmental impacts and if national environmental agencies will know how to apply them correctly. This study intends to serve as a basis for future actions regarding environmental management applied to the use of nanotechnology in Brazil.

Keywords: environment; management; nanotecnology; politics

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Authors: Carlos A. C. G. Neto, Natan C. G. e Silva , Thaís de O. Costa, Luciana R. B. Gonçalves, Maria V. P. Rocha

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β-galactosidases (E.C. 3.2.1.23) are enzymes that have attracted by catalyzing the hydrolysis of lactose and in producing galacto-oligosaccharides by favoring transgalactosylation reactions. These enzymes, when immobilized, can have some enzymatic characteristics substantially improved, and the coating of supports with multifunctional polymers is a promising alternative to enhance the stability of the biocatalysts, among which polyethylenimine (PEI) stands out. PEI has certain properties, such as being a flexible polymer that suits the structure of the enzyme, giving greater stability, especially for multimeric enzymes such as β-galactosidases. Besides that, protects them from environmental variations. The use of chitosan support coated with PEI could improve the catalytic efficiency of β-galactosidase from Kluyveromyces lactis in the transgalactosylation reaction for the production of prebiotics, such as lactulose since this strain is more effective in the hydrolysis reaction. In this context, the aim of the present work was first to develop biocatalysts of β-galactosidase from K. lactis immobilized on chitosan-coated with PEI, determining the immobilization parameters, its operational and thermal stability, and then to apply it in hydrolysis and transgalactolisation reactions to produce lactulose using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% (v/v) glutaraldehyde and then coated with 10% (w/v) PEI solution was evaluated using an enzymatic load of 10 mg protein per gram support. Subsequently, the hydrolysis and transgalactosylation reactions were conducted at 50 °C, 120 RPM for 20 minutes, using whey supplemented with fructose at a ratio of 1:2 lactose/fructose, totaling 200 g/L. Operational stability studies were performed in the same conditions for 10 cycles. Thermal stabilities of biocatalysts were conducted at 50 ºC in 50 mM phosphate buffer, pH 6.6 with 0.1 mM MnCl2. The biocatalyst whose support was coated was named CHI_GLU_PEI_GAL, and the one that was not coated was named CHI_GLU_GAL. The coating of the support with PEI considerably improved the parameters of immobilization. The immobilization yield increased from 56.53% to 97.45%, biocatalyst activity from 38.93 U/g to 95.26 U/g and the efficiency from 3.51% to 6.0% for uncoated and coated support, respectively. The biocatalyst CHI_GLU_PEI_GAL was better than CHI_GLU_GAL in the hydrolysis of lactose and production of lactulose, converting 97.05% of lactose at 5 min of reaction and producing 7.60 g/L lactulose in the same time interval. QUI_GLU_PEI_GAL biocatalyst was stable in the hydrolysis reactions of lactose during the 10 cycles evaluated, converting 73.45% lactose even after the tenth cycle, and in the lactulose production was stable until the fifth cycle evaluated, producing 10.95 g/L lactulose. However, the thermal stability of CHI_GLU_GAL biocatalyst was superior, with a half-life time 6 times higher, probably because the enzyme was immobilized by covalent bonding, which is stronger than adsorption (CHI_GLU_PEI_GAL). Therefore, the strategy of coating the supports with PEI has proven to be effective for the immobilization of β-galactosidase from K. lactis, considerably improving the immobilization parameters, as well as, the catalytic action of the enzyme. Besides that, this process can be economically viable due to the use of an industrial residue as a substrate.

Keywords: β-galactosidase, immobilization, kluyveromyces lactis, lactulose, polyethylenimine, transgalactosylation reaction, whey

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Authors: Carlos A. C. G. Neto, Natan C. G. Silva, Thais O. Costa, Luciana R. B. Goncalves, Maria v. P. Rocha

Abstract:

Galactosidases are enzymes responsible for catalyzing lactose hydrolysis reactions and also favoring transgalactosylation reactions for the production of prebiotics, among which lactulose stands out. These enzymes, when immobilized, can have some enzymatic characteristics substantially improved, and the coating of supports with multifunctional polymers in immobilization processes is a promising alternative in order to extend the useful life of the biocatalysts, for example, the coating with polyethyleneimine (PEI). PEI is a flexible polymer that suits the structure of the enzyme, giving greater stability, especially for multimeric enzymes such as β-galactosidases and also protects it from environmental variations, for example, pH and temperature. In addition, it can substantially improve the immobilization parameters and also the efficiency of enzymatic reactions. In this context, the aim of the present work was first to develop biocatalysts of β-galactosidase from Kluyveromyces lactis immobilized on PEI coated agarose, determining the immobilization parameters, its operational and thermal stability, and then to apply it in the hydrolysis of lactose and synthesis of lactulose, using whey as a substrate. This immobilization strategy was chosen in order to improve the catalytic efficiency of the enzyme in the transgalactosylation reaction for the production of prebiotics, and there are few studies with β-galactosidase from this strain. The immobilization of β-galactosidase in agarose previously functionalized with 48% (w/v) glycidol and then coated with 10% (w/v) PEI solution was evaluated using an enzymatic load of 10 mg/g of protein. Subsequently, the hydrolysis and transgalactosylation reactions were conducted at 50 °C, 120 RPM for 20 minutes, using whey (66.7 g/L of lactose) supplemented with 133.3 g/L fructose at a ratio of 1:2 (lactose/fructose). Operational stability studies were performed in the same conditions for 10 cycles. Thermal stabilities of biocatalysts were conducted at 50 ºC in 50 mM phosphate buffer, pH 6.6, with 0.1 mM MnCl2. The biocatalysts whose supports were coated were named AGA_GLY_PEI_GAL, and those that were not coated were named AGA_GLY_GAL. The coating of the support with PEI considerably improved immobilization yield (2.6-fold), the biocatalyst activity (1.4-fold), and efficiency (2.2-fold). The biocatalyst AGA_GLY_PEI_GAL was better than AGA_GLY_GAL in hydrolysis and transgalactosylation reactions, converting 88.92% of lactose at 5 min of reaction and obtaining a residual concentration of 5.24 g/L. Besides that, it was produced 13.90 g/L lactulose in the same time interval. AGA_GLY_PEI_GAL biocatalyst was stable during the 10 cycles evaluated, converting approximately 80% of lactose and producing 10.95 g/L of lactulose even after the tenth cycle. However, the thermal stability of AGA_GLY_GAL biocatalyst was superior, with a half-life time 5 times higher, probably because the enzyme was immobilized by covalent bonding, which is stronger than adsorption (AGA_GLY_PEI_GAL). Therefore, the strategy of coating the supports with PEI has proven to be effective for the immobilization of β-galactosidase from K. lactis, considerably improving the immobilization parameters, as well as the enzyme, catalyzed reactions. In addition, the use of whey as a raw material for lactulose production has proved to be an industrially advantageous alternative.

Keywords: β-galactosidase, immobilization, lactulose, polyethylenimine, whey

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Authors: Yang Yang, Luciana Magalhães Rebelo Alencar, Martha Sahylí Ortega Pijeira, Beatriz da Silva Batista, Alefe Roger Silva França, Erick Rafael Dias Rates, Ruana Cardoso Lima, Sara Gemini-Piperni, Ralph Santos-Oliveira

Abstract:

Radium-²²³ dichloride ([²²³Rₐ]RₐCl₂) is an alpha particle-emitting radiopharmaceutical currently approved for the treatment of patients with castration-resistant prostate cancer, symptomatic bone metastases, and no known visceral metastatic disease. [²²³Rₐ]RₐCl₂ is bone-seeking calcium mimetic that bonds into the newly formed bone stroma, especially osteoblastic or sclerotic metastases, killing the tumor cells by inducing DNA breaks in a potent and localized manner. Nonetheless, the successful therapy of osteosarcoma as primary bone tumors is still a challenge. Nanomicelles are colloidal nanosystems widely used in drug development to improve blood circulation time, bioavailability, and specificity of therapeutic agents, among other applications. In addition, the enhanced permeability and retention effect of the nanosystems, and the renal excretion of the nanomicelles reported in most cases so far, are very attractive to achieve selective and increased accumulation in tumor site as well as to increase the safety of [²²³Rₐ]RₐCl₂ in the clinical routine. In the present work, [²²³Rₐ]RₐCl₂ nanomicelles were produced, characterized, in vitro evaluated, and compared with pure [²²³Rₐ]RₐCl2 solution using SAOS2 osteosarcoma cells. The [²²³Rₐ]RₐCl₂ nanomicelles were prepared using the amphiphilic copolymer Pluronic F127. The dynamic light scattering analysis of freshly produced [²²³Rₐ]RₐCl₂ nanomicelles demonstrated a mean size of 129.4 nm with a polydispersity index (PDI) of 0.303. After one week stored in the refrigerator, the mean size of the [²²³Rₐ]RₐCl₂ nanomicelles increased to 169.4 with a PDI of 0.381. Atomic force microscopy analysis of [223Rₐ]RₐCl₂ nanomicelles exhibited spherical structures whose heights reach 1 µm, suggesting the filling of 127-Pluronic nanomicelles with [²²³Rₐ]RₐCl₂. The viability assay with [²²³Rₐ]RₐCl₂ nanomicelles displayed a dose-dependent response as it was observed using pure [²²³Rₐ]RₐCl2. However, at the same dose, [²²³Rₐ]RₐCl₂ nanomicelles were 20% higher efficient in killing SAOS2 cells when compared with pure [²²³Rₐ]RₐCl₂. These findings demonstrated the effectiveness of the nanosystem validating the application of nanotechnology in targeted alpha therapy with [²²³Ra]RₐCl₂. In addition, the [²²³Rₐ]RaCl₂nanomicelles may be decorated and incorporated with a great variety of agents and compounds (e.g., monoclonal antibodies, aptamers, peptides) to overcome the limited use of [²²³Ra]RₐCl₂.

Keywords: nanomicelles, osteosarcoma, radium dichloride, targeted alpha therapy

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