Insights on the DNA stability in aqueous solutions of ionic liquids

Deoxyribonucleic acid (DNA) carries the genetic information essential for the growth and functioning of living organisms, playing a significant role in life sciences research. However, the long-term storage and preservation of DNA, while ensuring its bioactivity, are still current challenges to overcome. In this work, aqueous solutions of ionic liquids (ILs) were investigated as potential preservation media for double stranded (dsDNA). A screening of several ILs, by combining the cholinium, tetrabutylammonium, tetrabutylphosphonium, and 1-ethyl-3-methylimidazolium, cations with the anions bromide, chloride, dihydrogen phosphate, acetate, and glycolate, was carried out in order to gather fundamental knowledge on the molecular features of ILs that improve the dsDNA stability. Different IL concentrations and the pH effect were also addressed. Circular dichroism (CD) spectroscopy was used to evaluate the conformational structure and stability of dsDNA. IL-DNA interactions were appraised by UV-Vis absorption spectrophotometry and 31P nuclear magnetic resonance (NMR) spectroscopy. The results obtained demonstrate that pH has a significant effect towards the dsDNA stability. Amongst the ILs investigated, cholinium-based ILs are the most promising class of ILs to preserve the dsDNA structure, in which electrostatic interactions between the cholinium cation and the DNA phosphate groups play a significant role as demonstrated by the 31P NMR data, being more relevant at higher IL concentrations. On the other hand, the denaturation of dsDNA mainly occurs with ILs composed of more hydrophobic cations and able to establish dispersive interactions with the nucleobases environment. Furthermore, the IL anion has a weaker impact when compared to the IL cation effect to interact with DNA molecules. The experimental data of this work provide relevant fundamental knowledge for the application of ILs in the preservation of nucleic acids, being of high relevance in the biotechnology field.publishe

A new strategy for RNA isolation from eukaryotic cells using arginine affinity chromatography

The relevance of RNA in many biological functions has been recognized, broadening the scope of RNA research activities, from basic to applied sciences, also aiming the translation to clinical fields. The preparation and purification of RNA is a critical step for further application, since the quality of the template is crucial to ensure reproducibility and biological relevance. Therefore, the establishment of new tools that allows the isolation of pure RNA with high quality is of particular importance. New chromatographic strategies for RNA purification were considered, exploiting affinity interactions between amino acids and nucleic acids. In the present study, a single arginine-affinity chromatography step was employed for the purification of RNA from a total eukaryotic nucleic acid extract, thus eliminating several steps compared with current RNA isolation procedures. The application of this process resulted in a high RNA recovery yield of 96 ± 17% and the quality control analysis revealed a high integrity (28S:18S ratio = 1.96) in RNA preparations as well as a good purity, demonstrated by the scarce detection of proteins and the reduction on genomic DNA contamination to residual concentrations. Furthermore, the performance of the new RNA isolation method was tested regarding the applicability of the isolated RNA in modern molecular biology techniques. Hence, this new affinity approach will simplify the isolation and purification of RNA, which can bring great improvements in biomedical investigation.info:eu-repo/semantics/publishedVersio

Desenvolvimento de formulações e de um processo de pintura à pistola para obtenção de efeito cromado

Mestrado em Engenharia QuímicaO processo de metalização em vácuo permite a obtenção de revestimentos semelhantes ao efeito cromado, sendo utilizado essencialmente na indústria automóvel e de decoração. Neste processo, um substrato é revestido com uma camada fina de um metal através de vaporização. O processo apresenta elevadas taxas de revestimento e é ambientalmente inofensivo, no entanto, o revestimento de peças com área elevada é limitado às dimensões da câmara de vácuo e, o de peças irregulares resulta em revestimentos deficientes e irregulares. Na presente dissertação, como alternativa à metalização em vácuo, desenvolveu-se um processo de pintura, para obtenção de um efeito semelhante, por aplicação à pistola. Neste sentido, foi concebido um sistema de pintura constituído por um fundo de poliéster, um revestimento acrílico base de alto brilho, um revestimento de alumínio com efeito cromado e por um revestimento de poliuretano de topo de dois componentes de alto brilho e transparente. Este sistema de pintura foi aplicado em amostras de MDF (painel de aglomerado de fibras de densidade média) e ABS (acrilonitrilo butadieno estireno), excluindo-se a aplicação do fundo poliéster neste último. O MDF sofreu previamente um processo de lixagem e aplicação de um fundo branco ou de poliéster transparente. Numa primeira fase, foram realizados estudos sobre a influência da cor do fundo (no caso do MDF) e do revestimento acrílico base no efeito desejado. Posteriormente, desenvolveram-se formulações com diferentes pigmentos de alumínio por forma a selecionar o que permitisse a maximização do efeito cromado e, por último, foi formulado um verniz de alto brilho de topo transparente de dois componentes para proteção do revestimento de alumínio, que não sofresse amarelecimento nem afetasse em demasia o efeito cromado, e que garantisse dureza e resistência ao risco. Por fim, algumas amostras de MDF revestidas com este esquema de pintura foram estudadas por espectroscopia de ultravioleta-visível, obtendo-se resultados a nível da refletância total e da refletância difusa. A realização deste trabalho permitiu desenvolver um sistema de pintura e um revestimento de alumínio que maximizasse o efeito cromado. O sistema de pintura final é constituído por um fundo de poliéster branco ou transparente, um revestimento acrílico negro de alto brilho, um revestimento de alumínio com o pigmento metalizado em vácuo LPG046 e um revestimento de poliuretano de topo de dois componentes de alto brilho e transparente, com valor de brilho de 157 GU, dureza suficiente e baixa resistência ao risco. Através do estudo de espectroscopia verificou-se que o revestimento de topo aumenta a refletância difusa em cerca de 18%, o que se traduz numa diminuição do efeito cromado.Vacuum metallizing process allows obtaining coatings similar to chrome effect, being mainly used in the automotive and decoration industries. In this process, a substrate is coated with a thin layer of a metal through vaporization. This process has high coating rates and is environmentally harmless, however, the coating of pieces with high area is limited to the dimensions of the vacuum chamber and, the coating of irregular pieces leads to an uneven and irregular coating. In this work, as an alternative to vacuum metalizing, it was developed a painting system to achieve a similar effect, through spray gun application. As such, it was developed a painting system that consists in a polyester background coat, a high gloss acrylic base coat, an aluminum chrome effect coating and two component high gloss and clear polyurethane top coat. The referred paint system was applied to MDF (medium density fiberboard) and ABS (acrylonitrile butadiene styrene) samples, with exclusion of the application of the polyester background coat is the latter case. MDF was previously submitted to a sanding process and coated with a white or clear polyester coating. In a first stage, the influence of the background coat (for MDF) and acrylic base coat color, on the desired effect, were studied. Afterwards, formulations with different aluminum pigments were tested in order to select the one that allowed the maximization of the chrome effect and, finally, it was formulated a high gloss two component clear top coat for protection that would not suffer yellowing nor affect, in a great extent, the chrome effect and ensure hardness and scratch resistance. Finally, some MDF samples coated with this paint system were studied by ultraviolet-visible spectroscopy, aiming to obtain results of total reflectance and diffuse reflectance. This work allowed us to develop a paint system and an aluminum coating that would maximize chrome effect. The final paint system is constituted by a white or clear polyester background coat, a high gloss acrylic black coating, an aluminum coating with the LPG046 vacuum metallized pigment and a high gloss two component clear top coat, with a gloss value of 157 GU, sufficient hardness and low scratch resistance. Based on the spectroscopy study it was found that the top coat increases the diffuse reflectance by about 18%, which leads to a reduction of the chrome effect

New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli

RNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.info:eu-repo/semantics/publishedVersio

Supported ionic liquids used as chromatographic matrices in bioseparation: an overview

Liquid chromatography plays a central role in biomanufacturing, and, apart from its use as a preparative purification strategy, either in biopharmaceuticals or in fine chemicals industries, it is also very useful as an analytical tool for monitoring, assessing, and characterizing diverse samples. The present review gives an overview of the progress of the chromatographic supports that have been used in the purification of high-value products (e.g., small molecules, organic compounds, proteins, and nucleic acids). Despite the diversity of currently available chromatographic matrices, the interest in innovative biomolecules emphasizes the need for novel, robust, and more efficient supports and ligands with improved selectivity. Accordingly, ionic liquids (ILs) have been investigated as novel ligands in chromatographic matrices. Given herein is an extensive review regarding the different immobilization strategies of ILs in several types of supports, namely in silica, Sepharose, and polymers. In addition to depicting their synthesis, the main application examples of these supports are also presented. The multiple interactions promoted by ILs are critically discussed concerning the improved selectivity towards target molecules. Overall, the versatility of supported ILs is here considered a critical point to their exploitation as alternatives to the more conventional liquid chromatographic matrices used in bioseparation processes.publishe

Percepções dos alunos acerca da diversidade étnico-racial no currículo do ensino técnico integrado ao médio

Esse artigo apresenta os resultados de três rodas de conversa realizadas com alunos do ensino técnico integrado ao médio do Instituto Federal de Rondônia (IFRO), Campus Porto Velho Calama, nas quais foram discutidas as percepções dos alunos acerca das temáticas referentes às relações étnico-raciais. A metodologia proposta é de natureza qualitativa e caráter exploratório e participante, a partir de uma percepção decolonial e afrocentrada dos processos de produção do conhecimento. Como resultados de nossas análises constatamos que os alunos consideram importante o trabalho em torno das referidas temáticas, reconhecem o processo de apagamento que ainda se mantém acerca das mesmas e propõem possibilidades pedagógicas integradas voltadas para a interdisciplinaridade

Pequenos leitores e grandes desafios no ensino fundamental i: contribuições teóricas para uma prática ressignificada

O presente artigo discute questões relacionadas à recepção do texto literário por parte de leitores no Ensino Fundamental I, bem como os desafios presentes nesta etapa. Tais desafios muitas vezes requerem da escola uma alteração dos percursos traçados e das estratégias utilizadas visando a ressignificação das suas práticas. Considerando que algumas abordagens do texto literário em sala de aula muitas vezes prejudicam a interação, o diálogo da criança com o texto, consequentemente prejudicando o exercício da construção de sentidos para a leitura, este artigo discute ainda questões que contribuam com a elaboração de um projeto de leitura viável que favoreça o ressignificar do trabalho com o texto literário e a recepção deste texto com leitores em formação. Sendo o texto literário espaço de interlocução, de diálogo, de troca de ideias faz-se necessária a superação de práticas que engessem o texto literário com propostas que fecham as múltiplas interpretações sugeridas por ele.Abstract This article discusses issues related to the reception of the literary text by readers in Elementary School, as well as the challenges present at this stage. Such challenges often require the school to change the paths traced and the strategies used in order to give new meaning to its practices. Considering that some approaches to the literary text in the classroom often impair the interaction, the child's dialogue with the text, consequently impairing the exercise of constructing meanings for reading, this article also discusses issues that contribute to the development of a viable project for reading that favors the redefinition of the work with the literary text and the reception of this text with readers in training. As the literary text is a space for interlocution, dialogue, and exchange of ideas, it is necessary to overcome practices that engender the literary text with proposals that close the multiple interpretations suggested by it.Este artículo aborda cuestiones relacionadas con la recepción del texto literario por parte de los lectores de la Enseñanza Básica, así como los desafíos presentes en esta etapa. Tales desafíos requieren muchas veces que la escuela cambie los caminos trazados y las estrategias utilizadas para redefinir sus prácticas. Considerando que algunas aproximaciones al texto literario en el aula a menudo perjudican la interacción, el diálogo del niño con el texto, afectando consecuentemente el ejercicio de la construcción de sentidos para la lectura, este artículo también discute cuestiones que contribuyen a la elaboración de un proyecto de viabilidad de la lectura que favorezca la resignificación de la obra con el texto literario y la recepción de este texto con lectores en formación. Siendo el texto literario un espacio de interlocución, diálogo e intercambio de ideas, es necesario superar las prácticas que asfixian al texto literario con propuestas que cierren las múltiples interpretaciones que éste sugiere.Résumé Cet article traite des enjeux liés à la réception du texte littéraire par les lecteurs de l'école élémentaire, ainsi que des défis présents à cette étape. De tels défis obligent souvent l'école à modifier les chemins tracés et les stratégies utilisées afin de redéfinir ses pratiques. Considérant que certaines approches du texte littéraire en classe nuisent souvent à l'interaction, au dialogue de l'enfant avec le texte, nuisant par conséquent à l'exercice de la construction de sens pour la lecture, cet article aborde également les questions qui contribuent à l'élaboration d'un projet de une lecture qui favorise la resignification de l'œuvre avec le texte littéraire et la réception de ce texte auprès des lecteurs en formation. Puisque le texte littéraire est un espace d'interlocution, de dialogue et d'échange d'idées, il est nécessaire de dépasser les pratiques qui étouffent le texte littéraire par des propositions qui ferment les multiples interprétations suggérées par celui-ci.info:eu-repo/semantics/publishedVersio

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.info:eu-repo/semantics/publishedVersio

Multimodal ionic liquid-based chromatographic supports for an effective RNA purification

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the biomanufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA purif ication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C 3 C 3NH2 Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromatographic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.publishe

Integrated platform resorting to ionic liquids comprising the extraction, purification and preservation of DNA

The large-scale production of therapeutically targeted-deoxyribonucleic acid (DNA) has passed through several challenges, postponing the tangible implementation of an effective, economic and sustainable manufacturing system. Such challenges comprise the need to develop an integrative downstream process able to extract, purify and long-term preserve DNA, whilst reducing the risk of degradation by endonucleases that would compromise their effectiveness as therapeutic products. In this work, three-phase partitioning (TPP) systems formed by the application of aqueous biphasic systems (ABS) composed of several biocompatible cholinium-based ionic liquids (ILs), are proposed for the separation of double stranded DNA (dsDNA) from the endonuclease deoxyribonuclease I (DNase I). By taking advantage of the tailor-made properties of ILs, dsDNA can be completely extracted to the IL-rich phase, whereas DNase I is precipitated at the ABS interphase. The ABS/TPP formed by IL cholinium glycolate ([N 111(2OH) ][Gly]) fulfills the aim of this work, i.e. at ensuring the technical viability of IL-based ABS/ TPP for the “one-pot” extraction, purification and long-term preservation of dsDNA. The results reveal the potential of this system to be applied in the bioprocessing of DNA, particularly relevant when envisioning DNA- based therapeutic products.publishe