Thermostable DNA repair enzymes for novel biotechnological applications

SNAP-tag® is a powerful tool with wide applications; however, this technology has some limitations: this technology is suitable only for mesophilic model organisms and mild reaction conditions, and the synthesis of ad hoc substrates needs tedious and complex purification procedures. This thesis is directed to get over these issues and to expand the biotechnological application potential of the SNAP-tag®. This aim was pursued by two strategies: acting on the enzyme, by the exploitation of homologous activities from extremophilic sources; and acting on the substrate, by the set-up of a novel approach. Acting on the enzyme: In part of my thesis (Chapter I and Chapter III), I addressed my work to the identification and characterization of a novel AGT from the hyperthermophilic organism P. furiosus, in order to expand the biotechnological potential of the SNAP-technology from "mesophilic" applications to in vivo and in vitro studies in extreme model organisms or in those processes that require non-moderate reaction conditions, and to the characterization of a novel immobilization and labelling tool. Acting on the substrate: In another part of my thesis (Chapter II), I focused my activity to the set-up of a novel approach to the SNAP-tag® reaction, by a chemo-enzymatic approach, which splits the reaction in two fast and extremely specific steps. The enzymatic reaction is performed by use of a selected azide-based BG-substrate, which allows the chemical step with a DBCO-based molecule via cycloaddition (or click-chemistry) reaction. I demonstrate that all the problems related to purification procedures can be bypassed by the utilization of a universal azide-derivative substrate, which is sufficiently exposed to the solvent to link any DBCO-molecules without affecting the reaction rate of the SNAP-tag®

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications

CONSUMER BEHAVIOR AND FACTORS THAT AFFECT SATISFACTION AND RISK PERCEPTION OF PURCHASES OF OWN BRAND FOOD PRODUCTS

In view of increased competition, combined with political, economic, social and technological uncertainties, the Brazilian retail sector has been going through a transformation process led, on the one hand, by market pressure itself, and on the other hand, by the permanent struggle for survival. From this point of view, foreign and Brazilian retailers saw the own brand segment as a viable marketing alternative for maximizing their turnover and meeting consumer needs and wishes. In that sense, this research aimed to identify factors that affect risk perception of purchases of own brand food products. Using an exploratory and quantitative approach, structured electronic questionnaires were applied using Google Docs. SPSS software was used for data analysis with a convenience sample of 250 individuals. This allowed the application of multivariate analysis techniques. Factors that were generated from applied analyses allowed us to conclude that risk elements are perceived differently, according to each analyzed category.In view of increased competition, combined with political, economic, social and technological uncertainties, the Brazilian retail sector has been going through a transformation process led, on the one hand, by market pressure itself, and on the other hand, by the permanent struggle for survival. From this point of view, foreign and Brazilian retailers saw the own brand segment as a viable marketing alternative for maximizing their turnover and meeting consumer needs and wishes. In that sense, this research aimed to identify factors that affect risk perception of purchases of own brand food products. Using an exploratory and quantitative approach, structured electronic questionnaires were applied using Google Docs. SPSS software was used for data analysis with a convenience sample of 250 individuals. This allowed the application of multivariate analysis techniques. Factors that were generated from applied analyses allowed us to conclude that risk elements are perceived differently, according to each analyzed category

The use of isoxazoline and isoxazole scaffolding in the design of novel thiourea and amide liquid-crystalline compounds

The authors wish to thank the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001 for the fellowships and Conselho Nacional de Pesquisa Cientifica (CNPq, edital Universal) for the financial support grant n. 403075 2016-5.A series of novel thiourea and amide liquid crystals containing 5-membered isoxazoline and isoxazole rings were synthetized and the liquid crystal properties studied. Thioureas were obtained using a condensation reaction of benzoyl chlorides, arylamines and ammonium thiocyanate. The amides, on the other hand, were the byproduct of a quantitative reaction which used potassium cyanate as the starting material. Thiourea and amide derivatives were predominantly SmA mesophase inductors. A nematic mesophase was observed only for thioureas and amides containing an isoxazole ring. Additionaly, the liquid crystal behavior was also dependent on the relative position of nitrogen and oxygen atoms on the 5-membered heterocycle.publishersversionpublishe

A segmentation proposal to the consumers’ brazilian beer sector / Proposta de segmentação do mercado brasileiro de cerveja

The objective of this research was to identify the different segments of beer consumers based on the behavior presented during the purchase decision process. A field survey was carried out with a convenience sample of 422 respondents, using questionnaires, available in the authors' relationship networks. In the data analysis, the statistical technique cluster analysis was applied, which allowed the identification of four consumer segments: high involvement consumers, medium consumption consumers, occasion buyers, and low consumption consumers. Results obtained from the investigation show that consumers with high levels of involvement (Group 1) seek a lot of information before consumption. For consumers of average consumption (Group 2), the search for knowledge and references on beers is limited to previous experiences they had and to friends, and they choose beers mainly by the taste and by the country of origin. Occasional consumers (Group 3) usually buy the same brands of beer, but use flavor and brand as a selection criterion. Finally, low-consumption consumers (Group 4) had a consumption of beer for special occasions. They often do not seek information about the product, and they do not do any kind of word-of-mouth marketing. The Main theoretical contribution is the new segmentation proposal method originated from the research. The relevance of this study is related to the new segmentation framework proposed segments that could be used to future studies in other competitive markets

Folding-upon-Repair DNA Nanoswitches for Monitoring the Activity of DNA Repair Enzymes

We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O6 -methyl-guanine (O6 -MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O6 -MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors

First thermostable CLIP-tag by rational design applied to an archaeal O6-alkyl-guanine-DNA-alkyl-transferase

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8. The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag varian

A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms

A SOCIALIZAÇÃO FEMININA E A IDEALIZAÇÃO DOS RELACIONAMENTOS: GÊNESE DE FRUSTRAÇÕES, CONFLITOS E VIOLÊNCIAS

Violence against women arises from patriarchal culture and involves society and family and intimate relationships. Based on bibliographical research, the article intends to discuss the historical construction of female subordination and the permanence of a culture that, even though it is in the 21st century and, counting on the advances achieved by feminist movements from the 19th century onwards, women are induced to silence and conformity with their secondary roles, in all social spheres of greater recognition. It is verified, through a Brazilian historiographical review and in “invisible” practices, that male domination occurs as naturalized and based on the sexual division of work and care.As violências contra mulheres decorrem da cultura patriarcal e envolvem a sociedade e as relações familiares e de intimidade. Com base em pesquisas bibliográficas, o artigo pretende discutir a construção histórica da subordinação feminina e a permanência de uma cultura que, mesmo estando no século XXI e, contando com os avanços conquistados pelos movimentos feministas a partir do século XIX, as mulheres são induzidas ao silenciamento e ao conformismo com seus papéis secundários, em todas as esferas sociais de maior reconhecimento. Verifica-se, através de revisão historiográfica brasileira e nas práticas “invisíveis” que a dominação masculina ocorre como naturalizada e pautada na divisão sexual de trabalhos e cuidados

Efectos económicos de la pandemia por COVID-19 sobre el sector restaurantero en la ciudad de Xalapa, Veracruz

The COVID-19 pandemic has generated various economic effects on the restaurant sector in Mexico, due to confinement and sanitary protocols to prevent the spread of the disease. Among the economic problems were structural changes, increased costs due to changes in spaces and cleaning standards, a drop in demand for food preparation services, and consumer distrust. This work has a cross-sectional and descriptive approach, with a simple random sample made up of 391 consumers and 16 restaurants. The characteristics, preferences and behavior of the restaurant sector in the municipality of Xalapa, Ver., were analyzed, comparing their perception of this activity before and during the pandemic.La pandemia por COVID-19 ha generado diversos efectos económicos sobre el sector restaurantero en México, debido al confinamiento y a los protocolos sanitarios para prevenir el contagio de la enfermedad. Entre los problemas económicos se presentaron cambios estructurales, incremento de costos por modificación de espacios y estándares de limpieza, caída en la demanda del servicio de preparación de alimentos y desconfianza del consumidor. Este trabajo tiene un enfoque transversal y descriptivo, con una muestra aleatoria simple compuesta por 391 consumidores y 16 restaurantes. Se analizaron las características, preferencias y comportamiento del sector restaurantero del municipio de Xalapa, Ver., comparando su percepción sobre esta actividad antes y durante la pandemia