Expressão gênica em grãos de café submetidos a diferentes tratamentos pós-colheita-uma análise preliminar.

O conhecimento sobre como os tipos de processamento pós-colheita interferem na qualidade final de bebida é essencial para que cafés com qualidades especiais possam ser desenvolvidos. Dentre as características que podem ser diretamente influenciadas pelo processamento, a composição química do grão é a de maior interesse. Fatores como a regulação da expressão de genes e a atividade de enzimas específicas são determinantes para esta composição. Neste contexto, o objetivo deste estudo preliminar foi avaliar como os processamentos por via seca e úmida afetam o padrão de expressão dos genes isocitrato liase (ICL), ?-mananase (MAN) e ?-galactosidase (GAL). Frutos cereja foram submetidos a condições de secagem natural, despolpamento e desmucilamento. A expressão dos genes foi quantificada pela metodologia de RT-PCR em tempo real. A análise comparativa da expressão dos 3 genes indicou que no processamento via seca a expressão de ICL e MAN nas sementes foi maior do que em sementes de frutos processados por via úmida. Para o gene GAL, não foram observadas diferenças expressivas no perfil de expressão em sementes dos frutos submetidos aos diferentes tratamentos. Esses resultados diferiram dos obtidos em outros estudos já descritos na literatura, em que, apesar de ter sido avaliada a expressão de genes, foram utilizadas etapas de processamento distintas das utilizadas neste trabalho. Esses resultados revelaram, portanto, que há necessidade de se padronizarem os processamentos experimentais em pesquisas visando avaliar efeitos dos tratamentos pós-colheita sobre a qualidade do café

Expressão de genes relacionados ao metabolismo de nitrogênio, fósforo de potássio em cafeeiros submetidos ao estresse biótico.

As plantas freqüentemente são ameaçadas por agentes externos que podem ser fatores bióticos e/ou abióticos. Para cada tipo de ameaça há um requintado tipo de resposta que caracteriza o processo de defesa. Dentre os processos de defesa enquadram-se a ativação ou repressão de genes relacionados como quitinases, glucanaes, lipoxigenases, transferases, entre outros. Quaisquer modificações bioquímicas que ocorram durante os ataques dos patógenos são indispensáveis para a compreensão deste mecanismo biológico. Correlacionar os mecanismos biológicos de regulação e transporte de íons com os diferentes períodos de infestação e infecção é uma dessas maneiras de elucidar os processos que ocorrem durante a infestação e infecção. Neste estudo foram analisados os perfis de expressão de 5 genes (HAK-5, KEA, PAP-1, PII, NTR) relacionados com o transporte e regulação de potássio e nitrogênio com diferentes estímulos bióticos e em diferentes etapas de cada um desses processos. Os resultados demonstraram que há padrões diferenciais de regulação entre as plantas suscetíveis e resistentes para todos os genes analisados. Estas análises preliminares indicam que a regulação da absorção e/ou transporte de nutrientes exerce um importante papel durante as respostas de defesa em cafeeiros contra o bicho mineiro

Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity. (C) 2013 Elsevier B.V. All rights reserved.9196774Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages.

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential Expression of genes associated to fruit development and maturation processes. GEne expression was characterized by both semi?quantitative and quantitative RT?PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene ??galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish?green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.Made available in DSpace on 2016-04-29T07:02:31Z (GMT). No. of bitstreams: 1 Geneexpressionprofileduringcoffee.pdf: 663560 bytes, checksum: 2d013d6fb18beee636668143cb50727c (MD5) Previous issue date: 2016-04-28201

Theoretical insight into the mechanism for the inhibition of the cysteine protease cathepsin B by 1,2,4-thiadiazole derivatives

Several cellular disorders have been related to the overexpression of the cysteine protease cathepsin B (CatB), such as rheumatic arthritis, muscular dystrophy, osteoporosis, Alzheimer's disease, and tumor metastasis. Therefore, inhibiting CatB may be a way to control unregulated cellular functions and prevent tissue malformations. The inhibitory action of 1,2,4-thiadiazole (TDZ) derivatives has been associated in the literature with their ability to form disulfide bridges with the catalytic cysteine of CatB. In this work, we present molecular modeling and docking studies of a series of eight 1,2,4-thiadiazole compounds. Substitutions at two positions (3 and 5) on the 1,2,4-thiadiazole ring were analyzed, and the docking scores were correlated to experimental data. A correlation was found with the sequence of scores of four related compounds with different substituents at position 5. No correlation was observed for changes at position 3. In addition, quantum chemistry calculations were performed on smaller molecular models to study the mechanism of inhibition of TDZ at the active site of CatB. All possible protonation states of the ligand and the active site residues were assessed. The tautomeric form in which the proton is located on N2 was identified as the species that has the structural and energetic characteristics that would allow the ring opening of 1,2,4thiadiazole.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides.

Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways

The First Anti-Snakebite and Hepatoprotective Characterization of a Trypsin Kunitz-like Inhibitor (EcTI) from the Plant <i>Enterolobium contortisiliquum;</i> A Case of Two Soul Mates Meeting

Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms

Optimization of lipid extraction from the oleaginous yeasts Rhodotorula glutinis and Lipomyces kononenkoae

The constant growing demand for vegetable oil for biodiesel and food is raising many environmental concerns about the sustainability of its production based on crops. Oleaginous yeasts show great potential to end with those concerns due to their high lipid productivity in small areas. To evaluate their productivity in lipids, an efficient and reproducible extraction process should be used. As no standard extraction process is available for the extraction of yeast lipids, an optimized extraction process is presented. In this work, the lipids extraction process for the yeasts Rhodotorula glutinis and Lipomyces kononenkoae is optimized using bead beating for cell rupture and introducing adaptations of the two most used extraction methods (Bligh and Dyer and Folch). For Rhodotorula g. the optimum extraction conditions are obtained by the Bligh and Dyer method applying 4.8 cycles of 47 s with 0.7 g of glass beads. For Lipomyces k. the optimum extraction conditions make use of the Folch method applying seven cycles of 42 s with 0.54 g of glass beads. These results reinforce the idea that, for each yeast, different extraction processes may be needed to correctly determine the lipid yield. The extraction procedure was further evaluated with less harmful solvents. Toluene was tested as a possible substitute of chloroform, and ethanol as a possible substitute of methanol. With the optimized extraction process, better results for Lipomyces k. were obtained using toluene and ethanol, while for Rhodotorula g. toluene proved to be a valid substitute of chloroform but ethanol is far less effective than methanol.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), FCT Doctoral grant (SFRH/BD/80490/2011) attributed to Bruno Vasconcelos and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio