Mestrado em Bioquímica
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- ItemPurificação e caracterização bioquímica de uma protease do muco do peixe escorpião scorpaena plumieri(Universidade Federal do Espírito Santo, 2024-10-09) Pirovani, Milenna Machado; Gonçalves, Juliana Barbosa Coitinho; Figueiredo, Suely Gomes de; https://orcid.org/0000-0002-5363-8329; Paula, Heberth de; Menezes, Thiago Nunes deProteases are the efficient executioners of a common chemical reaction, the hydrolysis of peptide bonds. These enzymes appear in all domains of life, including plants, animals, and microorganisms. Due to their involvement in various important biological processes (e.g., blood coagulation, blood pressure regulation, protein processing, and antimicrobial activity), as well as their high specificity, these enzymes have clinical and industrial biotechnological potential. Recently, using a proteomic approach – ‘shotgun’ modality – our research group identified 228 proteins in the epidermal mucus of the Brazilian scorpionfish, Scorpaena plumieri, including various classes of proteases. In this study, a gelatinolytic protease from S. plumieri skin mucus (named Sp-MGP) was purified to homogeneity through a combination of two chromatographic steps: conventional gel filtration (Sephacryl S-200 column) and anion exchange HPLC (SynChropak AX300 column). The purification process was monitored by zymography (SDS-PAGE gelatin). Physicochemical studies indicated that the purified enzyme is an approximately 79-kDa monomeric glycoprotein, as estimated by SDS-PAGE under both reducing and non-reducing conditions. Zymogram analysis of Sp-MGP showed gelatinolytic zones of activity in the gel, with optimal activity at pH 6.0 to 9.0, which is typical of serine proteases. The enzyme stored in aqueous solutions at room temperature (25–30°C), 4°C, -25°C, and -80°C for 60 days did not lose its activity. Sp-MGP was also active on the thrombin (S-2238) and kallikrein (S-2266) synthetic substrates. The activity on S2238 was inhibited by aprotinin and benzamidine. These data reaffirm the suggestion that the purified enzyme is a serine protease, specifically of the thrombin-like and kallikrein-like subclasses. The information generated represents the initial step in the study of a novel protease and may contribute to exploring its biotechnological potential, in addition to providing knowledge about the diversity of molecules in fish skin mucus.
- ItemEstudo do potencial antitumoral do extrato hidrolisado da espécie euphorbia tirucalli l. em modelos in vitro em câncer de ovário(Universidade Federal do Espírito Santo, 2024-07-30) Wanzeler, Ranna Batista; Rangel, Leticia Batista Azevedo; https://orcid.org/0000-0002-7810-0621; Tavares, Marcella Porto; Soares, Karla LirioCancer is a group of diseases characterized by the disordered proliferation of cells, which can be influenced by various factors. Ovarian cancer (OC) is the second most common gynecological neoplasm and the fifth most common cause of cancer-related death in women, primarily due to the asymptomatic growth of tumors. Cancer treatment involves various approaches, such as surgery, chemotherapy, and radiotherapy. However, chemoresistance is a challenge in the treatment of malignant neoplasms, leading to increased research on therapeutic combinations that include natural compounds. One substance that has been gaining attention is euphol, a compound found in the latex of the Euphorbia tirucalli plant. There are reports of its use in folk medicine to treat conditions such as inflammation, asthma, cough, warts, and malignant tumors. Thus, this study aimed to evaluate the antitumor potential of the hydrolyzed extract of Euphorbia tirucalli in vitro models using OC cell lines (A2780, ACRP, and OVCAR-3). Metabolic cell viability (MCV) experiments and IC₅₀ calculations were performed for OVCAR-3, A2780, and ACRP cell lines. Clonogenic assay, cell migration, and cell cycle analysis were performed only with OVCAR-3 cell line. MCV assay results showed that the A2780 and ACRP cell lines were not sensitive to the Euphorbia hydrolyzed extract, while the MCV of OVCAR-3 cell line had a significant decrease. Subsequent assays demonstrated that the hydrolyzed extract interferes with both migration and colony formation, drastically reducing viability. Finally, the cell cycle assay performed by flow cytometry showed that the cells were arrested in the G1 phase, corroborating other studies. Therefore, these results suggest a potential antitumor effect of the hydrolyzed extract of Euphorbia tirucalli.
- ItemAvaliação Físico-Química Da Interação De Betaciclodextrina Com Isoformas Alfa- E Psi- Tripsina(Universidade Federal do Espírito Santo, 2024-02-26) Oliveira, Daniel de Jesus de; Rosa, Dayanne Pinho; https://orcid.org/0000-0002-1483-5848; http://lattes.cnpq.br/7576001371663181; Santos, Alexandre Martins Costa; https://orcid.org/0000-0002-8801-8875; http://lattes.cnpq.br/4144105396879016; https://orcid.org/0009-0007-0082-9547; http://lattes.cnpq.br/5786173815850998; Araujo, Marlonni Maurastoni ; https://orcid.org/0000-0002-6064-3126; http://lattes.cnpq.br/6052184072658200; Cicilini, Maria Aparecida ; https://orcid.org/0000-0003-2751-117X; http://lattes.cnpq.br/7082425279468970Cyclodextrins are cyclic polysaccharides with a structure composed of glycopyranose. Studies have shown that cyclodextrins can form promising interactions with proteins, which has several applications in biotechnology and biochemistry, such as the solubilization of hydrophobic compounds and the protection of compounds sensitive to thermal, oxidative degradation, among others. In this work, in order to evaluate the influence of this polysaccharide on conformational stability and protein activity, physicochemical tools were used, adopting bovine trypsin as an enzymatic model of study, due to the knowledge of its structure and enzymatic activity. This enzyme belongs to the class of serine proteases and has several isoforms, α-trypsin, β-trypsin and ψ-trypsin being the most studied. The results showed that protease amidasic activity increased up to about 76% in commercial trypsin, 82% in alpha isoform, and 45% in psi isoform, suggesting that the addition of β-cyclodextrin optimized the amidsis activity of the trypsin isoforms tested. Fluorescence and absorption spectroscopy revealed changes in the structural properties of trypsin due to the interaction with βcyclodextrin, affecting the activity and conformational stability of trypsin. The formation of supramolecular states of trypsin isoforms in the presence of β-cyclodextrin by means of dynamic light scattering (DLS) was also evaluated, in which an increase in the size and dispersivity of the molecule suggestive of the formation of supramolecular states was verified. The joint evaluation of these results suggested that there was the formation of stabilizing interactions between β-cyclodextrin and trypsin isoforms through the formation of supramolecular states, with possible formation of inclusion complexes. Thus, it is suggested that β-cyclodextrin may interact with trypsin and affect its proteolytic activity and stability and thus the influence of the osmolyte can be understood at the molecular level.
- ItemO efeito da FNDC5 e Irisina em linhagens de células de câncer de mama MCF-7 e MDA-MB-231(Universidade Federal do Espírito Santo, 2023-09-01) Fortes Junior, Pedro Florencio da Cunha; Daltoe, Renata Dalmaschio; https://orcid.org/0000000285710428; http://lattes.cnpq.br/0931389996547944; https://orcid.org/0009-0007-4315-0315; http://lattes.cnpq.br/5958617857750748; Sousa, Nuno Manuel Frade de; http://lattes.cnpq.br/0299657415152651; Madeira, Klesia Pirola; https://orcid.org/0000-0002-9442-9961; http://lattes.cnpq.br/2179692838971480; Gonçalves, Juliana Barbosa Coitinho; https://orcid.org/000000025892050X; http://lattes.cnpq.br/3448669742301744Cancer is the second most prevalent disease and causes of deaths worldwide. Among the most prevalent cancers among women is breast cancer; It is estimated that 2.3 million new cases appear annually around the world. In this context, there is growing concern in the scientific field about the advancement of this disease. A myokine was discovered in 2012 that appears to reduce the proliferation of cancer cells, increasing the expression of pro-apoptotic proteins, decreasing the action of cell synthesis and proliferation mechanisms and other pro-inflammatory factors, in addition to regulating energy homeostasis. The objective of this work was to evaluate the effect of FNDC5 and Irisin molecules on human breast cancer lines. The recombinant molecules Irisin (glycosylated) and FNDC5 were used. The molecules were evaluated at concentrations of 0,625 nmol/L, 1,25 nmol/L, 2.5 nmol/L, 5 nmol/L, 10 nmol/L, 20 nmol/L and 40 nmol/L and the cells were treated48 hours. After this period, the MTT reagent was added at a concentration of 5 mg/mL, and incubation was carried out for 3 hours. After this period, the supernatant was discarded, 100 μl of DMSO reagent was added to all wells and, after 15 minutes, the absorbances were read at 540 nm. The estimated values of the 50% inhibitory concentration of cell proliferation (IC50) were calculated. The calculations were carried out using the GraphPad Prism software in version 8.0. Regarding the data from the MTT experiments, for normal data, one-way ANOVA with Dunnett's post hoc was used, and non-normal data were analyzed by Kruskall-Walli with Dunn's post hoc. The results showed that Irisin exerted cytotoxicity on the MDA-MB-231 strain at concentrations of 1.25 nmol/L (p<0.01), 5 nmol/L (p<0.01), 10 nmol/L (p< 0.0001), 20 nmol/L (p<0.05) and 40 nmol/L (p<0.01) with MCV values reaching 77.66%, 78.75%, 67.25%, 77, 83% and 75.5% respectively. In the MCF-7 line treated with Irisin, a reduction in viability was observed at a concentration of 2.5 nmol/L (p<0.05), a concentration at which the observed MCV was 83.58%. In turn, the FNDC5 molecule did not cause a statistically significant reduction in metabolic cell viability in the tested lines. Our data show that irisin can play an important role in treatment, with deleterious effects on the cell viability of cancer cells.
- ItemDesenvolvimento de um biossensor para detecção de Salmonella em alimentos(Universidade Federal do Espírito Santo, 2023-07-06) Silva, Gabrielle Batista Landim; Oliveira, Jairo Pinto de; https://orcid.org/0000000175951183; http://lattes.cnpq.br/2228283301316218; https://orcid.org/0009-0004-1921-6060; http://lattes.cnpq.br/2195941577218775; Xavier, Andre da Silva; https://orcid.org/0000000292510301; http://lattes.cnpq.br/5661020509713522; Nascimento, Isabella Sampaio do; https://orcid.org/0000-0002-4711-123X; http://lattes.cnpq.br/8257371551653352Salmonellosis is a disease transmitted by contaminated food and is one of the main causes of infections worldwide. To ensure public health, early detection of pathogens is crucial. However, current detection methods are laborious and time-consuming, impacting the entire food chain. In this study, we developed a biosensor for rapid detection of Salmonella in food samples. The detection system is based on lateral flow technology with gold nanoparticles as an optical transducer and antibodies as antigen recognition molecules, with results obtained in just 15 minutes after inserting the strip into the sample. The developed biosensor has selectivity in relation to other microorganisms such as Escherichia coli and Staphylococcus aureus, sensitivity, with a visible detection limit of 10³ CFU.ml⁻¹ and quantitative of 1 CFU.ml⁻¹, values below existing commercial tests and 100% of repeatability. It is fast and reliable and can be a favorable alternative for Salmonella screening in food to ensure food safety.