Mestrado em Bioquímica
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- ItemEstratégias para quantificar Ocratoxina A em amostras de café: estudo de interferentes, pré-tratamentos e análise computacional(Universidade Federal do Espírito Santo, 2025-07-29) Mancini, Isabela Fracalossi; Santos, Gabriel Fernandes Souza dos ; https://orcid.org/0000-0002-8798-6428; http://lattes.cnpq.br/2884729106673212; Oliveira, Jairo Pinto de; https://orcid.org/0000-0001-7595-1183; http://lattes.cnpq.br/2228283301316218; https://orcid.org/0009-0006-2698-6957; http://lattes.cnpq.br/5521119724911165; Guimarães, Marco Cesar Cunegundes; https://orcid.org/0000-0003-2146-0180; http://lattes.cnpq.br/0261991057482057; Antunes, Paulo Wagnner Pereira; http://lattes.cnpq.br/5259147170249880Ochratoxin A (OTA) is a mycotoxin produced by fungi of the Aspergillus and Penicillium genera that can be found in various food products and is particularly relevant in the context of coffee production. Due to its high toxicity, each region has its own regulations to ensure safe maximum consumption limits of this substance in coffee beans. The quantification of OTA is commonly performed using methodologies that involve immunoaffinity columns (IACs) as a pre-treatment step, followed by high performance liquid chromatography with fluorescence detection (HPLC-FL). However, components of the coffee matrix can directly influence the efficiency of OTA recognition by IAC antibodies. Therefore, the present study investigated individually the influence of some coffee markers (caffeine, caffeic acid, chlorogenic acid, cafestol, acrylamide, and melanoidins) on OTA detection, as well as their effects in different types of coffee. The assays were performed by pre-treating the samples using IACs followed by HPLCFL detection, and also employing rapid tests (Lateral Flow Immunoassay, LFIA). The results showed that nearly all interferents were capable of reducing OTA recovery in the tests. Caffeine and caffeic acid yielded the lowest recoveries in the HPLC-FL tests, with values of 65.17% and 69.39%, respectively. In the LFIA tests, chlorogenic acid and melanoidins resulted in the lowest recoveries, with only 48.45% and 35.74%, respectively. Computational molecular docking studies were carried out, and the in silico results were compared with the tests performed. The findings indicate that the reduction in toxin recovery may be related to possible interactions with OTA, mainly through hydrogen interactions or π-π bonds.
- ItemPurificação e caracterização de lectina(s) de Xanthosoma sagittifolium (L.) Schott (taioba): hemaglutinina(s) com potencial efeito citotóxico(Universidade Federal do Espírito Santo, 2025-03-19) Paiva, Ana Paula Passos de; Gonçalves, Juliana Barbosa Coitinho; https://orcid.org/0000-0002-5892-050X; http://lattes.cnpq.br/3448669742301744; https://orcid.org/0009-0000-7612-0090; http://lattes.cnpq.br/1797554469669361; Oliveira, Jairo Pinto de; https://orcid.org/0000-0001-7595-1183; http://lattes.cnpq.br/2228283301316218; Costa, Mariana Amalia Figueiredo; https://orcid.org/0000-0002-8539-3063; http://lattes.cnpq.br/2806425060879128Lectins are carbohydrate-binding proteins that appear in all domains of life and, due to their ability to bind specifically and reversibly to carbohydrate glycoconjugates through specific sites/domains - being known as agglutinins - they play an essential role in various biological processes. The antimicrobial and antitumor potential of these proteins has been explored for the development of alternative drugs and/or adjuvants. The presence of lectins in the leaves and roots of various plant species has been described, which are associated with inflammatory responses, antioxidant capacity, antiproliferative and pro-apoptotic effects of these plants. This work aimed to purify and characterize – biochemically and functionally – hemagglutinating fractions - lectin(s) - from Xanthosoma sagittifolium (L.) – Taioba – an Unconventional Food Plant (UFP). The purification process was monitored by hemagglutination and SDS-PAGE assays (presence of bands with a molecular mass between 12-14 kDa - the monomeric unit of lectins). Hemagglutinating fractions were obtained by sequenced saline precipitation ((NH4)2SO4 15% and 35%) from the protein extract of leaves and roots. The fraction of the leaf extract precipitated at 15% was selected for the continuation of the purification process – molecular exclusion chromatography in an HPLC system – from which two partially purified hemagglutinating fractions were obtained. The protein profile of these fractions showed bands with the molecular mass of the monomeric unit and its multiples, suggesting the formation of supramolecular arrangements, a common characteristic of lectins. It was shown that the hemagglutinating fractions (P15 and P35 from leaves) have mannose binding specificity and do not differentiate between blood types (ABO). P15 from the taioba leaf also showed an optimum pH between 7,5 and 7,9 and an optimum temperature of 37°C, being dependent on the magnesium ion. Using the resazurin-based cell viability assay, it was shown that the hemagglutinating fractions - from molecular exclusion - were cytotoxic in a dose-dependent manner (~ 0. 1 µg to 53 µg), 1 µg to 53 µg) for the cancerous cell lines of rat glioma (C6), human colon carcinoma (RKO) and primary malignant human ovarian adenocarcinoma (TOV-21G), except for the human glioblastoma tumor line (U373), reducing viability by up to 78%. Preliminary tests did not identify any antifungal activity for Trichoderma reesei. In this work, two hemagglutinating fractions with cytotoxic potential were partially purified, which represents the first steps towards their complete biochemical andfunctional characterization, as well as the exploration of their biotechnological potential.
- ItemPotencial efeito neuroprotetor do canabidiol em um modelo pré-clínico da doença de parkinson(Universidade Federal do Espírito Santo, 2024-10-18) Toledo, João Gabriel Assis; Pires, Rita Gomes Wanderley; https://orcid.org/0000-0002-4739-8349; https://orcid.org/http://lattes.cnpq.br/8356031036198869; https://orcid.org/0009-0008-9696-8679; http://lattes.cnpq.br/1148485070900089; Hollais, André Willian; https://orcid.org/0000-0002-2991-8646; http://lattes.cnpq.br/0068789063590867; Silva, Sarah Martins Presti da; https://orcid.org/0000-0003-2579-4866; http://lattes.cnpq.br/0768873502799973Parkinson's disease (PD) is the second most common neurodegenerative disease, second only to Alzheimer's disease. PD is characterized by the death of dopaminergic neurons in the substantia nigra pars compacta, leading to a decrease in dopamine release in the striatum region. The increase in cases of death and disability from PD has increased more than any other neurodegenerative disease, however, there is still no cure or treatment that reduces the progression of PD, making the search for new treatment methods and drug candidates that promote such effects imperative. The gold standard medication for the treatment of Parkinson's disease, levodopa, brings with it some problems with prolonged use, such as levodopa-induced dyskinesia, which is often worse than the disease itself. In this context, Cannabidiol (CBD), a non-psychoactive compound extracted from the Cannabis sativa plant, has shown promise, as there are several studies demonstrating its potential neuroprotective effect in different neurological diseases and also a possible indication for the treatment of levodopa-induced dyskinesia. Swiss mice were treated for 21 days with rotenone and two hours later cannabidiol was administered, and at the end of the treatment motor tests (open field and rotarod) were performed for biochemical analyses of the content of dopamine, serotonin and their metabolites by HPLC, in addition to the expression of tyrosine hydroxylase by Western Blotting. From the results obtained, there was no statistical difference in the results found in the open field test, however there was a significant difference in the latency to fall from the rotarod test apparatus, however, this difference was not found with the biochemical parameters both by Western Blotting and by HPLC. The data found in this work suggest a behavioral alteration that was not reflected in biochemical alterations and new experiments should be carried out with other doses of both rotenone and CBD.
- 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.