DSpace Coleção:https://repositorio.ufms.br/handle/123456789/24672024-03-29T06:37:58Z2024-03-29T06:37:58ZDesenvolvimento de um novo peptídeo com atividade antimicrobiana (bactérias e leveduras) e antiproliferativa em células neoplásicas: análises in silico, in vitro e in vivohttps://repositorio.ufms.br/handle/123456789/71522023-11-27T20:46:26Z2023-01-01T00:00:00ZTítulo: Desenvolvimento de um novo peptídeo com atividade antimicrobiana (bactérias e leveduras) e antiproliferativa em células neoplásicas: análises in silico, in vitro e in vivo
Abstract: Antimicrobial resistance (AMR) acquired by microorganisms, whether naturally or due to the indiscriminate use of antimicrobials, and multidrug resistance (MRD) acquired by microorganisms and also by cancer cells, has become a serious public health problem throughout the world. world. Microorganisms, in addition to being able to overcome the effects of antimicrobials, also present biofilm formation as a form of resistance, which makes the treatment of microbial infections even more difficult. This highlights the importance of discovering and/or developing new antimicrobial and anticancer agents. Among the classes of biomolecules, peptides have stood out due to their broad spectrum of biological activities. With this in mind, in this study a new antimicrobial peptide (AMP) was developed, based on a peptide encrypted in the sequence of Inga laurina seed trypsin inhibitor (ILTI). To obtain the AMP, the ILTI sequence was fragmented in silico, obtaining 169 fragments, and the fragment that presented the greatest antimicrobial potential was chosen for study. Changes in the positioning of amino acid residues and exchange of amino acid residues in the selected sequence were carried out, thus obtaining a AMP with 19 amino acid residues called KWI-19. In silico evaluations of the physicochemical characteristics of the peptide sequence obtained showed desirable parameters for a AMP. Once this was done, homology modeling was carried out, the validation of the lower energy model (more stable) and subsequently the synthesis of the peptide using solid phase methodology (SPSP), followed by its purification by high performance liquid chromatography (HPLC) and determination of its mass by mass spectrometry using the technique matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF). The evaluation of the cytotoxicity of the peptide in vitro and in vivo was carried out and it was found that the peptide presented a hemolytic concentration in 50% of erythrocytes at a concentration of 6.54 μmol L-1. Regarding the in vivo acute toxicity test on Galleria mellonella larvae, no toxic effects were observed. In vitro tests were carried out with Gram-positive and negative bacteria, and with yeasts belonging to the Candida genus. KWI-19 inhibited bacterial growth with minimum inhibitory and bactericidal concentrations (MIC and MBC) ranging from 1.25 to 10 μmol L-1. Of the yeast species tested, KWI-19 inhibited growth at minimum inhibitory and fungicidal concentrations (MIC and CFM) ranging from 2.5 to 20 μmol L-1. Due to the results obtained, the species of Pseudomonas aeruginosa, Staphylococcus saprophyticus and C. tropicalis were chosen for further studies. For these species, we carried out death kinetic assays, where it was found that KWI-19 inhibits bacterial and fungal growth, in MBC and MFC, from 30 min for S. saprophyticus, in 120 minutes for P. aeruginosa and in 60 min for C. tropicalis. Assays to determine the peptide's mode of action were carried out, and in the nucleic acid release assay, in bacteria, it was observed that there is a greater amount of DNA and RNA present in the extracellular environment, when the strains were treated with KWI-19 . In the crystal violet uptake assay, it was not possible to observe a statistical difference between bacteria treated and not treated with KWI-19. To investigate the mode of action of KWI-19 in yeast, assays were carried out using the SYTOXTM Green probe and another using ergosterol and sorbitol. In these assays it can be seen that KWI-19 acts on the yeast plasma membrane. The effects of KWI-19 on inhibiting biofilm formation and eradicating mature biofilm from selected strains were also evaluated and biofilm viability was quantified with the total number of viable colony forming units (CFU), where KWI-19 inhibited and eradicated part of the biofilm from all study strains. Cell viability assays with the murine melanoma line B16F10-Nex2 were performed and the peptide showed an IC50 of 22.1 μmol L-1, where it was verified, by flow cytometry, that the main cell death process activated by KWI-19 is necrosis.
Keywords: encrypted peptide, antibacterial, antifungal, antibiofilm, anticancer.
Tipo: Tese2023-01-01T00:00:00ZProdução, purificação e caracterização bioquímica de pectinases de Aspergillus japonicus e Thermoascus aurantiacus: aplicação da enzima de A. japonicus na clarificação de sucos de frutashttps://repositorio.ufms.br/handle/123456789/58592023-05-10T19:43:37Z2023-01-01T00:00:00ZTítulo: Produção, purificação e caracterização bioquímica de pectinases de Aspergillus japonicus e Thermoascus aurantiacus: aplicação da enzima de A. japonicus na clarificação de sucos de frutas
Abstract: The development of innovative and sustainable alternatives to add value to agricultural and food waste (lignocellulosic biomass) is needed worldwide. Lignocellulosic biomass is a source of renewable resources because it is an organic matter made up of a complex matrix of polysaccharides, including cellulose, hemicellulose and pectin. Pectinases are a complex group of enzymes that degrade pectic substances widely used by industries, mainly in the food industry. In this context, the general objective of this study was the production of pectinases by two fungi (Aspergillus japonicus and Thermoascus aurantiacus) using low-cost substrates (agribusiness residues/products), as well as the purification and biochemical characterization of the enzymes, followed by immobilization and application of pectinase from A. japonicus in the clarification of fruit juices. In this context, the general objective was to study the production of pectinases by the fungi Aspergillus japonicus and Thermoascus aurantiacus using low-cost substrates and to analyze their applications in the clarification of fruit juices. The best pectinase production by T. aurantiacus was in SSC with cassava flour from Rondonópolis/MT with 26.7 U/g of dry substrate (or 2.67 U/mL). And for A. japonicus it was observed that the highest production of pectinase was with passion fruit peel in SSC (30 U/g of dry substrate or 3 U/mL).
For T. aurantiacus, 96 hours of growth was the best time, while for A. japonicus it was 48 hours. In evaluating the effects of pH and temperature on enzyme activity, T. aurantiacus had pH 4.0 and 70 °C as optimal parameters, and A. japonicus pH 4.0 and 60 °C. In the stability evaluation, pectinase from A. japonicus (crude extract) was completely stable for 4 hours at all tested pHs. About thermostability, pectinase from A. japonicus remained stable for 6 hours at 25 °C, and after 24 hours of testing it still maintained 74% of the initial activity. At 50 °C the enzyme maintained 71% activity for 6 hours. The pectinase (crude extract) of A. japonicus was evaluated in the clarification of 13 pulps and was superior to the commercial pectinase (Pectinex) in all evaluated fruits. The best clarification obtained using 3 U/mL of crude pectinase from A. japonicus was in mango (Haden) with 85.56%, while Pectinex clarified only 50.89%. With apple (Argentina), pectinase from A. japonicus was 5 times more efficient in clarification than Pectinex, with 66.32% and 12.67% clarification, respectively. T. aurantiacus pectinase was purified in two chromatographic steps (DEAE-fractogel and Sephacryl S-200), with specific activity of 75.7 U/mg protein, resulting in a 10-fold purification with 21% enzyme recovery. And pectinase from A. japonicus was also semi-purified in two chromatographic steps (DEAE-fractogel and fenil-sepharose), resulting in a 2.9-fold purification with 81% enzyme recovery and a specific activity of 7.9 U/mg protein, exhibiting a molecular weight of about 40 kDa (named as PGAj). In mass spectrometry (LC-MS/MS) of PGAj a polygalacturonase of 29.99 kDa was the most abundant. The optimum pH and temperature for PGAj activity were pH 4.0 and 55 °C, respectively. Furthermore, the PGAj enzyme retained over 90% of its initial activity for 4 hours at pH 4.0, 5.0 and 6.0. The enzyme maintained 83% of residual activity after 20 min at 50 °C. In the specificity test, PGAj had citrus pectin as the preferred substrate, followed by apple pectin and polygalacturonic acid. For the juice clarification tests by PGAj, 13 pulps were also used, where the best result was obtained with mango pulps (Palmer and Tommy), with 65% and 41%, while Pectinex clarified only 49% and 21%, respectively. Then the white guava, the banana “nanica” and the gala apple, had 40%, 11% and 9.4% of clarification, respectively. The best result of immobilization obtained was with 2% sodium alginate, using 0.1 M CaCl2. In reuse tests, immobilized PGAj maintained 100% activity after 6 reaction cycles using 1% pectin as substrate. It is concluded that crude and semi-purified pectinase from A. japonicus showed potential for application in beverage industries, to contribute to an efficient and economical production of clearer fruit juices.
Tipo: Tese2023-01-01T00:00:00ZEfeito de dissulfetos de diarila sobre formas epimastigotas e tripomastigotas de Trypanosoma cruzihttps://repositorio.ufms.br/handle/123456789/58302023-04-24T13:37:21Z2023-01-01T00:00:00ZTítulo: Efeito de dissulfetos de diarila sobre formas epimastigotas e tripomastigotas de Trypanosoma cruzi
Abstract: This work aims to evaluate the effect of synthetic compounds diaryl disulfides in replicative and infective forms of the etiological agent of Chagas Disease (CD), the protozoan Trypanosoma cruzi. CD is an endemic disease in Latin America, representing a serious public health problem, as it affects about 6 million people worldwide. In this perspective, the search for new compounds with anti-Trypanosoma activities that present low cytotoxicity and may represent an alternative for the treatment of this disease has been the objective of numerous research groups. There are reports in the literature of compounds of diaryl disulfides with antiproliferative activity against tumor cells, as well as action on protozoa. Therefore, this study proposes to evaluate the in vitro effect of Diaryl Disulfide compounds on the replicative and infective forms of T. cruzi. Biological assays of viability, growth, morphological analysis for epimastigotes and viability of trypomastigotes were carried out. The in silico analysis was also carried out to evaluate the ADMET properties of the compounds. During the analysis of the ADMET properties it was observed that the compounds present good absorption and do not present hepatotoxicity or mutagenicity. In the viability assay, it was observed that the compounds have biological activity on the epimastigotes forms of T. cruzi, with compounds D and E being the most active (3.124 and 5.091µM, respectively). The growth curve showed that from 24h of exposure, compound D caused a 50% inhibition and at 96h of exposure, this inhibition was 19.64%, demonstrating a reduction of activity according to the time of exposure. The recovery test was performed with compounds B, C and D showed that after 96h the inhibition values were between 26% and 40%, indicating a trypanostatic action. The morphological analysis performed with compounds B and D indicated that these compounds cause morphological changes that affect the viability of the parasite. The viability assay with trypomastigotes treated with compound D indicated that the compound has action on the infective form of the parasite, with an IC50 of 10.90 µM.
Keywords: Diaryl Disulfides, Trypanosoma cruzi, Chagas Disease.
Tipo: Dissertação2023-01-01T00:00:00ZCaracterização bioquímica e biológica da peçonha de Tityus confluenshttps://repositorio.ufms.br/handle/123456789/58272023-04-20T19:47:46Z2023-01-01T00:00:00ZTítulo: Caracterização bioquímica e biológica da peçonha de Tityus confluens
Abstract: Scorpions are arthropods from Scorpiones, habit several ecosystems, and are venomous animals with a venom-inoculating structure, causing accidents in humans. These accidents are considered a public health problem in several countries, such as Brazil, however, only 50 species of the more than 2700 that have already been described are responsible for accidents, and in Brazil, all are from the genus Tityus. Venoms are complex mixtures, composed of bioactive molecules, with promising potential for prospecting new biotechnological and pharmacotherapeutic products. However, few studies about the venom of several scorpions, such as Tityus confluens, occur in the Brazilian savanna. The present study aimed to characterize biochemically and biologically the venom from Tityus confluens. The protein and chromatographic profile of the venom was verified, as well as the enzymatic activities, such as phospholipase, amylolytic and proteolytic, the effect of the venom on the activity of membrane ATPases, in addition to evaluating the cytotoxicity on normal and tumor cells. The venom showed a diversity of components by the electrophoretic and chromatographic profile, showing protein bands with molecular masses between 50 -100 kDa, bands smaller than 10kDa, and eluted fractions between 18 - 25 min of RT and the largest fraction at 41 min of RT, possibly corresponding to peptides. The venom caused an increase in the activity of ATPase enzymes when incubated at 30 and 50 min, demonstrated amylolytic activity, did not present phospholipase and proteolytic activities, and was not cytotoxic against normal and neoplastic cells. The results obtained in this study allow an initial characterization of the composition of the venom of Tityus confluens, expanding the view of scorpion venoms and further studies on these compounds are necessary to elucidate the potential molecules for bioprospecting.
Tipo: Dissertação2023-01-01T00:00:00Z