Breast cancer. considered the main malignant neoplasm that affects women in the Brazil and in the World. Molecular subtypes are categorized into luminal and non-luminal. Non-luminal tumors have an unfavorable prognosis, high recurrence rate and few treatment possibilities, and those available do not significantly increase patient survival. Therefore, the need to prospect new, more effective and specific treatment strategies for non-luminal subtypes. The objective proposed in this work is the in silico prediction of tumor antigenic multi-epitope vaccine candidates for non-luminal subtypes. The choice of targets for the development of multi-epitope vaccines was performed based on the analysis of 170 RNA expression samples from patients with breast cancer of the invasive ductal carcinoma type and non-luminal subtypes (HER2 and basal) and 80 samples of breast tissue standard available for download in public banks. In these data, the most differentially expressed genes were obtained using the limma package and considering p<0.05 and |logFC| >1 for each subtype. These genes were compared to each other using the Venn diagram. The enriched pathways of genes unique to each subtype were evaluated in KEGG and Reactome. In addition, the protein interaction of the differentially expressed transcripts and the subcellular localization in the cell membrane were evaluated in the Cytoscape 3.9.1 software by STRING plugging. Amino acid sequences of selected target proteins, in canonical FASTA format, were obtained from the UniProt database. Each sequence was evaluated for its tumor antigenicity by the Vaxijen server v.2.0. Then be. The prediction of the linear epitopes of the B lymphocytes, which stimulate the humoral response, was carried out by the ABCpred server. In the epitopes of class I and class II of T lymphocytes, which trigger the cellular response, the analyzes will be performed by the IEDB server. All selected epitopes will be evaluated for antigenicity, allergenicity, immunogenicity and toxicity by servers. They will also be modeled and evaluated for affinity with experimentally validated software alleles. With all epitopes selected be. The estimate of population coverage of MHC class I and II epitopes was performed by the IEDB servers. After being. The prediction of the epitopes that stimulate the production of interferon-gamma cytokines was performed by the IFN epitope software. The construction of the multi-epitope vaccine involved the use of several tools that enabled structural modifications to increase immunogenicity, evaluation of cross-reactivity, evaluation of physicochemical properties, allergenicity, antigenicity and toxicity, prediction of secondary and tertiary structure, refinement and validation, assessment of immunogenicity and receptor binding affinity. Also, be. Optimization of in silico cloning of the multi-epitope vaccine was performed to ensure in vivo expression. At this stage of the study, proteins PPP4C and PAK4 in the Her2 subtype, and ENO1 and SEPHS1 in the Basal subtype, were identified as potential targets for predicting the epitopes that will compose multi-epitope vaccines. These genes present pathways associated with important events in the tumor development process, as well as interaction with other proteins that play a regulatory role. It is expected to find, from all analyzes carried out, promising tumor antigenic multi-epitope vaccine candidates for the immunotherapeutic treatment of non-luminal subtypes.

The application of enzymes in biotechnological processes allows for a significant reduction in costs with a consequent reduction in energy consumption. In this context, proteases are the most produced biomolecules and represent 60% of all enzymes sold in the world. The importance of using these molecules is due to factors such as high catalytic activity, selectivity, specificity, as well as production variability. Given the demand to produce studies aimed at obtaining enzymes with an industrial bias, the aim of this work is to obtain a protease of biotechnological interest from the seeds of Leucaena leucocephala, following its characterization and application. The project's starting point was to investigate the enzymatic activity in the crude extract of L. leucocephala seeds. Through this research, it was possible to describe a new milk coagulant. The optimal enzyme activities were determined at 40ºC and pH 9.0. In stability tests, the enzyme remained stable over a wide range of pH (5.0 to 9.0) and temperatures (40ºC to 60ºC). Furthermore, the enzyme's catalytic activity improved in the presence of metal ions (Na+, K+, Mn2+, Mg2+ and Ca2+). The enzyme has demonstrated stability and compatibility with detergents, reducing agents and organic solvents. This highlights the biotechnological potential of this enzyme in various industrial processes. When the enzyme extract was subjected to different chromogenic substrates conjugated with p-nitroaniline, the hydrolysis showed specificity for BApNA. Furthermore, the enzyme was inhibited by PMSF (95.80%) followed by Benzamidine (84.22%). Therefore, the enzyme belongs to the serine protease class, an endopeptidase that preserves the presence of the amino acids serine, histidine and aspartic acid in its catalytic site. The aqueous extract of L. leucocephala seeds was subjected to toxicity testing using a bioindicator. It was possible to demonstrate that no toxic environment was generated after 24 hours of exposure of the crustaceans, Artemia salina, to different concentrations of the aqueous extract.

Ecossistemas de alta diversidade como os manguezais são promissores à bioprospecção de moléculas de interesse biotecnológico. Biossurfactantes são moléculas tensoativas, com baixa toxicidade, capaz de reduzir a tensão superficial, podendo tolerar diferentes temperatura, salinidade ou pH. Entretanto, a bioprospecção de leveduras em manguezais para a obtenção de biossurfactantes ainda é incipiente no Brasil. Nesse estudo, leveduras de sedimentos de manguezais do litoral de Alagoas (rios Santo Antônio e Tatuamunha) foram avaliadas quanto ao potencial de produção de biossurfactantes/bioemulsificantes. Nesse sentido, as leveduras foram submetidas a triagem na literatura para a identificação de espécies ainda não descritas para a produção de biossurfactante. Em seguida, 39 isolados de espécies não reportadas foram submetidas ao teste de emulsão IE₂₄%, onde 6 linhagens de P. pseudolambica com os maiores IE₂₄% foram selecionadas. A avaliação consistiu na determinação da tensão superficial, teste Parafilm® M, ensaio de deslocamento de óleo e colapso da gota. Foi analisado o IE₂₄% a partir de cultivos de P. pseudolambica em caldo YEPD com óleo de soja. Também foram avaliados os efeitos de diferentes faixas de temperaturas, salinidades e pH sob os biossurfactantes/emulsificantes, assim como a capacidade de emulsificação em diferentes hidrocarbonetos. Além disso, avaliou-se a produção de lipases, e, posteriormente, foram caracterizados os grupos funcionais de amostras da biomassa celular, via Espectroscopia vibracional de infravermelho médio com transformada de Fourier (FTIR). As linhagens de P. pseudolambica demonstraram capacidade de reduzir a tensão superficial da água entre 36.69 mN/m a 42.47 mN/m, tolerância a condições de pH ácidos, emulsões variando entre 51% e 63%, e resistência a altas temperaturas (90, 100, 110, 120 e 150 ºC). Os cultivos celulares apresentaram capacidade de emulsionar diferentes hidrocarbonetos e atividade de lipase. A análise espectroscópica (FTIR) das amostras de biomassa celular revelou a existência de diversos grupos funcionais típicos de uma estrutura complexa de natureza de carboidrato e lipídio. Portanto, essas descobertas inéditas contribuem à compreensão do potencial de produção de biossurfactantes/bioemulsificantes e lipases de P. pseudolambica oriunda de manguezais da região Nordeste do Brasil, ainda não relatado na literatura, revelando uma eventual possibilidade de uso biotecnológico a ser melhor explorado.

Chikungunya virus (CHIKV) is an arbovirus transmitted by Aedes sp. mosquitoes that causes Chikungunya fever (CHIKF) and could be progress to Rheumatic Chronic Syndrome (RSC). The synovial joints are affected during this infection, and fibroblast- like synoviocytes (FLS) are the major cell type in the composition of synovial tissue. FLS in pathologies such as rheumatoid arthritis (RA), assumes an aggressive profile, causing severe tissue damage in the inflammatory process. This damage can occur due to changes in cellular biomechanical properties. Thus, the aim of this work was to understand biomechanical, biochemical and molecular changes in human fibroblast-like synoviocytes (HFLS) after CHIKV infection in vitro. Determination of the MOI was performed by MTT assay and viral infection was confirmed by flow cytometry, RT-PCR and infection of Vero E6 cells with HFLS supernatant. The following were evaluated: morphological changes and Young`s modulus by atomic force microscopy (AFM); cytoskeleton changes by fluorescence analysis of F-actin labeling; biochemical changes were determined by Raman spectroscopy; relative gene expression analysis by RT-qPCR. Also, the quantification of chemokines was measured in the supernatant using the CBA methodology. As result, HFLS were infected in vitro since 46.8% of the cells were CHIKV-positive after 48h of infection in the intracellular flow cytometry analysis. In addition, viral RNA in the cell supernatant was detected by RT-PCR. The detection of plaque-forming units (PFUs) in the monolayer of infected Vero E6 cells indicated the presence of viable viral particles in the HFLS supernatant. AFM analysis shows significant changes in cell morphology and nanoindentation analysis detected a 107.46% increase in Young's elastic modulus, suggesting alterations in the cytoskeleton. Fluorescence cytoskeleton staining indicated disruption of F-actin filaments after 12h of infection. Raman spectroscopy and PCA analysis show a total variance of 65% between uninfected and CHIKV-infected HFLS. In addition, the major alterations detected were related to nucleotides, amide I, amide III, β-sheets, lipids and collagen, with signatures in 795, 977, 1031, 1223, 1290, 1444, 1495, 1635, 1660 and 1709 cm-1. Relative gene expression shows an increase in the expression of MMP1, VEGFA and UGDH. Also, an increase in the IP-10 and a reduction in MCP-1 chemokine levels were detected in the supernatant 48 after viral infection. In summary, the results presented in this work help elucidate the cellular and molecular changes involved in the CHIKV-HFLS interaction, thus contributing to developing new therapeutic strategies for this arbovirus.

The use of macromolecules obtained from plant tissues has great economic and pharmacological potential and this is due to the presence of bioactive compounds. Among these, lectins have been intensely explored due to the various biotechnological actions they may have, especially the antifungal, anticoagulant and antitumor activities. Thus, the present work aimed to purify, characterize and evaluate the antitumor, antimicrobial and hemostatic activity of lectins from Genipa americana L. (jenipapo), Rhizophora mangle L. (red mangrove) and red propolis from Alagoas. Lectins were isolated initially by extraction in 50mM Tris-Hcl pH 8.0 for G. americana seeds and bark, 0.15M NaCl for R. mangle seeds and 0.15M NaCl with 30% ethanol for red propolis. Then, salt fractions were made with ammonium sulfate and the fractions that showed better activity were submitted to molecular exclusion chromatography (Sephacryl S-100), for isolation of lectins from G. americana (GaBL); molecular exclusion chromatography (Sephacryl S-100) followed by a cation exchanger (DEAE Sepharose) for the lectin from G. americana seeds (GaSL); or chitin column chromatography to isolate lectins from R. mangle and red propolis. Through a single chromatographic step a lectin (GaBL) was isolated and by SDS-PAGE (10%) it was observed that GaBL has an approximate molecular mass of 242.5 kDa. The effect of lectin on blood clotting was evaluated through activated partial thromboplastin time (APTT), prothrombin time (PT). GaBL showed anticoagulant activity only in the intrinsic pathway, where the APTT was prolonged.

In the cytotoxic test, GaBL was not toxic to 3T3 fibroblast cell lines at concentrations below 50μg/mL. The antitumor activity evaluated used human skin cancer (A431), melanoma (B16) and tongue squamous cell carcinoma (SCC9) cell lines. Cell proliferation and cell migration were decreased in all cell lines evaluated in contact with 10 µg/ml of GaBL decreased invasion of SCC9 cells. Apoptosis was higher in lectin treated B16 and SCC9 cells. GaBL's positive regulation of E-cadherin and suppression of Col1A1 in all strains tested indicated less cancer development. The lectin from G. americana seeds, GaSL, had its hemagglutinating activity (AH) strongly inhibited by rhamnose, but was not affected by divalent ions or EDTA.  GaSL is classified as a thermostable lectin, with best activity at a temperature below 80°C and in the pH range 5.0 to 6.0. In the face of antifungal activity, GaSL, when tested with the fungi Candida albicans, Staphylococcus aureus and Cryptococcus neoformans, was shown to inhibit all fungal strains at concentrations below 12.5 μg/mL. The lectin from R. mangle seeds is also thermostable, but unlike the others, it was inhibited more strongly by arabinose and casein, had better activity at pH 8.0 and 9.0 and had its AH reduced in the presence of Mn2+, Mg2+ and Ca2+ ions, not altered by EDTA and stimulated in the presence of Zn2+. The lectin from propolis was also strongly inhibited by casein, is thermostable, showed better AH in the pH 5.0 to 6.0 range and was reduced in the presence of Mn2+, Mg2+, slightly stimulated by Zn2+ and not affected by EDTA and Ca2+. In view of the antifungal activity PVAL proved to be a promising biotechnological tool, when tested with the fungi Candida albicans, Staphylococcus aureus and Cryptococcus neoformans. PVAL at concentrations of 12.5 μg/mL was able to strongly inhibit Candida albicans and Staphylococcus aureus and 25 μg/mL Cryptococcus neoformans. The effect of PVAL on blood clotting was evaluated through activated partial thromboplastin time (APTT), prothrombin time (PT). Where PVLA promoted expressive prolongation of APTT, while for TP it promoted strong inhibition. In view of the above data, we can highlight that GaBL has high antitumor potential against cancer of the upper aerodigestive tract and has a notorious homeostatic activity. GaSL and PVAL have remarkable antifungal activity and can be a promising biotechnological tool, the latter having an important role in the homeostatic processes of coagulation, being a strong protractor of APTT and TP inhibitor. Finally, we emphasize that all the lectins studied have structural characteristics that favor the biotechnological study involving human health.

The marine environment has become promising in the search for chemical substances with biotechnological, pharmaceutical and agricultural potential. These complex substances are often produced by microorganisms associated with marine invertebrates (corals and sponges), especially filamentous fungi. This group of microorganisms has shown potential because they have peculiar biochemical routes capable of synthesizing characteristic or species-specific metabolites. These natural products provide antifungal, antimicrobial, antioxidant, anti-inflammatory, antiviral properties, among others. Therefore, the objective of this work was to make a systematic review on marine fungi and secondary metabolites with biological activities and to investigate the biotechnological potential of filamentous fungi associated with marine invertebrates in Maceió / AL with antifungal activity against infectious agents. The systematic review was carried out by searching for keywords such as “marine fungi”, “antifungal”, “secondary metabolite” and “natural products” in the Pubmed and Science Direct databases, obtaining 19 articles published between the years 2015 and 2020 that describe 84 metabolites produced by marine fungi. The biotechnological potential was verified from the production of crude extracts from 16 fungi, collected in the coral reef of Praia de Ponta Verde, Maceió-AL. To obtain the fungal extracts, they were grown. After cultivation of 7 days in Potato Dextrose Agar (BDA), followed by a second cultivation of 14 days in liquid medium composed of 1% glucose, 0.1% yeast extract, 0.1% potassium chloride, sodium chloride 0.1%, the fungi supernatant and mycelium were extracted with ethyl acetate, generating an organic and aqueous fraction. Subsequently, they were analyzed using the Minimum Inhibitory Concentration Determination (MIC) technique to analyze the sensitivity of microorganisms to antimicrobials. Crude extracts from ten marine fungi showed antifungal activity against the yeast Candida haemulonii, five against Candida albicans, two against Cryptococcus gatti and 10 against Cryptococcus neoformans. Two of the extracts of the active fungi were fractionated with methanol, acetonitrile and water and tested again by the CIM method. The fungi C. neoformans ATCC 40283 and C. albicans ATCC 90028 were inhibited by all tested fractions, presenting MIC between 0.5-2.0 µ/mL, demonstrating that the bioactive compounds derived from marine microorganisms have antimicrobial and biotechnological potential.

Diversas plantas são utilizadas pelo homem para o uso terapêutico e propagadas ao longo das gerações. Atualmente há um crescimento em pesquisas buscando identificar e isolar substâncias ativas presentes nesses organismos visando a criação de novos fármacos. A produção dos princípios ativos desses organismos está relacionada ao seu mecanismo de defesa contra agentes externos. Essas substâncias são normalmente classificadas em metabólitos primários e secundários. As lectinas são proteínas do metabolismo primário que podem assumir diversos papéis biológicos, incluindo atividades antibacteriana, antifúngica, anti-inflamatória, antitumoral e cicatrizante. Essas atividades estão relacionadas à capacidade que as lectinas têm de se ligarem a carboidratos presentes na superfície celular de organismos alvos, impedindo o seu desenvolvimento e/ou induzindo uma resposta celular. Abarema cochliacarpos (Gomes) Barneby & Grimes, popularmente conhecida como barbatimão, é uma planta utilizada na medicina popular para tratar úlceras, feridas, infecções na pele, gastrite, dentre outras. Este estudo tem como objetivo investigar a presença de lectina em entrecascas de A. cochliacarpos e realizar a purificação e caracterização parciais da mesma. O extrato foi preparado e filtrado em carvão ativado na tentativa de remover compostos que não são de interesse. Uma alíquota desse filtrado foi aplicada em uma coluna cromatográfica de quitina e as proteínas de interesse foram eluídas com ácido acético 1 M. Em seguida, a amostra foi dialisada para remover a solução de eluição e sua atividade hemaglutinante foi avaliada (512). A partir daí denominou-se por AcBL (lectina de entrecascas de A. cochliacarpos). Não foi possível confirmar a purificação da lectina através de eletroforese SDS-PAGE em condições redutoras e não redutoras; o gel mostrou a presença de duas bandas proteicas nas duas condições. A dosagem de proteínas e de fenóis foi feita em todas as etapas da purificação, bem como foram realizados os testes de caracterização. AcBL foi inibida por caseína, apresentou termoestabilidade em ampla faixa de temperatura, demonstrou ser uma proteína com caráter ácido-neutro e teve sua atividade reduzida na presença de EDTA, íons cálcio e magnésio. O desenvolvimento deste trabalho contribui para o isolamento de novas proteínas, apresentando relevância regional ao investigar lectinas de uma planta medicinal do Nordeste brasileiro, podendo representar um novo biomaterial com elevado potencial biotecnológico.