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    Obtenção estrutural de um antiagiogênico das vísceras da tilápia (oreochromis niloticus)
    (Universidade Federal de São Paulo (UNIFESP), 2014-01-29) Souza Junior, Airton Araujo de [UNIFESP]; Nader, Helena Bonciani Nader [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Heparan sulfate (HS) and heparin are GAGs that share similar structure, yet, HS contains reduced levels of O- and N-sulfation together with lower levels of epimerized glucuronic acid; iduronic acid. It is a ubiquitously distributed in cell surface of mammals as well as in other vertebrates and invertebrates, hence, it shows a high biotechnological relevance due to its and structural diversity which leads an extensive ?interactome? and modulatory effects on several biological/pharmacological processes. Thus, the comprehension of HS structures in different cells/tissues is crucial for the total elucidation structure-function relationships. The search for heparan-like compounds in waste material from fish farming is both economical and environmental attractive since it adds value to waste products and solves issues related to waste handling, consolidating tilapia culture as a valuable new source of ecofriendly biotechnological agents. Accordingly, this work aimed on the isolation and structural characterization of a heparan sulfate from Tilapia (Orechromis niloticus) waste material, besides its potential effects on coagulation and angiogenesis. The purified compound showed the same electrophoretic behavior, in different buffer systems, of heparan sulfate from bovine sources and the structural analysis, both chromatographic and spectroscopic, proved that the Tilapia compound has strong structural similarity with HS from bovine pancreas. Also, it was demonstrated that the isolated compound has low anticoagulant activity and inhibited capillary tube formation of endothelial cells on 2D angiogenesis assay in Matrigel. Together, our data suggests the HST is a promising bioproduct that could be used to target angiogenesis in pathophysiological processes.
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    Quitosana e quitosanas de baixo peso molecular quimicamente sulfatadas: produção, padrão de sulfatação, comportamento hemostático e interação proteica
    (Universidade Federal de São Paulo (UNIFESP), 2015-06-24) Sabry, Diego de Araujo [UNIFESP]; Sassaki, Guilherme Lanzi Sassaki [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The incidence of cardiovascular diseases still is a major cause of death around the world, only in 2012 it was the causa mortis of about 17.5 million people. Currently, the main drug used against cardiovascular diseases is heparin. However, its use presents some complicating factors, such as: mechanism of action, contamination risk and obtaining costs. These factors indicate the need to search for new compounds that may replace heparin, among which the chitosan has been outstanding. The use of chitosan has attracted much attention of researchers due to its physicochemical and biological properties. In addition, its chemical modification is considerably increasing its applications in the cosmetic, food, pharmaceutical and medical industries. In this study, the commercial chitosan has been chemically modified by sulfation. The sulfated chitosan obtained by different methods have been structurally characterized, and their sulfation pattern determined by NMR spectroscopy. The sulfation method 1 (sulfuric acid:clorossulfonic acid) was the most efficient, yielding 99%, producing a chitosan sulfate (CS) consisting of glucosamine (GlcNH2) and N-acetyl-glucosamine (GlcNAc), 3,6-disulfated, with 14.2 and 85.8% of sulfate content in positions 3 and 6, respectively. Subsequently, low molecular weight chitosans (LMWC) were produced by chitosanolysis with HCI 12M, and separated by centrifugation. The LMWC were subjected to sulfation, producing supernatant sulfate (LMWC-SS), yielding 17%, and precipitate sulfate (LMWC-PS), yielding 67.3%, both composed by 2N- and 3,6-di-O-sulfate. LMWC-SSshowed 12.1, 16.5, 13.6 and 2.3% of sulfate content, respectively, in positions 2, 3, 6 of the deacetylated unit (O) and 6 of the acetylated unit (A). As for LMWC-PS, the percentage of sulfate was 13.8, 13.4 and 15.3% in the positions 2, 3 and 6, respectively. Oespite showing the same sulfation pattern, LMWC-SS and LMWC-PS have different molecular weights, 3.2 and 11.2 kOa, respectively. Both CS and LMWCS were tested for anticoagulant (aPTT, PT and TT) and antithrombotic activity. At a dose of 333.33 IJg/mL in aPTT test, whereas CS prolonged the clotting time of plasma up to 5 times, LMWC-SS and LMWC-PS prolonged 6 and 13 times, respectively. CS showed no activity at the PT test. However, LMWC-SS considerably prolonged the PT clotting time, while LMWCPS at a lower concentration, prolonged the clotting time even more. In TT test, however, only LMWC-PS acted, prolonging thrombin time more than 7 times clotting time of plasma at the dose of 26.7 IJg/mL. Assessing the antithrombotic potential in vivo, LMWC-SS does not reduce thrombus formation and CS has a moderate action. However, LMWC-PS reduced by 77% the formed thrombus, in a concentration of 10mg/kg animal. The identification of the proteins which interacted with CS and LMWCS showed that CS interacted with IgG and IgM, LMWC-SS with IgG and apolipoprotein A-li (ApoA2) and, LMWC-PS interacted with IgG, IgM, ApoA 1, ApoA2 andfibrinogen. Standingthatthe anticoagulant and antithrombotic activities of LMWCS can be both the synergistic action with ApoA 1 and 2 as by direct action on fibrinogen. These results suggest that this difference in anticoagulant mechanism of action should be related to molecular weight, as both LMWCS feature sulfate groups in the same positions.