Mestrado em Bioquímica

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http://repositorio.ufes.br/handle/10/17562

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Navegando Mestrado em Bioquímica por Assunto "615.1"

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    Caracterização bioquímica e estrutural da proteína catecol o-metiltransferase (COMT) como potencial alvo para drogas contra paracoccidioidomicose
    (Universidade Federal do Espírito Santo, 2018-03-28) Cruz, Fabiano Torres; Santos, Alexandre Martins Costa; Gonçalves, Juliana Barbosa Coitinho; Campos, Fabiana Vasconcelos; Costa, Mariana Amália Figueiredo
    Paracoccidioidomycosis (PCM), a systemic mycosis caused by fungi of the genus Paracoccidioides, is endemic in Latin America, with Brazil being the most affected country. Currently, treatment with traditional antifungals is usually long, costly, restricted to a few classes of drugs and with cases of resistance already reported. The catechol O-methyltransferase (COMT) protein, which has no structure solved in Paracoccidioides or other fungi, catalyzes the methylation of a catechol substrate using S-adenosylmethionine and Mg2+ ions as cofactors and appears to participate in important processes in pathogenic fungi. Thus, its biochemical and structural characterization could contribute to the development of new and more effective drugs for the treatment of PCM. To study COMT, its coding sequence was cloned into the pET-28aTEV vector, which adds an N-terminal histidine-tag (His-tag) to the expressed protein. The recombinant vector was then inserted into the strains of Escherichia coli BL21, ArcticExpress, Rosetta and Rosetta-gami. Recombinant COMT was expressed in the soluble fraction of ArcticExpress and it was subjected to nickel affinity chromatography followed by gel filtration (Ultrahydrogel and Superose 12 columns). Western blot with anti-Histag antibody showed the two major bands observed on SDS-PAGE (30 and 60 kDa) of the fraction obtained after affinity. The gel filtration profile showed three main peaks (fractions 1, 2 and 3) which, on SDSPAGE, showed the presence of a prominent band near 60 kDa. Samples from the three fractions were used in an activity assay to evaluate the catechol consumption at 35 ºC and pH 7.5, and fraction 2 showed a higher activity/protein ratio. Theoretical calculations of the masses of gel filtration chromatography peaks indicate that COMT behaves as a dimer. Fraction 2, although not pure, was subjected to thermal denaturation test, where a process of aggregation or denaturation was detected at temperatures above 320 K (~47 ° C). Circular dichroism of fraction 2 indicated an αhelix and β-sheet profile different from other COMT. An in silico model has also been generated and the structural data obtained can contribute as a guide for future biochemical characterization.
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    O extrato etanólico da planta medicinal Combretum leprosum previne déficits cognitivos em um modelo murino de isquemia cerebral global
    (Universidade Federal do Espírito Santo, 2019-05-03) Ferreira, Glenda Gomes; Pires, Rita Gomes Wanderley; Silva, Cristina Martins e; Gonçalves, Juliana Barbosa Coitinho; Rodrigues, Lívia Carla de Melo
    Stroke (AVE) is described as one of the main causes of death according to the World Health Organization (WHO). The AVE is subdivided into ischemic (AVEi) and hemorrhagic (AVEh), the first being more prevalent. The interruption of blood in a particular region of the brain in the AVEi creates two distinct regions in the tissue: the "ischemic nucleus" and the "ischemic penumbra". The cells of the ischemic nucleus are first affected from necrosis due to the direct lack of basic supplies for the cell survival, while cells in the penumbra region die from apoptosis, being this region feasible of intervention. Currently, the main pharmacological therapeutic approach involves the use of Alteplase, a thrombolytic drug that is extremely restricted. Thus, new approaches are needed to therapeutic spectrum, in addition to reducing the side effects. To this end, medicinal plants of the Brazilian flora are a powerful source for the development of medicines for the treatment of stroke. Given the above, our work evaluated the possible effect of the treatment with the ethanolic extract of the medicine plant Combretum leprosum (EECL) in preventing cellular and cognitive damage caused by AVEi in mice. Herein, in the present study, we used male mice of the Mus species musculus and Swiss lineage, 8-10 weeks (CEUA 53/2017 - UFES), which were submitted to a global ischemia / reperfusion (BCCAo) model and treated orally with EECL at the dose of 100mg / kg. The animals were randomly divided into four experimental groups: vehicle sham (saline + tween20) (SV), sham extract (saline + tween 20 + EECL) (SE), vehicle ischemia (IV) and ischemia extract (IE). After anesthesia recovery, the animals received two doses of EECL or vehicle at intervals of 3 hours between doses and this regimen was repeated 24 hours later. Subsequently, the dose became unique until the end of the experiments. The quantification of the infarct area showed a reduction of the ischemic area by 33%. Additionally, our data shows that the treatment decrease the cognitive deficits related to spatial memory and object recognition. The improvement found in the treatment of AVEi is possibly related to the increase of cells immunopositive for the neuronal marker NeuN in the Immunofluorescence assay in animals of the IE group, as well as the decrease of the expression of the marker of activated astrocytes GFAP in the same group. Overall, this report provides promising evidence of the potencial use of EECL in the treatment of stroke.