Mestrado em Bioquímica
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- ItemCaracterização bioquímica e farmacológica da Sp-CTx- Toxina Multifuncional da Peçonha do Peixe Escorpião Scorpaena Plumieri(Universidade Federal do Espírito Santo, 2017-03-30) Malacarne, Pedro Felipe; Figueiredo, Suely Gomes de; Pires, Rita Gomes Wanderley; Guimarães, Marco Cesar Cunegundes; Trindade, Juliana Bambrilla CarnielliCytolysins are substances elaborated by many different organisms that exhibit toxic activities on cells, causing its dissolution through cytolysis. Fish venom cytolysins are multifunctional proteins that display neurotoxic, cardiotoxic and inflammatory effects, described as "protein lethal factors". A pore-forming labile hemolytic cytolysin called Sp-CTx (Scorpaena plumieri - Cytolytic Toxin) (glycoprotein, 2 subunits of 65 kDa) was recently purified from the venom of the scorpionfish Scorpaena plumieri. The aim of this work was to establish optimal storage conditions to maintain the activities of this toxin and to continue its biochemical and pharmacological characterization. For this, SpV (Scorpaena plumieri Venom) and Sp-CTx were stored in different conditions (pH, temperature and in the presence of co-solutes), for different periods of time. Afterwards, their hemolytic activity on rabbit erythrocytes was compared to the activity of the freshly extracted venom and purified toxin. We also evaluated the effects of different cations and membrane lipids on the hemolytic activity of Sp-CTx. It was demonstrated that storage of Sp-CTx at -4 ºC in pH 7,4 with 500mM NaCl, 1M (NH4)2SO4, 60mM of threalose and 10% glycerol, and at -80 and -196 ºC with glycerol 10% were effective in maintaining the hemolytic activity. Furthermore, the hemolytic activity is calcium dependent and completely inhibited by EDTA. Cholesterol and phosphatidylglycerol reduced the hemolytic activity of Sp-CTx which suggests that it is a barrel-pore-forming toxin and that the pore formation is dependent of electrostatic interactions with membrane lipids. Through western blot, using the “DIG Glycan Differentiation” kit, sugar motifs of Sp-CTx were evaluated, which suggested the presence of: galactose (1-4) N-acetylglucosamine and sialic-acid (2-3) galactose in N- and/or O- glycan complexes. Using the mouse hindpaw test it was shown that Sp-CTx induced intensive nociception and dose-dependent edematogenic responses in the mice footpad Sp-CTx. The local induced edema was considerably reduced by previous administration of aprotinin and HOE-140. These data suggest the involvement of the kinin– kallikrein system in the inflammatory response induced by Sp-CTx.
- ItemEstudo da citotoxicidade do ácido rosmarínico em modelos in vitro de câncer humano(Universidade Federal do Espírito Santo, 2017-09-29) Souza, Marcele Lorentz Mattos de; Santos, Adair Roberto Soares dos; Rangel, Letícia Batista Azevedo; Silva, Cristina Martins e; Silva, Ian VictorCancer is one of the leading causes of death worldwide. Every year 8.2 million people die from the disease. Among the most incident tumors in the population are lung cancer and breast cancer. According to the World Health Organization (WHO), the number of new cancers will continue to rise despite the huge investment in fighting the disease. Added to that, the chemotherapeutic regimen of first choice for the treatment of these diseases has as limitation the high systemic toxicity and both the innate and acquired chemoresistance of the tumor cells. Therefore, various natural substances with potential antitumor activity have been studied, among which rosmarinic acid (RA). RA is a naturally occurring phenolic compound. It is an ester derived from the caffeic and 3,4- dihydroxyphenylacetic acids found in various plant species, being originally isolated in 1958 from the rosemary plant (Rosmarinus officinalis). RA modulates the immune system, has action antimicrobial, neuroprotective, inhibits cell proliferation and induces apoptosis (PETERSEN; SIMMONDS, 2003). Motivated by the diverse activities and benefits of RA, we performed a screening in a panel of lung lines (H460/ A549), ovarian (SKOV-3, A2780, OVCAR3) and breast (MCF-7/ MDAMB-231). The results verified the cytotoxic efficacy of RA in triple negative breast cancer MDAMB-231 and luminal A MCF-7 with a reduction of more than 80% in cellular viability in monotherapy and increased sensitivity to conventional chemotherapeutic with drastic reduction of IC50 in combination therapy with paclitaxel and cisplatin. The in vitro cytotoxicity data obtained so far suggest that RA may be a promising chemotherapeutic agent in the fight against cancer, especially, breast cancer.
- ItemEstudo dos efeitos antineoplásicos da atorvastatina em monoterapia e em terapia combinada com quimioterápicos convencionais em modelos in vitro de câncer de mama(Universidade Federal do Espírito Santo, 2017-10-10) Santos, Diandra Zipinotti dos; Rangel, Letícia Batista Azevedo; Bem, Daniela Amorim Melgaço G. do; Daltoé, Renata DalmaschioBreast cancer is the second most common kind of cancer in the world and the most common among women. Its frequency rate is becoming alarming, being this way a major challenge to global health. It is a complex and heterogeneous disease, composed of multiple subgroups with different biological and morphological characteristics and it shows different clinical manifestations and patterns of response to existing therapies. The metabolic reprogramming has been increasingly recognized as a characteristic of cancer cells. These changes may affect the availability of structural lipids for membrane synthesis, lipid synthesis and degradation that contribute to energetic homeostasis and cellular signaling functions. According to what has been mentioned above and due to the reduced effectiveness of chemotherapy currently in use for certain subgroups of cancer, substances that interfere with lipid metabolism, such as statins, appear as promising auxiliary strategies in the fight against cancer. In this study, atorvastatin alone and in combination with other chemotherapeutics had been evaluated in breast cancer cell lines. The results have shown that the tested drug have antitumor activity on all lineages of breast cancer, being the most effective inhibitor of MDA-MB-231 breast cancer cell growth. In addition, the antineoplastic effect of atorvastatin appears to be associated with G1 phase cell cycle arrest, as well as autophagy induction Moreover, atorvastatin altered the effectiveness of drugs used in conventional clinic, but most often at higher concentrations than those considered safe and used in the clinic.
- ItemModulação do sistema colinérgico pelo sistema endocanabinoide em tarefas apreendidas e reapreendidas : efeito do agonista canabinoide WIN-2(Universidade Federal do Espírito Santo, 2016-05-11) Ferreira, Tamara Andrea Alarcón; Silva, Cristina Martins e; Pires, Rita Gomes Wanderley; Ribeiro, Angela Maria; Harres, Vanessa BeijaminiThe Cannabis sativa plant, commonly known as marijuana, contains compounds named cannabinoids, and the 9-tetrahydrocannabinol (9 - THC) is the most active component. After cloning and characterization of cannabinoid receptors, it was found that they bind to 9 - THC and to endogenous ligands, named endocannabinoids (ECs), capable of modulating multiple neurotransmitter systems, and emerging as important regulators of various physiological brain functions. The effect of endogenous and exogenous cannabinoids in the processes of acquisition, consolidation and recovery of information, both in learning and memory is still controversial. Moreover, there is evidence that the processes of acquisition and consolidation have distinct biological basis with probable involvement of hippocampal and cortical cholinergic system in these differences. In order to understand the molecular basis of cognitive processes that involve the participation of the central cholinergic system by the activation of cannabinoid receptors, we proposed the use of a cannabinoid agonist, WIN 55.212-2 (WIN-2). For this, Swiss mice were treated with WIN-2 at a dose of 2 mg/kg and submitted to testing in the aquatic Morris maze to evaluate aspects of acquisition and consolidation of the task. We observed that the cognitive impairment caused by chronic treatment with WIN-2 is mainly related to short term memory in acquisition of spatial task process, while consolidating remained unchanged. This cognitive impairment in the acquisition may be related to a possible increase in the concentration of 2-AG (2-arachidonylglycerol) and decreased of AEA (anandamide) in the prefrontal cortex. Although the behavioral changes observed in this study are subtle, we verified modulation of cholinergic by endocannabinoid system, since treatment with WIN-2 in the memory consolidation period resulted in a decreased basal release of acetylcholine (ACh) in hippocampus. However, this decrease was not associated with cognitive deficits observed. Therefore, this study confirms the importance of both systems studied in the modulation of cognitive processes and hopefully, in the future, help in developing treatments with pharmacological and non-pharmacological approaches, to seek reduce the cognitive deficits caused by drug abuse, as well as neurodegenerative diseases such as Alzheimer's disease.