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Research Interests: Biology, Cell Biology, Medicine, Extracellular Matrix, Mice, and 15 moreAnimals, Astrocytes, Glycosaminoglycan, Glycosaminoglycans, Brazilian, Chondroitin Sulfate, Midbrain, Extracellular, Secretion, Sulfates, Mesencephalon, Heparan Sulfate, Medical and Health Sciences, Sulfation, and Cell culture techniques
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Research Interests: Biochemistry, Microbiology, Biology, Cell Biology, Oxidative Stress, and 14 moreMedicine, Experimental parasitology, Interactome, Ciencias Biológicas, Antioxidant Activity, Peroxiredoxin, Trypanosoma, Trypanosoma Cruzi, Oxidative phosphorylation, Veterinary Sciences, Enseñanza - Aprendizaje Ciencias Naturales Y Exactas, Reactive Oxygen Species (ROS), Mitochondrion, and Atividade antioxidante
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Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from... more
Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from host proteins. Although extensive research has been performed examining hydroperoxide cytosolic antioxidant defense centered on trypanothione, the mechanisms of mitochondrial antioxidant defense are not yet known. The aim of this study was to elucidate the partners of TcMPx antioxidant pathway and to determine the influence of the cellular context (physiological versus oxidative stress). Through co-precipitation coupled with a mass spectrometry approach, a variety of proteins were detected under physiological and oxidative stress conditions. Interestingly, functional category analysis of the proteins identified under physiological conditions showed that they were involved in the stress response, oxidoreduction, thiol transfer, and metabolic processes; this profile is distinct under oxidative stress conditions likely due to structural alterations. Our findings help to elucidate the reactions involving TcMPx and most importantly also reveal that this protein is present throughout the cell and that its interaction partners change following oxidative stress exposure. The involvement and significance of the proteins found to interact with TcMPx and other possible functions for this protein are discussed widening our knowledge regarding T. cruzi mitochondrial antioxidant defenses.
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Research Interests: Mass Spectrometry, Biology, Medicine, Mice, Animals, and 12 moreVenom, Gel Permeation Chromatography, High Performance Liquid Chromatography, High Pressure Liquid Chromatography, Snake Venom, Thrombosis, Species Specificity, Amino Acid Sequence, Base Sequence, Toxicon, Molecular Sequence Data, and Pharmacology and pharmaceutical sciences
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Fibrilina-1 (FBN-1) e um importante componente da rede de microfibrilas da matriz extracelular (MEC). As microfibrilas estao presentes nas fibras elasticas que sao responsaveis pela resiliencia de tecidos como pulmoes, pele e grandes... more
Fibrilina-1 (FBN-1) e um importante componente da rede de microfibrilas da matriz extracelular (MEC). As microfibrilas estao presentes nas fibras elasticas que sao responsaveis pela resiliencia de tecidos como pulmoes, pele e grandes vasos. Mutacoes no gene da fibrilina-1 estao associadas a Sindrome de Marfan, doenca autossomica dominante, caracterizada por uma desordem do tecido conjuntivo. Pacientes com esta Sindrome apresentam anomalias no sistema esqueletico e trato cardiovascular. Dados da literatura relacionam a menor quantidade de FBN-1 na MEC com a atividade exacerbada do TGF-? promovendo a quase totalidade das alteracoes fenotipicas encontradas. Estudos preliminares em nosso laboratorio com camundongos que possuem menor quantidade de FBN-1 de um camundongo normal tem demonstrado que necessitam do dobro do tempo para a formacao de trombo em um modelo de trombose arterial. As plaquetas tem fundamental importância neste processo, quando sao ativadas secretam varias moleculas que determinam a formacao dos trombos. Realizamos um estudo comparativo do proteoma de plaquetas e da arteria aorta de camundongos selvagens e deficientes em FBN-1, atraves da tecnica de eletroforese bidimensional (2DE) juntamente com a espectrometria de massas a fim de encontrar diferencas no perfil proteico que justificassem tais sintomas. Diversas proteinas plaquetarias foram encontradas apenas no grupo controle, como endoplasmina, fator de von Willebrand, calpaina, dentre outras; assim como a proteina vinculina foi encontrada apenas no grupo deficiente em FBN-1. Todas as proteinas encontradas podem ser de grande interesse para o esclarecimento a respeito do maior tempo de formacao de trombo e os sintomas relacionados a Sindrome de Marfan. Abstract
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As glicoproteínas das microfibrilas 1, MAGP-1 e a Fibrilina-1, são importantes constituintes da rede de microfibrilas que compõem as fibras elásticas, juntamente com a elastina, que proporcionam força e elasticidade para os tecidos. Esses... more
As glicoproteínas das microfibrilas 1, MAGP-1 e a Fibrilina-1, são importantes constituintes da rede de microfibrilas que compõem as fibras elásticas, juntamente com a elastina, que proporcionam força e elasticidade para os tecidos. Esses componentes são abundantes na matriz extracelular de tecidos que estão sob constante estresse mecânico. Além de funcionar como um molde para a deposição de tropoelastina, formando a fibra elástica madura, as microfibrilas atuam na sinalização e interação das células com o meio extracelular. Uma das principais funções das microfibrilas é o controle da disponibilidade de fatores de crescimento, em especial da família TGF-β. Dados do nosso laboratório demonstram que a deficiência de MAGP-1 ou de Fibrilina-1 levam a uma alteração da formação do trombo. Esta alteração desaparece quando os animais são tratados com losartan, anti-hipertensivo que está associado com a diminuição da atividade de TGF-β. Dados com fibroblastos de derme de camundongos deficien...
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Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from... more
Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from host proteins. Although extensive research has been performed examining hydroperoxide cytosolic antioxidant defense centered on trypanothione, the mechanisms of mitochondrial antioxidant defense are not yet known. The aim of this study was to elucidate the partners of TcMPx antioxidant pathway and to determine the influence of the cellular context (physiological versus oxidative stress). Through co-precipitation coupled with a mass spectrometry approach, a variety of proteins were detected under physiological and oxidative stress conditions. Interestingly, functional category analysis of the proteins identified under physiological conditions showed that they were involved in the stress response, oxidoreduction, thiol transfer, and metabolic processes; this profile is distinct under oxidative stress conditions likely due to structural alterations. Our findings help to elucidate the reactions involving TcMPx and most importantly also reveal that this protein is present throughout the cell and that its interaction partners change following oxidative stress exposure. The involvement and significance of the proteins found to interact with TcMPx and other possible functions for this protein are discussed widening our knowledge regarding T. cruzi mitochondrial antioxidant defenses.
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Research Interests: Biochemistry, Chemistry, Biology, Glycobiology, Medicine, and 15 moreBiological Sciences, Animals, Glycosaminoglycan, Sea Urchins, Marine invertebrates, High Pressure Liquid Chromatography, Sea Urchin, Charge Density, Larval Development, Embryos, Marine Invertebrate, Blastula, Strongylocentrotus purpuratus, Dermatan sulfate, and Medical and Health Sciences
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Background Plasma free hemoglobin (PFHb) can be elevated by hemolysis in several diseases, including malaria, sepsis and hemolytic anemias such as sickle cell disease (SCD). When PFHb is not neutralized, it can trigger vascular and organ... more
Background Plasma free hemoglobin (PFHb) can be elevated by hemolysis in several diseases, including malaria, sepsis and hemolytic anemias such as sickle cell disease (SCD). When PFHb is not neutralized, it can trigger vascular and organ dysfunction, leading to adverse clinical effects. As an example, increased PFHb in SCD is proposed to reduce nitric oxide (NO) bioavailability and induce vascular oxidative stress and inflammation. Vaso-occlusive (VO) processes in SCD may be triggered by the adhesion of leukocytes (WBC), and subsequently red blood cells, to the vessel wall. Hydroxyurea (HU), used as a fetal Hb-inducing drug in SCD, may also exert some of its effects by acting as an NO donor. We induced hemolytic processes in mice to observe WBC recruitment and inflammatory processes; the ability of HU to affect such processes was also investigated. Methods Hemolysis was induced in wild-type C57BL/6 mice (2-4 months old) by injecting water (H2O). For intravital microscopy (IVM), crem...
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Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite... more
Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with d...
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Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We... more
Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in β2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in...
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Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the... more
Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment w...
MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed,... more
MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-β in the arterial thrombosis process. We analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity. Our results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation.
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Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells... more
Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.
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Hemolysis, and consequent cell-free (CF) hemoglobin (Hb) release, impairs vascular nitric oxide (NO) bioavailability and causes oxidative and inflammatory processes. Hydroxyurea, commonly used as a therapy in sickle cell disease (SCD),... more
Hemolysis, and consequent cell-free (CF) hemoglobin (Hb) release, impairs vascular nitric oxide (NO) bioavailability and causes oxidative and inflammatory processes. Hydroxyurea, commonly used as a therapy in sickle cell disease (SCD), induces fetal Hb production, and can act as a NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice, and determined the abilities of a NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 min, accompanied by NO metabolite generation. The administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, ...
Research Interests: Sickle Cell Anemia, Inflammation, Transgenic Mice, Humans, Mice, and 15 moreNitric oxide, Animals, Blood, Viscosity, Male, Clinical Sciences, Primary Cell Culture, Hydroxyurea, Imidazoles, Leukocytes, Hemoglobins, Hemolysis, Nitric oxide donors, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in... more
Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.
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The endothelium plays a crucial role in regulating vascular tone, inflammatory responses, thrombosis, and atherosclerosis. Part of these activities is regulated by the presence of glycosaminoglycans (GAGs) in the vessel wall and in the... more
The endothelium plays a crucial role in regulating vascular tone, inflammatory responses, thrombosis, and atherosclerosis. Part of these activities is regulated by the presence of glycosaminoglycans (GAGs) in the vessel wall and in the extracellular matrix (ECM) surrounding it. GAGs in atherosclerosis can help regulate atherogenesis through their ability to retain lipoproteins in the vessel wall. Prolonged retention of lipoproteins
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We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single... more
We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The pro...
Research Interests: Kinetics, Snake venoms, Fibrinogen, Platelet aggregation, Mice, and 13 moreAnimals, Viperidae, Male, Blood Coagulation, Biomed, High Pressure Liquid Chromatography, Cattle, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Serine Proteases, Molecular weight, Amino Acid Sequence, Molecular Sequence Data, and Blood platelets
SummaryShedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation... more
SummaryShedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte- derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that...