929 resultados para Matriz extracelular Teses
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El cncer se ha considerado una enfermedad definida y dirigida por la inestabilidad genmica, las alteraciones cromosmicas y las mutaciones genticas. Sin embargo, en la actualidad, la influencia de las clulas estromales no malignas del microambiente tumoral est claramente establecida. Los tumores son tejidos complejos, compuestos no slo por las clulas malignas, sino tambin, por clulas estromales genticamente estables, incluyendo a fibroblastos y macrfagos adems de la matriz extracelular que producen. Al igual que en rganos sanos, estos compartimentos del microambiente no son meros espectadores, sino que regulan crticamente la iniciacin tumoral, la progresin maligna y la metstasis. Ms an, los diferentes tipos estromales en diferentes contextos pueden exhibir capacidades promotoras u opuestas al tumor.
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A Diabetes Mellitus (DM) compreende um conjunto de desordens metablicas comuns caracterizadas por hiperglicemia, que afeta diferentes rgos do organismo. Ao longo do tempo, ocorrem danos microvasculares no glomrulo renal, retina e nervos perifricos, bem como doena macrovascular nas artrias. A composio da saliva tambm afetada pela DM, com consequncias na homeostasia oral. No entanto, o proteoma e o peptidoma salivar tm sido pouco explorados na DM tipo 1 e nas suas complicaes crnicas. Tendo em conta o crescente interesse na saliva como fluido diagnstico, o objetivo principal deste trabalho foi avaliar os eventos proteolticos subjacentes DM tipo 1 e s suas complicaes microvasculares, bem como, caracterizar as alteraes induzidas pela DM tipo 1 no proteoma e peptidoma salivar. A DM tipo 1 e particularmente as complicaes microvasculares associadas modulam o perfil proteoltico dos fluidos biolgicos, com diferenas significativas de atividade observadas na urina e saliva, atribudas principalmente ao complexo Metaloproteinase da Matriz (MMP)-9/lipocalina associada gelatinase de neutrfilos, aminopeptidase N, azurocidina e calicrena 1. O aumento da atividade proteoltica observado na saliva total dos diabticos resultou no aumento da percentagem de pptidos, principalmente de um nmero acrescido de fragmentos de colagnio do tipo I, refletindo possivelmente um estado inflamatrio crnico dos tecidos orais e periodontais. O peptidoma tambm corrobora uma maior suscetibilidade das protenas salivares, especificamente, das protenas ricas em prolina bsicas (bPRP) 1, bPRP2 e protenas ricas em prolina cidas (aPRP) protelise, evidenciando a gerao de fragmentos de protenas associadas ligao a bactrias. A anlise do proteoma salivar baseada em iTRAQ mostrou uma sobre-expresso de L-plastina, fator do adenocarcinoma do pncreas e das protenas S100-A8 e S100-A9, enfatizando a importncia do sistema imune inato na patognese da DM tipo 1 e das complicaes microvasculares associadas. A anlise integrada de todas as protenas expressas diferencialmente entre os pacientes diabticos com ou sem complicaes microvasculares e indivduos saudveis foi realizada com o STRING, onde se observam trs conjuntos funcionalmente ligados, um compreende a interao entre o colagnio tipo I, colagnio tipo II e MMP-9, um segundo conjunto envolve a MMP-2 e o colagnio de tipo I e um terceiro conjunto composto por protenas salivares e inflamatrias. Estes conjuntos esto associados com as vias Kegg de interao recetor-matriz extracelular, de adeso focal e migrao transendotelial dos leuccitos. Por outro lado, a anlise do proteoma e peptidoma salivar destacou potenciais biomarcadores para o diagnstico e prognstico da DM tipo 1 e das suas complicaes.
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Dissertao de Mestrado, Biologia Marinha, Especializao em Biotecnologia Marinha, Faculdade de Cincias do Mar e do Ambiente, Universidade do Algarve, 2008
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The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.
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Gilthead seabream is the most important farmed species in the Mediterranean, and knowledge on how common farming practices impact its quality is limited. As such, this Thesis aimed to evaluate how gilthead seabream flesh quality is affected by some of these practices. In Chapter 2, the influence of nutritional factors was evaluated, specifically the high replacement of traditional marine-derived ingredients, both fishmeal and fish oil, with vegetable sources. We have seen that the vegetable-based diets tested did not greatly impact seabream flesh quality, although some alterations were seen in the fatty acid profile of the muscle. However, and despite having caused no alterations in flesh texture, vegetable ingredients reduced the amount of sulphated glycosaminoglycans in the extracellular matrix, affected muscle pH and reduced the activity of proteolytic enzymes. Throughout this Thesis, we measured for the first time the activity of proteolytic enzymes in seabream muscle, and cathepsin B was found to play a pivotal role in post-mortem muscle degradation. In Chapter 3, we evaluated the effect of harvesting and slaughter stress on seabream quality, and contrary to what is seen in most farmed species, our results show that gilthead seabream muscle structure is highly resistant to changes caused by stressful events. Nonetheless, considering that welfare is an increasingly important quality criterion, the use of a zero-withdrawal anaesthetic as a rested harvest technique or even slaughter method could prove valuable to the industry. In Chapter 4, we used maslinic acid as a dietary supplement, to modulate the muscles energetic status pre-mortem. As a finishing strategy, maslinic acid failed to increase levels of glycogen and ATP in the muscle. However, supplementation resulted in higher muscle fibre diameter and lower cathepsin B activity, and maslinic acid is likely to be useful to promote growth in this species. In general our Thesis has generated new knowledge to a major challenge facing the aquaculture industry, which is to find a compromise between the trends towards intensive rearing and consumer demand for healthy, high quality seafood being ethically acceptable and having a low impact on the environment.
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Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor (TGF) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.
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The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.
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RESUMO: Actualmente, a nica possibilidade de cura para doentes com adenocarcinoma do pncreas (PDAC) a resseco cirrgica, no incio deste estudo, perguntamo-nos se os predictores clnico-patolgicos clssicos de prognostico poderiam ser validados em uma grande cohort de doentes com cancro do pncreas ressecvel e se outros predictores clnicos poderiam ter um papel na deciso de que doentes beneficiariam de resseco cirrgica. No captulo 2, observamos que at 30% dos doentes morrem no primeiro ano aps a resseco cirrgica, pelo que o nosso objectivo foi determinar factores pr-operatrios que se correlacionam com mortalidade precoce aps ressecao cirrgica com recurso a um instrumento estatisticamente validado, o Charlson-Age Comorbidity Index (CACI), determinamos que um CACI score superior a 4 foi preditivo de internamentos prolongados (p <0,001), complicaes ps-operatrias (p = 0,042), e mortalidade em 1 ano ps- resseco cirrgica (p <0,001). Um CACI superior a 6 triplicou a mortalidade no primeiro ano ps-cirurgia e estes doentes tm menos de 50% de probabilidade de estarem vivos um ano aps a cirurgia. No captulo 3, o nosso objectivo foi identificar uma protena de superfcie que se correlacionasse estatisticamente com o prognostico de doentes com adenocarcinoma do pncreas e permitisse a distino de subgrupos de doentes de acordo com as suas diferenas moleculares, perguntamo-nos ainda se essa protena poderia ser um marcador de clulas-estaminais. No nosso trabalho anterior observamos que as clulas tumorais na circulao sangunea apresentavam genes com caractersticas bifenotpica epitelial e mesenquimal, enriquecimento para genes de clulas estaminais (ALDH1A1 / ALDH1A2 e KLF4), e uma super-expresso de genes da matriz extracelular (colagnios, SPARC, e DCN) normalmente identificados no estroma de PDAC. Aps a avaliao dos tumores primrios com RNA-ISH, muitos dos genes identificados, foram encontrados co-localizando em uma sub-populao de clulas na regio basal dos ductos pancreticos malignos. Alm disso, observamos que estas clulas expressam o marcador SV2A neuroendcrino, e o marcador de clulas estaminais ALDH1A1/2. Em comparao com tumores negativos para SV2, os doentes com tumores SV2 positivos apresentaram nveis mais baixos de CA 19-9 (69% vs. 52%, p = 0,012), tumores maiores (> 4 cm, 23% vs. 10%, p = 0,0430), menor invaso de gnglios linfticos (69% vs. 86%, p = 0,005) e tumores mais diferenciados (69% vs. 57%, p = 0,047). A presena de SV2A foi associada com uma sobrevida livre de doena mais longa (HR: 0,49 p = 0,009) bem como melhor sobrevida global (HR: 0,54 p = 0,018). Em conjunto, esta informao aponta para dois subtipos diferentes de adenocarcinoma do pncreas, e estes subtipos co-relacionam estatisticamente com o prognostico de doentes, sendo este subgrupo definido pela presena do clone celular SV2A / ALDH1A1/2 positivo com caractersticas neuroendcrinas. No Captulo 4, a expresso de SV2A no cancro do pncreas foi validado em linhas celulares primrias. Demonstramos a heterogeneidade do adenocarcinoma do pncreas de acordo com caractersticas clonais neuroendcrinas. Ao comparar as linhas celulares expressando SV2 com linhas celulares negativas, verificamos que as linhas celulares SV2+ eram mais diferenciadas, diferindo de linhas celulares SV2 negativas no que respeita a mutao KRAS, proliferao e a resposta quimioterapia. No captulo 5, perguntamo-nos se o clone celular SV2 positivo poderia explicar a resistncia a quimioterapia observada em doentes. Observamos um aumento absoluto de clones celulares expressando SV2A, em mltiplas linhas de evidncia - doentes, linhas de clulas primrias e xenotransplantes. Embora, tenhamos sido capazes de demonstrar que o adenocarcinoma do pncreas uma doena heterognea, consideramos que a caracterizao gentica destes clones celulares expressando SV2A de elevada importncia. Pretendemos colmatar esta limitao com as seguintes estratgias: Aps o tratamento com quimioterapia neoadjuvante na nossa coorte, realizamos microdissecao a laser das amostras primarias em parafina, de forma a analisar mutaes genticas observadas no adenocarcinoma pancretico; em segundo lugar, pretendemos determinar consequncias de knockdown da expresso de SV2A em nossas linhas celulares seguindo-se o tratamento com gemicitabina para determinao do papel funcional de SV2A; finalmente, uma vez que os nossos esforos anteriores com um promotor - reprter e SmartFlare falharam, o prximo passo ser realizar RNA-ISH PrimeFlow seguido de FACS e RNA-seq para caracterizao deste clone celular. Em conjunto, conseguimos provar com vrias linhas de evidncia, que o adenocarcinoma pancretico uma doena heterognea, definido por um clone de clulas que expressam SV2A, com caractersticas neuroendcrinas. A presena deste clone no tecido de doentes correlaciona-se estatisticamente com o prognostico da doena, incluindo sobrevida livre de doena e sobrevida global. Juntamente com padres de proliferao e co-expresso de ALDH1A1/2, este clone parece apresentar um comportamento de clulas estaminais e est associado a resistncia a quimioterapia, uma vez que a sua expresso aumenta aps agresso qumica, quer em doentes, quer em linhas de clulas primrias.----------------------------- ABSTRACT: Currently, the only chance of cure for patients with pancreatic adenocarcinoma is surgical resection, at the beginning of my thesis studies, we asked if the classical clinicopathologic predictors of outcome could be validated in a large cohort of patients with early stage pancreatic cancer and if other clinical predictors could have a role on deciding which patients would benefit from surgery. In chapter 2, we found that up to 30% of patients die within the first year after curative intent surgery for pancreatic adenocarcinoma. We aimed at determining pre-operative factors that would correlate with early mortality following resection for pancreatic cancer using a statistically validated tool, the Charlson-Age Comorbidity Index (CACI). We found that a CACI score greater than 4 was predictive of increased length of stay (p<0.001), post-operative complications (p=0.042), and mortality within 1-year of pancreatic resection (p<0.001). A CACI score of 6 or greater increased 3-fold the odds of death within the first year. Patients with a high CACI score have less than 50% likelihood of being alive 1 year after surgery. In chapter 3 we aimed at identifying a surface protein that correlates with patients outcome and distinguishes sub-groups of patients according to their molecular differences and if this protein could be a cancer stem cell marker. The most abundant class of circulating tumor cells identified in our previous work was found to have biphenotypic features of epithelial to mesenchymal transition, enrichment for stem-cell associated genes (ALDH1A1/ALDH1A2 and KLF4), and an overexpression of extracellular matrix genes (Collagens, SPARC, and DCN) normally found in the stromal microenvironment of PDAC primary tumors. Upon evaluation of matched primary tumors with RNA-ISH, many of the genes identified were found to co-localize in a sub-population of cells at the basal region of malignant pancreatic ducts. In addition, these cells expressed the neuroendocrine marker SV2A, and the stem cell marker ALDH1A1/2. Compared to SV2 negative tumors, patients with SV2 positive tumors were more likely to present with lower CA 19-9 (69% vs. 52%, p = 0.012), bigger tumors (size > 4 cm, 23% vs. 10%, p= 0.0430), less nodal involvement (69% vs. 86%, p = 0.005) and lower histologic grade (69% vs. 57%, p = 0.047). The presence of SV2A expressing cells was associated with an improved disease free survival (HR: 0.49 p=0.009) and overall survival (HR: 0.54 p=0.018) and correlated linearly with ALDH1A2. Together, this information points to two different sub-types of pancreatic adenocarcinoma, and these sub-types correlated with patients outcome and were defined by the presence of a SV2A/ ALDH1A1/2 expressing clone with neuroendocrine features. In Chapter 4, SV2A expression in cancer was validated in primary cell lines. We were able to demonstrate pancreatic adenocarcinoma heterogeneity according to neuroendocrine clonal features. When comparing SV2 expressing cell lines with SV2 negative cell lines, we found that SV2+ cell lines were more differentiated and differ from SV2 negative cell lines regarding KRAS mutation, proliferation and response to chemotherapy. In Chapter 5 we aimed at determining if this SV2 positive clone could explain chemoresistance observed in patients. We found an absolute increase in SV2A expressing cells, with multiple lines of evidence, in patients, primary cell lines and xenografts. Although, we have been able to show evidence that pancreatic adenocarcinoma is a heterogeneous disease, our findings warrant further investigation. To further characterize SV2A expressing clones after treatment with neoadjuvant chemotherapy in our cohort, we have performed laser capture microdissection of the paraffin embedded tissue in this study and will analyze the tissue for known genetic mutations in pancreatic adenocarcinoma; secondly, we want to know what will happen after knocking down SV2A expression in our cell lines followed by treatment with gemcitabine to determine if SV2A is functionally important; finally, since our previous efforts with a promoter reporter and SmartFlare have failed, we will utilize a novel PrimeFlow RNA-ISH assay followed by FACS and RNA sequencing to further characterize this cellular clone. Overall our data proves, with multiple lines of evidence, that pancreatic adenocarcinoma is a heterogeneous disease, defined by a clone of SV2A expressing cells, with neuroendocrine features. The presence of this clone in patients tissue correlates with patients disease free survival and overall survival. Together with patterns of proliferation and ALDH1A1/2 co-expression, this clone seems to present a stem-cell-like behavior and is associated with chemoresistance, since it increases after chemotherapy, both in patients and primary cell lines.
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A hematopoiese representa uma cascata de eventos de proliferao e diferenciao celular precisamente regulada, onde uma populao de clulas tronco pluripotentes indiferenciadas origina todas as clulas sangneas. Durante o perodo embrionrio o principal rgo hematopoitico o fgado. A partir do desenvolvimento dos ossos longos, a hematopoiese deslocada para a medula ssea, sendo este, na vida adulta, o stio de produo das clulas sangneas. O microambiente da medula ssea, composto pelas clulas estromais, componentes de matriz extracelular e fatores de crescimento ou citocinas, desempenha importncia fundamental na proliferao e diferenciao das clulas progenitoras hematopoiticas. Em algumas condies patolgicas, na vida adulta, a hematopoiese pode ser observada em stios extramedulares, especialmente no fgado, que demonstra assim preservar um potencial hematopoitico. Este fenmeno descrito como hematopoiese extramedular e pode estar associado a reaes fibrogranulomatosas, como a esquistossomose mansnica. No presente estudo avaliou-se a hiptese de que os gangliosdios possam participar do microambiente carregado negativamente necessrio para o suporte da hematopoiese. Para isso, analisou-se o contedo, sntese e liberao (shedding) de gangliosdios de dois estromas extramedulares, GRWT e GR(IFN-Ro/o), que expressam GM-CSF de maneira semelhante, mas tm capacidades diferentes de suporte da mielopoiese in vitro. A capacidade de suporte da hematopoiese pelos dois estromas foi monitorada atravs da proliferao das clulas FDC-P1, uma linhagem precursora mielide. Observamos que os dois estromas sintetizam e liberam os mesmos gangliosdios, embora em propores diferentes. Tambm verificamos que a inibio da sntese de gangliosdios diminui a proliferao mielopoitica em ambos os estromas extramedulares.
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Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. They are extracted mainly from the extracellular matrix of brown algae and echinoderms. The brown alga Spatoglossum schrederi (Dictyotaceae) has three heterofucans named A, B and C. Our research group have been extracted non anticoagulant heterofucan from S. schrederi which possess antithrombotic activity in vivo. However, their toxicity in vitro and in vivo has not yet been determined. For the results in toxicity in vitro, we observed that the fucan A at 20, 500 and 1000 μg/plate showed no mutagenic activity in Kado test (Microsuspension), when the bacterial strains TA97a, TA98, TA100 and TA102, with and without S9 were used. The comet assay showed that fucan A (from 20 to 1000 μg/mL) did not cause any genotoxic effect on CHO cells. There was no damage to the DNA of these cells, as evidenced by the tail length and tail moment, which were similar to that found for the negative control. The fucan A from S. schrederi was administered at 20 μg/g of rat (dose which it showed high antithrombotic activity) during two months. After that, the animals were killed and examined. The data showed that fucan A did not cause any change in biochemistry and hematological parameters, as well as, in the morphology and size of the rat s organs analyzed. In conclusion, this study indicates that fucan is a compound with potential pharmacological that has no toxicity
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Marine algae are one of the major sources of biologic compounds. In extracellular matrix of these organisms there are sulfated polysaccharides that functions as structural components and provides protection against dehydration. The fraction 1.0 (F1.0) rich in sulfated galactans obtained from red seaweed Hypnea musciformis was physicochemical characterized and evaluated for pharmacologic activity through antioxidant activity, cytotoxic action on erythrocytes, anticoagulant, stimulatory action under antithrombotic heparan sulfate synthesis and their effects on cell proliferation and cycle cell progression. The main components of F1.0 were carbohydrates (49.70 0.10%) and sulfate (44.59 0.015%), presenting phenolic compounds (4.79 0.016%) and low protein contamination (0.92 0.001%). Fraction 1.0 showed polidisperse profile and signs in infrared analysis in 1262, 1074 and 930, 900 and 850 attributed to sulfate esters S=O bond, presence of a 3,6- anidrogalactose C-O bond, non-sulfated -D-galactose and a C-O-SO4 bond in galactose C4, respectively. The fraction rich in sulfated galactans exhibited strong antioxidant action under lipid peroxidation assay with IC50 of 0.003 mg/mL. Besides the inhibition of hemolysis induced by H2O2 in erythrocytes treated with F1.0, this fraction did not promote significant cytotoxity under erythrocytes membranes. F1.0 exhibited low anticoagulant activity causing moderate direct inhibition of enzimatic activity of thrombin. This fraction promoted stimulation around of 4.6 times on this synthesis of heparan sulfate (HS) by rabbit aortic endothelial cells (RAEC) in culture when was compared with non treated cells. The fraction of this algae displayed antiproliferative action under RAEC cells causing incresing on cell number on S fase, blocking the cycle cell progression. Thus F1.0 presented cytostatic and no cytotoxic action under this cell lineage. These results suggest that F1.0 from H. musciformis have antioxidant potential which is a great effect for a compound used as food and in food industry which could be an alternative to food industry to prevent quality decay of lipid containing food due to lipid peroxidation. These polysaccharides prevent the lipid peroxidation once the fraction in study exhibited strong inhibitory action of this process. Furthermore that F1.0 present strong antithrombotic action promoting the stimulation of antithrombotic HS synthesis by endothelial cells, being important for thrombosis preventing, by its inhibitory action under reactive oxygen species (ROS) in some in vitro methods, being involved in promotion of hypercoagulability state.
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Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 0.10 %) and sulfate (42.30 0.015 %), with phenolic compounds ( 3.86 0.016 %) and low protein contamination ( 0.58 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state
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Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP)
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Ameloblastoma and adenomatoid odontogenic tumor are odontogenic tumors arising from the odontogenic epithelium with distinct clinical behavior. In attempt to comprehend the interaction between the odontogenic tumor cells and the extracellular matrix, the present work evaluated and compared the immunohistochemical expression of the matrix metalloproteinases-1 (MMP-1), -2 (MMP-2) and -9 (MMP-9) in 20 cases of ameloblastoma and 10 adenomatoid odontogenic tumor. MMP-1 exhibited exuberant expression in the parenchyma and in the stroma of both studied tumors, while the MMP-2 showed varied expression with about of 80% and 60% of the neoplastic cells exhibiting positivity in the ameloblastoma and adenomatoid odontogenic tumor, respectively. With relation to the MMP-2 expression by the mesenchymal cells, it was observed that 65% of the ameloblastoma and 80% of the adenomatoid odontogenic tumor were positive. The immunoreactivity of MMP-9 was detected in all studied cases, although its expression had occurred predominantely in less than 50% of the parenchyma cells of the ameloblastoma, while in about of 60% of the adenomatoid odontogenic tumor more than 50% of cells were positive. The mesenchymal cells were positive to MMP-9 in 65% of the ameloblastoma and in 80% of the adenomatoid odontogenic tumor, respectively. Statistically significant difference was observed to the MMP-1 expression with relation to MMP-2 and MMP-9 in the ameloblastoma (p < 0.001). It was not possible to perform statistical analysis to the cases of adenomatoid odontogenic tumor, however there was a tendency toward a differential expression of the MMP-1 with relation to other studied MMPs. These results suggest that MMP-1, - 2 and -9 are implicated in the growth and progression of both tumors analyzed as well as the more pronounced participation of the stroma in the ameloblastoma could together to be related to the higher clinical aggressiveness
