Plasticidade dependente da experiência induzida por manipulação da matriz extracelular

The primary somatosensory cortex (S1) receives inputs from peripheral tactile receptors and plays a crucial role on many important behaviors. However, the plastic potential of this region is greatly reduced during adulthood, limiting functional recovery after injuries. This fact is due to the presence, in the brain parenchima, of structures and substances that have an inhibitory effect on plasticity, such as chondroitin sulfate proteoglicans (CSP) present in the perineuronal.nets (PNNs) surrounding a subset of neurons. Maturation of PNNs coincide with the closure of critical periods of plasticity in cortical areas, since CSP act to stabilize synaptic contacts. Removal of CSP is proven to be an effective therapeutic approach to restore plasticity and increase the odds of functional recovery after cortical lesion. In the present work, we removed CSP from the sensorimotor cortex of rats to restore plasticity and promote the compensatory morphofunctional regeneration of cortical circuits modified by removal of mystacial vibrissae during the critical period. Treatment with the CSP-digesting enzyme chondroitinase ABC proved efficient to restore plasticity in S1 circuits, as evidenced by morphological rearrangements and functional recovery.

Plasticidade dependente da experiência induzida por manipulação da matriz extracelular

The primary somatosensory cortex (S1) receives inputs from peripheral tactile receptors and plays a crucial role on many important behaviors. However, the plastic potential of this region is greatly reduced during adulthood, limiting functional recovery after injuries. This fact is due to the presence, in the brain parenchima, of structures and substances that have an inhibitory effect on plasticity, such as chondroitin sulfate proteoglicans (CSP) present in the perineuronal.nets (PNNs) surrounding a subset of neurons. Maturation of PNNs coincide with the closure of critical periods of plasticity in cortical areas, since CSP act to stabilize synaptic contacts. Removal of CSP is proven to be an effective therapeutic approach to restore plasticity and increase the odds of functional recovery after cortical lesion. In the present work, we removed CSP from the sensorimotor cortex of rats to restore plasticity and promote the compensatory morphofunctional regeneration of cortical circuits modified by removal of mystacial vibrissae during the critical period. Treatment with the CSP-digesting enzyme chondroitinase ABC proved efficient to restore plasticity in S1 circuits, as evidenced by morphological rearrangements and functional recovery.

Análise imuno-histoquímica das metaloproteinases da matriz ( MMP-1,MMP-2 e MMP-9) na doença periodontal

The tissular destruction found in periodontal diseases is caused mainly by components of the host that have its production stimulated by the products of the microorganisms present on the plaque. Matrix Metalloproteinases (MMPs), a class of enzymes involved both in physiologic and pathologic extracellular matrix degradation are considered the main responsible for the characteristic tissular loss in periodontal disease, and the understanding of how this happens can have a series of beneficial implications for prevention, diagnosis and treatment of this illness. The aim of this work was to study the immunohistochemical expression of MMP-1, MMP-2, and MMP-9 in fragments of gingival biopsies with clinical diagnose of periodontal disease. MMP-1 has expressed significantly more than the others MMPs in gingivitis both in the epithelium (p=0,0008) and connective tissue (p=0,0049). In periodontitis, both MMP-1 and MMP-9 has expressed significantly more than MMP-2 in the epithelium (p<0,0001) and in the connective tissue (p=0,0002). MMP-1 and MMP-9 presented more expression in periodontitis than in gingivitis but MMP-1 only in the connective tissue (p=0,03) and MMP-9 in the epithelium (p=0,003) and in the connective tissue (p=0,04). In conclusion, these results indicate that the MMP-1 presents high expression in every stages of the periodontal diseases, and increases its expression in the connective tissue when the gingivitis evolves to periodontitis. Therefore, it may have an important role in connective tissue degradation and bone loss observed in disease, since early, in gingivitis, until late stages, in periodontitis, of the periodontal disease. MMP-9 has expressed more in periodontitis than in gingivitis, both in epithelium and in connective tissue. It means that this enzyme may have some importance in the progression of gingivitis to periodontitis by acting in bone resorption observed in this desease

Análise toxicológica in vitro e in vivo de uma fucana antitrombótica da alga marrom Spatoglossum schdöederi

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 schröederi (Dictyotaceae) has three heterofucans named A, B and C. Our research group have been extracted non anticoagulant heterofucan from S. schröederi 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. schröederi 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

Propriedades antioxidante, anti-hemostástica e antiproliferativa de galactanas sulfatadas da alga vermelha hypnea musciformis (wulfen) j. V. Lamouroux

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 β-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.

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

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

Expressão imuno-histoquímica metaloproteinase -1, -2 e -9 em ameloblastoma sólido e tumor odontogênico adematóide

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

Expressão imuno-histoquimica de colagenase-1 e gelatinases A e B em mixomas odontogênicos e papilas de germes dentários

The odontogenic myxoma shares cellular and structural aspects with dental papilla, which has been implicated as probable origin of this neoplasm. The aim of the present study was to perform a comparative immunohistochemical analysis for the expression of collagenase-1 (MMP-1) and gelatinases A (MMP-2) and B (MMP-9) in odontogenic myxomas and dental papilla of teeth germs. Twelve cases of odontogenic myxomas and eight specimens of teeth germs were selected. It was taken into consideration the presence or absence of immunoreactivity, the pattern of immunohistochemical distribution of proteases within extracellular matrix, as well as, the number of cells revealing immunostaining for matrix metalloproteinases (MMPs). It was verified a significant difference (p<0,05) in relation to MMP-2 immunoexpression, which was observed only within extracellular matrix of myxomas. Nevertheless, MMP-1 labeling was revealed by most of the cases of odontogenic myxoma, at levels close to those observed in dental papilla. In relation to the pattern of distribution, a significant difference was obtained between specimens (p<0,05), with neoplasms predominantly exhibiting a focal pattern for MMP-1. The quantitative analysis of neoplastic cells labeled for MMPs denoted a significant difference (p<0,05), demonstrating a higher proportion of MMP-1 in comparison to MMPs-2 and -9. It can be concluded that immunohistochemical expression of MMP-1 at levels comparable to those observed in dental papilla and quantitatively superior in relation to MMPs-2 and -9, suggest an implication of this protease on extracellular matrix degradation of odontogenic myxomas. Moreover, the possibility of interactions with receptors involved in cellular adhesion, particularly with integrins, suggests a plausible function on local invasiveness of such neoplasms. Additionally, the presence of a descent immunoexpression gradient for these MMPs on odontogenic myxomas, associated to substrate specificity inherent in each enzyme, suggest the existence of a coordinated mechanism between interstitial collagenase and gelatinases A and B in order to allow an efficient degradation of extracellular matrix and local invasion by neoplastic cells

Associação entre polimorfismos funcionais nos genes da MMP-7 e MMP-9 e o perfil clinicopatológico do carcinoma epidermóide de língua

Matrix metalloproteinase-7 (MMP-7) and -9 (MMP-9) modulate important functions strictly related to the development, invasion and metastasis of several human cancers among them the squamous cell carcinoma of the tongue (SCCT). However, individual genetic factors such as the functional single nucleotide polymorphisms (SNPs) influence the pattern of protein expression of these MMPs and thus may be related to the variability observed in the clinical behavior of patients with SCCT. In this context, the present cross-sectional study aimed to evaluate the association between the frequency of the functional SNPs MMP-7 -181 A/G and MMP-9 -1562 C/T and the clinical (age, gender and metastasis) and pathological (malignancy histological grading and immunohistochemistry expression) features of SCCT cases. Genotyping of these SNPs were performed by PCR-RFLP on DNA samples from 71 cases of SCCT and 60 individuals without cancer who constitute the control group. Among the results of this research, it was observed that the frequency of the polymorphic alleles MMP-7 -181 G and MMP-9 -1562 T in SCCT patients was 28% and 12%, respectively, and the frequency of the heterozygotes A/G (PR = 2.00; p < 0.001) and C/T (PR = 1.54; p = 0.014) were significantly higher in the patient group than in the controls. The prevalence of patients carrying the combination of SNPs studied was significantly associated with SCCT cases (PR = 2.00; p = 0.011) and metastasis (PR = 2.00; p < 0.001). Furthermore, with the frequency of SNPs analyzed, the age, gender, histological grading and immunoreactivity of MMP-7 and MMP-9 formed clinical and pathological parameters relevant to the identification of population subgroups more related to the development of SCCT and metastasis. Based on these results, it is suggested that the protein expression levels of MMP-7 and -9 substantially influence the balance between their pro- and anticancer biological functions and hence the clinicopathological profile of the squamous cell carcinoma of the tongue

Análise da expressão imuno-histoquímica da ciclofilina A, EMMPRIN e MMP-7 na doença periodontal

Periodontal disease is an infectious disease resulting from the immunoinflammatory response of the host to microorganisms present in the dental biofilm which causes tissue destruction. The objective of this study was to evaluate the immunohistochemical expression of cyclophilin A (CYPA), extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 7 (MMP-7) in human specimens of clinically healthy gingiva (n=32), biofilm-induced gingivitis (n=28), and chronic periodontitis (n=30). Immunopositivity for CYPA, EMMPRIN and MMP-7 differed significantly between the three groups, with higher percentages of staining in chronic periodontitis specimens, followed by chronic gingivitis and healthy gingiva specimens (p < 0.001). Immunoexpression of CYPA and MMP-7 was higher in the intense inflammatory infiltrate observed mainly in cases of periodontitis. Analysis of possible correlations between the immunoexpression of EMMPRIN, MMP-7 and CYPA and between the expression of these proteins and clinical parameters (probing depth and clinical attachment loss) showed a positive correlation of CYPA expression with MMP-7 (r = 0.831; p < 0.001) and EMMPRIN (r = 0.289; p = 0.006). In addition, there was a significant positive correlation between probing depth and expression of MMP-7 (r = 0.726; p < 0.001), EMMPRIN (r = 0.345; p = 0.001), and CYPA (r = 0.803; p < 0.001). These results suggest that CYPA, EMMPRIN and MMP-7 are associated with the pathogenesis and progression of periodontal disease

Expressão imuno-histoquímica das integrinas α2ß1, α3ß, e α5ß1, em carcinoma epidermóide de lábio inferior e língua

The unpredictable biologic behavior of the oral squamous cells carcinoma has determined extensive research on the evolution of such tumor. Due to the existing relation between the outer cell matrix and the tumor cells, the integrins have been used as markers in the predictive study of the cell behavior. This study aims to analyze immunohistochemically the expression of the integrin α2β1, α3β1, and α5β1 connections for the collagen, the laminin and the fibronectin respectively in 15 cases of squamous cells carcinoma from the lower lip and 15 from the tongue, with different scores of malignance grading. A predominantly diffuse, cytoplasm and granular immunological marking was observed in the majority of the analyzed cases. According to the marking intensity, integrin α2β1 appeared positive in 80% of the lip and in 93,3% of the tongue cases. The immunological reactivity of integrin α3β1 was classified as positive in 60% of both the tongue and lip cases. For this integrin, 20% and 33.3% of the tongue and lip cases, respectively, were negative. In relation to integrin α5β1 the intensity was classified as positive in 53,3% of the cases and strongly positive in 46,7% of those located in the lip. In the tongue carcinomas, the intensity was positive in 46,7% of the cases and strongly positive in 53,3%. The statistic analysis did not show any significant differences or correlation of expression between these integrins nor between the anatomical sites or between different scores of malignancy grading. The expressive immunological marking of the integrins, α2β1, α3β1, and α5β1 in the studied cases of squamous cell carcinomas leads us to think of a great participation of these proteins in oral carcinogenesis; however, our results do not allow us to correlate its expression as an indicator of variations in the biological behavior of this neoplasia

Expressão imuno-histoquímica das integrinas a2ß1, a3ß1 e a5ß1 em adenoma pleomórfico de glândula salivar menor e maior e carcinoma adenóide cístico

Pleomorphic adenoma and adenoid cystic carcinoma (ACC) consist benign and malignant neoplasm from salivary gland, respectively. These neoplasms share some characteristics, such as cellular origin and considerable production of extracellular matrix, however, with distinct biological behavior. The aim of the present study was to compare the expression of D2E1, D3E1 e D5E1 integrins in pleomorphic adenoma from minor and major salivary glands and ACCs. Furthermore, it was investigated possible differences in the expression of these integrins according to histological subtypes of ACC. Fourteen cases of pleomorphic adenoma from major salivary gland, fourteen cases from minor salivary gland and ten cases of ACC were selected. It was taken into consideration the presence or absence, localization and intensity of integrin immunoexpression. The cases of pleomorphic adenoma were grouped in order to compare the expression between the distinct neoplasms. It was observed a highly significant difference (p<0,0001) in relation to D2E1 integrin between the neoplasms since pleomorphic adenoma showed a pronounced immunostaining. It was not possible to perform statistical tests considering the D2E1 integrin expression; nevertheless, it could be observed a tendency of higher staining in pleomorphic adenoma. For comparative reasons the cases ACCs were divided in two groups: solid and tubular/cribriform. It was not detected significant differences in regard to D2E1 integrin; and statistical analysis could not be realized in relation to D3E1 and D5E integrin expression. However, it was also verified a tendency of absence or reduced expression in the solid subtype. It can be concluded that the reduced D2E1 integrin expression observed in CACs may be related to a lesser degree of cell differentiation in this neoplasm and the reduced D5E1 integrin expression can be associated with aggressive biological behavior. Moreover, the absence and/or reduced expression of the studied integrins in solid ACC suggests a role in pathogenesis and more aggressive biological behavior of this histological subtype

Estudo da descelularização tecidual na produção de arcabouços biológicos para enxertos

The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds

Análise estrutural e avaliação do efeito condroitim sulfato extraído de tilápia (Oreochromis niloticus) em modelo de peritonite aguda

Chondroitin sulfate (CS) is a naturally glycosaminoglycan found in the extracellular matrix of connective tissues and it may be extracted and purified those tissues. CS is involved in various biological functions, which may be related to the having structural variability, despite the simplicity of the linear chain structure from this molecule. Researches in biotechnology and pharmaceutical field with wastes from aquaculture has been developed in Brazil. In recent decades, tilapia (Oreochromis niloticus), native fish from Africa, has been one of the most cultivated species in various regions of the world, including Brazil. The tilapia farming is a cost-effective activity, however, it generates large amount of wastes that are discarded by producers. It is understood that waste from tilapia can be used in research as a source of molecules with important biotechnological applications, which also helps in reducing environmental impacts and promote the development of an ecofriendly activity. Thus, nile tilapia viscera were subjected to proteolysis, then the glycosaminoglycans were complexed with ion exchange resin (Lewatit), it was fractionated with increasing volumes of acetone and purified by ion exchange chromatography DEAE-Sephacel. Further, the fraction was analyzed by agarose gel electrophoresis and nuclear magnetic resonance (NMR). The electrophoretic profile of the compound together the analysis of 1H NMR spectra and the HSQC correlation allow to affirm that the compound corresponds to a molecule like chondroitin sulfate. MTT assay was used to assess cell viability in the presence of CS tilapia isolated and showed that the compound is not cytotoxic to normal cells such as cells from the mouse embryo fibroblast (3T3). Then, this compound was tested for the ability to reduce the influx of leukocytes in model of acute peritonitis (in vivo) induced by sodium thioglycolate. In this context, it was done total and differential leukocytes counting in the blood and peritoneal fluid collected respectively from vena cava and the peritoneal cavity of the animals subjected to the experiment. The chondroitin sulfate for the first time isolated from tilapia (CST ) was able to reduce the migration of leukocytes to the peritoneal cavity of inflamed mice until 80.4 per cent at a dose 10µg/kg. The results also show that there was a significant reduction (p<0.001) of the population of polymorphonuclear leukocytes from peritoneal cavity in the three tested doses (0.1µg/kg; 1µg/kg and 10µg/kg) when it was compared to the positive control (just thioglycolate). Therefore, since the CST structure and mechanism of action has been completely elucidated, this compound may have potential for therapeutic use in inflammatory diseases

Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA)

The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.