Efeito do tratamento por plasma na proliferação de fibroblastos e esterilização de membranas de quitosana

Chitosan is being studied for use as dressing due their biological properties. Aiming to expand the use in biomedical applications, chitosan membranes were modified by plasma using the following gases: nitrogen (N2), methane (CH4), argon (Ar), oxygen (O2) and hydrogen (H2). The samples were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle, surface energy and water absorption test. Biological Tests were also performed, such as: test sterilization and proliferation of fibroblasts (3T3 line). Through SEM we observed morphological changes occurring during the plasma treatment, the formation of micro and nano-sized valleys. MFA was used to analyze different roughness parameters (Ra, Rp, Rz) and surface topography. It was found that the treated samples had an increase in surface roughness and sharp peaks. Methane plasma treatment decreased the hydrophilicity of the membranes and also the rate of water absorption, while the other treatments turned the membranes hydrophilic. The sterilization was effective in all treatment times with the following gases: Ar, N2 and H2. With respect to proliferation, all treatments showed an improvement in cell proliferation increased in a range 150% to 250% compared to untreated membrane. The highlights were the treatments with Ar 60 min, O2 60 min, CH4 15 min. Observing the results of the analyzes performed in this study, it appears that there is no single parameter that influences cell proliferation, but rather a set of ideal conditions that favor cell proliferation

Efeito de um extrato de chrysobalanus icaco na marcação de constituíntes sanguíneos com tecnécio-99m, na morfologia de hemácias, na topologia plasmidial e na ação do cloreto estanoso no dna plasmidial

The use of radionuclides has contributed for advances in Health Sciences, to research or to the diagnosis and/or treatment of diseases. These advances have been possible with the utilization of radiopharmaceuticals labeled with technetium-99m (99mTc). Stannous chloride (SnCl2) has the main reducing agent utilized to obtain radiopharmaceuticals labeled with technetium-99m. It has been reported that several natural or synthetic drugs are capable to alter the labeling of blood constituents with 99mTc, as well as the red blood cells morphology. The aim of this study was to evaluate possible alterations of Chrysobalanus icaco extract on the labeling of blood constituents with 99mTc, on the morphology of RBC of blood of Wistar rats, on the breakage of plasmid DNA and on the effects of stannous chloride on plasmid DNA. The results showed significant (P<0.05) alteration of the labeling of blood constituents with 99mTc, as well as, modification of the morphology and morphometry (perimeter/area ratio) of the RBC in presence of the extract. These data suggest that this abajeru extract could alter the labeling of blood constituents with 99mTc by its chelating/antioxidant action and/or effects on membrane structures. Moreover C. icaco extract altered the electrophoretic profile and decreased significantly (p<0.05) the effect of SnCl2 on plasmid DNA. The results obtained in this work could indicate a dose-dependent protective action against the SnCl2 and a genotoxic effect of C. icaco extract on plasmid DNA

Comparação de efeitos dos extratos de Hypericum perforatum (Hipérico) e de Mentha crispa (Hortelã) em diferentes modelos experiemtais

Several clinic evaluations have been possible with radiobiocomplexes labeled with technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere on the labeling of blood constituents with 99mTc, as well as on the biodistribution of radiobiocomplexes. Authors have also reported about the toxicity of several natural products. The aim of this study was to compare the effects of the Mentha crispa (hortelã) and of the Hypericum perforatum (hipérico) in different experimental models. On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc, both extracts were capable of to decrease the radioactivity percentage on the cellular compartment and on the fixation on plasma and cellular proteins. On the morphometry of the RBC, only the hortelã was capable to alter the shape and the perimeter/area ratio of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate (Na99mTcO4), the hortelã increased the Na99mTcO4 distribution in the kidney, spleen, liver and thyroid, meanwhile the hipérico decreased the Na99mTcO4 distribution in the bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the pancreas. On the bacterial cultures survival, the hipérico was capable of to protect the bacteria against the stannous chloride (SnCl2) effect. The hipérico did not alter the topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2 action. Probably, the effects presented by both extracts could be due to chemical compounds of the extracts that could alter the morphology of the RBC and the plasma membrane ions transport, and/or by phytocomplexes that could be formed with different effects dependent on the biological system considered

Avaliação de efeitos de um extrato aquoso de cinnamomum zeylanicum L. na marcação de constituintes sanguíneos com tecnécio-99m e na morfologia das hemácias de ratos wistar

Radiobiocomplexes are used to obtain images in nuclear medicine and employed in basic research. Blood constituents labeled with technetium-99m (99mTc) have also been employed as radiobiocomplexes and used also experimental model for evaluation of the biological effects of natural or synthetic drugs. The analysis of the morphology and the morphometrics parameters (perimeter/área ratio) can be used to evaluate the effects of drugs upon the structure of the membrane of red blood cells. Cinnamomum zeylanicum (cinnamon) is a spice used as herbal medicine to treat diseases. The aim of this study was to evaluate the effect of in vitro and in vivo treatment with an aqueous cinnamon extract on the labeling of blood constituents with 99mTc and on the morphology of red blood cells from Wistar rats. In the in vitro treatment, isolated blood sample from animals were incubated with cinnamon extract. In the in vivo treatment, blood samples were also withdrawn from animals treated with cinnamon extract. In both cases, the radiolabeling of blood constituents was done. The morphological analysis of red blood cells was also done. As control, blood or animals treated with NaCl 0.9%. The data obtained on the labeling of blood constituents with 99mTc experiments indicated that the in vitro treatment with cinnamon extract was capable to decrease signiicantly (p<0.05) the percentage of radioactivity in cellular compartments and on the fixation of cellular and plasma proteins. These effects were not observed on the in vivo treatment. The results obtained for the morphology of red blood cells suggest that the in vitro and in vivo treatments did not alter the morphology and the perimeter/area ratio. The in vitro treatment with aqueous cinnamon extract could affect the membrane structures related with the oxidation status of the stannous ion pertechnetate ion, altering the labeling of blood constituentes with 99mTc. This study was a multidisciplinary experimental research. It was developed with the contribution of the different Departments and Services of the Hospital Universitário Pedro Ernesto of the Universidade do Estado do Rio de Janeiro

Adesão, proliferação e genotoxicidade celular de celulose bacteriana modificada por plasma

Bacterial cellulose (BC) has a wide range of potential applications, namely as temporary substitute skin in the treatment of skin wounds, such as burns, ulcers and grafts. Surface properties determine the functional response of cells, an important factor for the successful development of biomaterials. This work evaluates the influence of bacterial cellulose surface treatment by plasma (BCP) on the cellular behavior and its genotoxicity potential. The modified surface was produced by plasma discharge in N2 and O2 atmosphere, and the roughness produced by ion bombardment characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell adhesion, viability and proliferation on BCP were analysed using crystal violet staining and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) method. Genotoxicity was evaluated using the comet and cytokinesis block micronucleus assay. The results show that the plasma treatment changed surface roughness, producing an ideal cell attachment, evidenced by more elongated cell morphology and improved proliferation. The excellent biocompatibility of BCP was confirmed by genotoxicity tests, which showed no significant DNA damage. The BCP has therefore great potential as a new artificial implant

Efeito de um extrato de Artemísia Vulgaris L. marcação in vitro de constituintes sanguíneos com tecnécio-99m em ratos wistar

Artemisia vulgaris L..is used in folk medicine and in Traditional Chinese Medicine (TCM). This medicinal plant has been utilized as anticonvulsive, analgesic, antispasmodic effect, rheumatic pains, menstrual dyspepsia, asthenia, epilepsy, hepatitis, fevers, anemia and to expel parasites. In nuclear medicine, blood constituents are labeled with technetium-99m (99mTc) and used as radiopharmaceuticals (radiobiocomplexes). Authors have been described that synthetic and/or natural drugs could modify the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the effects of an aqueous extract of Artemisia vulgaris L. on the labeling of blood constituents with 99mTc. Blood samples withdrawn of Wistar rats were incubated with Artemisia vulgaris L, stannous chloride and 99mTc, as pertechnetate ion. Aliquots of plasma (P) and blood cells (BC) were isolated. Aliquots of P and BC were also precipitated with trichloroacetic acid and soluble (SF) and insoluble (IF) fractions were separated. The radioactivity in each fraction was counted and the percentages of radioactivity (%ATI) were calculated. Artemisia vulgaris L. extract decreased significantly (p<0.05) the %ATI on BC and on IF-BC. The analysis of the results indicates that the extract could have substances that could interfere on the transport of stannous through the erythrocyte membrane altering the labeling of blood cells with 99mTc. Working in this study was a multidisciplinary group, with Phisical therapists, Biomedicals, Physicals, Pharmacists, Biologists, Statistics and Physicians.

Análise cintilográfica da deposição pulmonar de radioaerossol após associação da nebulização através dos dispositivos mesh e jato com a ventilação não invasiva em indivíduos normais e com pneumopatias obstrutivas crônicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Liofilização como ferramenta de preservação de sêmen humano

Liofilização é um método alternativo de preservação de espermatozóide, com vantagens sobre o criopreservado pelo baixo custo de armazenamento, facilidade no transporte pela não necessidade de containers e baixas taxas de contaminação. Esse estudo foi realizado no intuito e testar a eficácia de 5 açúcares na proteção do material cromossômico (DNA) de espermatozoides humanos liofilizados após reidratação. Para isso foram utilizados espermatozoides de indivíduos classificados como normozooespérmicos segundo OMS. Em eppendorfs foram colocados 1ml de sêmen que foram diluídos (1:1) em 1 ml de solução de Glicose, Lactose, Maltose, Manitol e Sorbitol, separadamente, nas concentrações 0.2M, 0.4M, 0.5M e 0.6M. Essas amostras foram analisadas em duas fases a primeira após congelamento observando a porcentagem de células com danos de membrana e outra análise após a liofilização, utilizando o Teste Cometa para detectar as taxas de fragmentação de DNA dos espermatozoides recuperadas após reidratação. O processo de liofilização de espermatozoides não foi capaz de recuperar a motilidade dos mesmos, mas foi capaz de manter 62.30 ± 13.76% de membrana plasmática intacta na presença de 0.6M manitol. As substâncias que melhor preservaram o DNA dessas células foram 0.2 lactose, 0.2M e 0.6M manitol e sorbitol, com apenas 4% de danos ao DNA. Esses resultados sugerem uma boa proteção ao DNA de espermatozoides humanos liofilizados, mas ainda requer testes adicionais para avaliar a fertilização e estudos que garantam a viabilidade da prole

Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica

Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL

Produção e avaliação de nanoemulsão catiônica como possível vetor não-viral para pIRES2-EGFP

Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355®, 1.2 % of Tween 80®, 0.8 % of Span 80®, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 μS/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ºC. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/μL); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier

Anodização para obtenção de membranas cerâmicas

This a study on the achievement of alumina membranes by the method of anodizing. From this method got up a layer of aluminum oxide on the anodic metal, who presented the basic properties necessary for the application as a support for the production and acquisition of nanomaterials, such as porosity nano and resistance to high temperature, and other properties, as resistance to corrosion, and chemical, high ranking of the structure and pore size of the pores. The latter, ranging from 10 to 100nm depended on the electrolyte used, which in this study was the H2SO4. To remove all remaining aluminum, it is a bath of dissolution with HCl and CuCl where the residual aluminum has been withdrawn, and the deep pores were opened after chemical treatment with NaOH. After the dissolution, the membranes were calcined at temperatures of 300, 600 and 900° C, and sintered at temperatures of 1200 and 1300º C to win mechanical strength, porosity and observe the desired crystallization. Then went through analyses of composition through X-ray diffraction and morphology of the microstructure through a scanning electron microscope. The method was effective for obtaining alumine membranes applied in the processes of production of materials in nano

Otimização do processo de recuperação do cromo de efluentes de curtumes por microemulsões no extrator Morris

The tanning industries are those which transform animal hide or skin into leather. Due to the complexity of the transformation process, greater quantities of chemicals are being used which results in the generation of effluents with residual solids. The chromium in the residual waters generated by tanning tend to be a serious problem to the environment, therefore the recovery of this metal could result in the reduction of manufacturing costs. This metal is usually found in a trivalent form which can be converted into a hexavalent compound under acidic conditions and in the presence of organic matter. The present study was carried out with the objective to recover chromium through an extraction/re-extraction process using micro emulsions. Micro emulsions are transparent and thermodynamically stable system composed of two immiscible liquids, one forming the continuous phase and the other dispersed into micro bubbles, established by an interfacial membrane formed by surface active and co-surface active molecules. The process of recovering the chromium was carried out in two stages. The first, an extraction process, where the chromium was extracted in the micro emulsion phase and the aqueous phase in excess was separated. In the second stage, a concentrated acid was added to the micro emulsion phase rich in chromium in order to obtain a Winsor II system, where the water that formed in the micro emulsion phase separates into a new micro emulsion phase with a higher concentration of chromium, due to the lowering of the hydrophiles as well as the ionisation of the system. During the experimental procedure, a study was initiated with a synthetic solution of chromium sulphate passing onto the effluent. A Morris extractor was used in the extraction process. Tests were carried out according to the plan and the results were analysed by statistical methods in order to optimise the main parameters that influence the process: the total rate of flow (Q), stirring speed (w) and solvent rate (r). The results, after optimization, demonstrated that the best percentuals in relation to the chromium extraction (99 %) were obtained in the following operational conditions: Q= 2,0 l/h, w= 425 rpm and r= 0,375. The re-extraction was carried out at room temperature (28 °C), 40 °C and 50°C using hydrochloric acid (8 and 10 M) and sulphuric acid (8 M) as re-extracting agents. The results obtained demonstrate that the process was efficient enough in relation to the chromium extraction, reaching to re-extraction percentage higher than 95 %.

Avaliação da eficiência de filtros anaeróbios na remoção de coliformes fecais e ovos de helmintos

The technology of anaerobic reactors for sanitary wastewater treatment has been extensively developed in Brazil, and today it is practically consolidated. They present several advantages, such as low construction and operating costs, and low sludge production, the anaerobic reactors are an attractive alternative to minimize problematic lack of basic sanitation in urban areas, and also of the rural areas. The anaerobic filters have been widely used in Brazil. It produces an effluent with low concentration of organic matter and solids suspended, besides conserving the nutrients, therefore, it is good for use in irrigation, but the practice must be associated with knowledge of the pathogens presence. The main objective of this study was to evaluate the efficiency of anaerobic filters in removal faecal coliforms and helminth eggs, and to verify if the effluent can be used for agricultural purposes, according to the World Organization of Health (WHO, 1989). The protocol used to enumerate helminths eggs was the modified Bailenger method, (Ayres and Mara, 1996) recommended by WHO for evaluation of raw effluent and treated effluent. The membrane filtration method was utilized to determine the concentrations of faecal coliforms. Three different systems of sewer treatment composed by anaerobic filters were analyzed. The results, in a general analysis, showed that all the researched systems reached a larger removal than 93% to helminth eggs, resulting in an effluent with smaller average than 1 egg/L. One of these systems, Sistema RN, reached a larger removal than 99%, confirming the good performance of the anaerobic filters in removal helminths eggs. Even with low concentrations of eggs in the influent, the filters were capable to remove this parameter efficiently. About faecal coliforms, it was observed for all the researched systems an effluent with 106 CFU/100mL. The high concentrations to faecal coliforms in the effluent just allow reuse for restricted irrigation, in agreement with the guidelines of WHO. Although the researched systems have not removed faecal coliforms efficiently, the results indicated a good efficiency of the anaerobic filters in removal helminth eggs

Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

Imunoexpressão das claudinas -1 e -7 em ameloblastomas e germes dentais humanos

We investigated the immunohistochemistry expression of claudins -1 and -7 in ameloblastoma and in human dental germs on the pattern of distribution (focal, regional or diffuse), the cells that expressed (if central or peripheral) and the location of that expression in the cell components recital membrane, cytoplasm and nucleus. Among the 29 cases of ameloblastoma, 24 were type solid and 6 unicystic. In 7 mandibular specimens of human fetuses found dental germs from the stage of bud to the crown. We note that the pattern of expression in the dental germs was variable for claudinas studied according to the cell type and stage of differentiation and was invariate only in the cells of stellate reticulum. In epithelium internal of enamel organ, claudin-1 has been decreasing with the progression of differentiation as to claudina-7 that was found in the cells of the peripheral papilla. For ameloblastoma the expression was more significant than that observed in dental germs. Fisher s exact test no found association between the expression of claudinas cells in central and peripheral and the type of ameloblastoma (solid or unicystic). Thus, in general the claudin-1 was positive in the central cell of 93,1% of the cases and in peripheral cells of 51,7%. The claudin-7 was expressed in the cells of all cases central and peripheral cells from 89,7%. For both claudins the distribution was predominantly diffuse cells both in central and peripheral cells. Given our findings it is suggested that the expression of claudins may be indicative of the involvement of these molecules in morphogenetics events culminating with the dental development and that possibly influence the development of neoplastic ameloblastoma