Modelagem dos efeitos da irradiação luminosa no cérebro de camundongos e rastreamento de neurônios durante experimentos de microscopia de fluorescência

The fluorescent proteins are an essential tool in many fields of biology, since they allow us to watch the development of structures and dynamic processes of cells in living tissue, with the aid of fluorescence microscopy. Optogenectics is another technique that is currently widely used in Neuroscience. In general, this technique allows to activate/deactivate neurons with the radiation of certain wavelengths on the cells that have ion channels sensitive to light, at the same time that can be used with fluorescent proteins. This dissertation has two main objectives. Initially, we study the interaction of light radiation and mice brain tissue to be applied in optogenetic experiments. In this step, we model absorption and scattering effects using mice brain tissue characteristics and Kubelka-Munk theory, for specific wavelengths, as a function of light penetration depth (distance) within the tissue. Furthermore, we model temperature variations using the finite element method to solve Pennes’ bioheat equation, with the aid of COMSOL Multiphysics Modeling Software 4.4, where we simulate protocols of light stimulation tipically used in optogenetics. Subsequently, we develop some computational algorithms to reduce the exposure of neuron cells to the light radiation necessary for the visualization of their emitted fluorescence. At this stage, we describe the image processing techniques developed to be used in fluorescence microscopy to reduce the exposure of the brain samples to continuous light, which is responsible for fluorochrome excitation. The developed techniques are able to track, in real time, a region of interest (ROI) and replace the fluorescence emitted by the cells by a virtual mask, as a result of the overlay of the tracked ROI and the fluorescence information previously stored, preserving cell location, independently of the time exposure to fluorescent light. In summary, this dissertation intends to investigate and describe the effects of light radiation in brain tissue, within the context of Optogenetics, in addition to providing a computational tool to be used in fluorescence microscopy experiments to reduce image bleaching and photodamage due to the intense exposure of fluorescent cells to light radiation.

Estrutura e dinâmica do fitoplânctone bacterioplâncton em cultivos de camarão no Rio Grande do Norte-Brasil: impacto sobre o ambiente natural

This work aimed to study the structure and dynamic of Phytoplankton and Bacterioplankton in a complete cycle of shrimp cultivation (Litopenaeus vannamei) and determine the environmental factors responsible for the structural changes of these communities. The study was realized in a saltwater shrimp farm (Macaíba, RN), between September/2005 and February/2006, and in a freshwater shrimp farm (Ceará Mirim, RN), between May/2007 and September 2007. The samplings were collected weekly in saltwater farm and every fifteen days in freshwater farm. Total phosphorus, chlorophyll a and environmental parameters (pH, dissolved oxygen, salinity, temperature, depth and water transparency) were measured. Qualitative and quantitative analysis of the phytoplankton and bacterioplankton were carried out. The Shannon-Wiener ecologic indexes of diversity and the Pielou equitability indexes were calculated to the phytoplankton. Bacterial density was determined by epifluorescence microscopy. The data were statistically analyzed by Pearson correlation and t-Test. Chlorophycea were predominat in salt water and in the captation/drainage point (24 to 99%). Diatoms had higher wealth. The species Choricystis minor had the highest occurrence (100%) and dominance (90-100%), thus showing its adaptation to the high temperatures, salinity and low water transparency conditions. Filamentous Cyanobacteria like Oscillatoria sp., Pseudoanabaena sp. and Phormidium sp. had constant levels. The negative correlation between chlorophycea and water transparency, and the positive correlation between chlorophyll a and salinity, showed that the phytoplankton was well adapted to the low transparency and to the high salinity. The bacterioplankton was negatively correlated with the total phosphorus and salinity. In freshwater, Cyanobacteria were predominant (>80%), presenting some producers of toxins species like Microcystis sp., Aphanizomenon sp., Cylindrospermopsis raciborskii e Anabaena circinalis. Cyanobacterial density and total phosphorus and chlorophyll a concentrations exceeded the maximum value allowed by legislation. The means of total phosphorus varied from 264 to 627 Wg.L-1 and the means of chlorophyll a oscillated between 22 and 182 Wg.L-1. The phytoplankton species were selected by low availability of the light, high pH, temperature and high availability of total phosphorus. The bacterioplankton showed high densities (5,13 x 107 to 8,50 x107 Bac.mL-1). The studied environments (ponds and rivers) presented a high level of trophic state based on the high concentrations of chlorophyll a and total phosphorus and cyanobacteria dominance. The composition of species in the ponds and rivers was similar, as well as high concentrations of total phosphorus and chlorophyll a, highlighting the pollution caused by the discharges of the farms in natural environment

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Utilização da Berberina na Identificação dos Mastócitos do Molusco Anomalocardia brasiliana

Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates

Bioprospecção de extratos metanólicos de macroalgas marinhas do litoral do Rio Grande do Norte: avaliação das atividades anticoagulante, antioxidante e antiproliferativa

Seaweeds are organisms known to exhibit a variety of biomolecules with pharmacological properties. The coast of Rio Grande do Norte has over 100 species of seaweeds, most of them not yet explored for their pharmacological potential. Sugars and phenolic compounds are the most studied of these being assigned a range of biological properties, such as anticoagulant , antiinflammatory, antitumor and antioxidant activities. In this work, we obtained methanolic extracts from thirteen seaweeds of the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; D. mertensis; Sargassum filipendula; Spatoglossum schröederi; Acanthophora specifera; Botryocladia occidentalis; Caulerpa cupresoides; C. racemosa; C. prolifera; C. sertularioides e Codium isthmocladum). They were evaluated as anticoagulant and antioxidant drugs, as well as antiproliferative drugs against the tumor cell line HeLa. None of the methanolic extracts showed anticoagulant activity, but when they were evaluated as antioxidant drugs all of extracts showed antioxidant activity in all tests performed (total antioxidant capacity, sequestration of superoxide and hydroxyl radicals, ferric chelation and reductase activity), especially the algae D. mentrualis, D. cilliolata and C. prolifera, who had the greatest potential to donate electrons.In addition, the ability of iron ions chelation appears as the main antioxidant mechanism of the methanolic extracts of these seaweeds mainly for the extract of the C. racemosa seaweed, which reached almost 100% activity. In the MTT assay, all extracts showed inhibitory activity at different levels againts HeLa cells. Moreover, D. cilliolata (MEDC) and D. menstrualis (MEDM) extracts showed specific activity to this cell line, not inhibiting the viability of 3T3 normal cell line, so they were chosen for detailing the antiproliferative mechanism of action. Using flow cytometry, fluorescence microscopy and in vitro assays we demonstrated that MEDC and MEDM induced apoptosis in HeLa cells by activation of caspases 3 and 9 and yet, MEDC induces cell cycle arrest in S phase. Together, these results showed that the methanolic extracts of brown seaweed D. menstrualis and D. cilliolata may contain agents with potential use in combatting cells from human uterine adenocarcinoma. This study also points to the need for more in-depth research on phytochemical and biological context to enable the purification of biologically active products of these extracts

Estrutura e dinâmica do fitoplânctone bacterioplâncton em cultivos de camarão no Rio Grande do Norte-Brasil: impacto sobre o ambiente natural

This work aimed to study the structure and dynamic of Phytoplankton and Bacterioplankton in a complete cycle of shrimp cultivation (Litopenaeus vannamei) and determine the environmental factors responsible for the structural changes of these communities. The study was realized in a saltwater shrimp farm (Macaíba, RN), between September/2005 and February/2006, and in a freshwater shrimp farm (Ceará Mirim, RN), between May/2007 and September 2007. The samplings were collected weekly in saltwater farm and every fifteen days in freshwater farm. Total phosphorus, chlorophyll a and environmental parameters (pH, dissolved oxygen, salinity, temperature, depth and water transparency) were measured. Qualitative and quantitative analysis of the phytoplankton and bacterioplankton were carried out. The Shannon-Wiener ecologic indexes of diversity and the Pielou equitability indexes were calculated to the phytoplankton. Bacterial density was determined by epifluorescence microscopy. The data were statistically analyzed by Pearson correlation and t-Test. Chlorophycea were predominat in salt water and in the captation/drainage point (24 to 99%). Diatoms had higher wealth. The species Choricystis minor had the highest occurrence (100%) and dominance (90-100%), thus showing its adaptation to the high temperatures, salinity and low water transparency conditions. Filamentous Cyanobacteria like Oscillatoria sp., Pseudoanabaena sp. and Phormidium sp. had constant levels. The negative correlation between chlorophycea and water transparency, and the positive correlation between chlorophyll a and salinity, showed that the phytoplankton was well adapted to the low transparency and to the high salinity. The bacterioplankton was negatively correlated with the total phosphorus and salinity. In freshwater, Cyanobacteria were predominant (>80%), presenting some producers of toxins species like Microcystis sp., Aphanizomenon sp., Cylindrospermopsis raciborskii e Anabaena circinalis. Cyanobacterial density and total phosphorus and chlorophyll a concentrations exceeded the maximum value allowed by legislation. The means of total phosphorus varied from 264 to 627 Wg.L-1 and the means of chlorophyll a oscillated between 22 and 182 Wg.L-1. The phytoplankton species were selected by low availability of the light, high pH, temperature and high availability of total phosphorus. The bacterioplankton showed high densities (5,13 x 107 to 8,50 x107 Bac.mL-1). The studied environments (ponds and rivers) presented a high level of trophic state based on the high concentrations of chlorophyll a and total phosphorus and cyanobacteria dominance. The composition of species in the ponds and rivers was similar, as well as high concentrations of total phosphorus and chlorophyll a, highlighting the pollution caused by the discharges of the farms in natural environment

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Utilização da berberina na identificação dos mastócitos do molusco Anomalocardia brasiliana

Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates

Caracterização e correlação do fenômeno pró-zona com títulos de sororeatividade do VDRL e reação de imuno- fluorescência indireta em soros de pacientes com sífilis

AZEVEDO, Luciana Karla Araújo de, et al. Caracterização e correlação do fenômeno pró-zona com títulos de sororeatividade do VDRL e reação de imuno-fluorescência indireta em soros de pacientes com sífilis. Revista Brasileira de Análises Clínicas, Rio de Janeiro, v. 38, n. 2, p. 183-187, 2006.

Avaliação da dolomita funcionalizada para remoção de H2S em gás natural

The natural gas is an alternative source of energy which is found underground in porous and permeable rocks and being associated or not to the oil. Its basic composition includes methane, other hydrocarbon and compounds such as carbon dioxide, nitrogen, sulphidric gas, mercaptans, water and solid particles. In this work, the dolomite mineral, a double carbonate of calcium and magnesium whose the chemical formula is CaMg(CO3)2, was evaluated as adsorbent material. The material was characterized by granulometric analysis, X-ray fluorescence, X-ray diffraction, thermogravimetric analysis, differential thermal analysis, specific surface area, porosity, scanning electronic microscopy and infrared spectroscopy. Then the material was functionalized with diethanolamine (dolomite+diethanolamine) and diisopropylamine (dolomite+diisopropylamine). The results indicated that the adsorbents presented appropriate physiochemical characteristics for H2S adsorption. The adsorption tests were accomplished in a system coupled to a gas chromatograph and the H2S monitoring in the output of the system was accomplished by a pulsed flame photometric detector (PFPD). The adsorbents presented a significant adsorption capacity. Among the analyzed adsorbents, the dolomite+diethanolamine presented the best capacity of adsorption. The breakthrough curves obtained proved the efficiency of this process

Desenvolvimento de formulações para produção de grés porcelanato a partir de matérias-primas da região da bacia do Parnaíba

Brazil is a great ceramic raw materials productor because of the its big number of clay deposits, in various areas of the ceramic industry. Although, the majority of the natural reservations are unknown or not studied yet, so there is no scientific technical dates that can guide their usage and industrial application, as well as the racional and optimazed way of usage by the industrial sector. The state of Maranhão has a gigant mineral wealth as esmectite, bentonite, kaolin, clays, feldspates, marine salt, iron and others, but produce only products with small agregated value compared to the porcelanato, one of the most expensives ceramic cover tiles, the reason for that is the low water absorption (lower than 0,5%), beside present amazing tecnicals features, like mechanical resistence. The main objective of the work is to do the characterization of four clays, with the finallity of find an application by the results and develop formulations to produce porcelanato using these raw materials from Timon-MA. For this were made the raw materials characterization using X ray fluorecence; X ray diffraction; Differencial thermal analysis; Dilatometric analysis and Tecnological properties, planing three formulations that were sinterized at six different temperatures: 1150, 1170, 1190, 1210, 1230 and 1250ºC for 7 minutes. After the sinteratization, the samples were submitted to tension resistance analysis. Were attained two formulations with the requested properties to produce porcelanato

Aproveitamento de resíduos minerais na formulação de argamassas para a construção civil

Current environmental concerns include the excessive consumption and inefficient use of non-renewable natural resources. The construction industry is considered one of the largest consumers of natural raw materials, significantly contributing to the environmental degradation of the planet. The use of calcareous quarry (RPPC) and porcelain tile polishing residues (RPP) as partial replacements of the cement in mortars is an interesting alternative to minimize the exploration of considerably large amounts of natural resources. The present study aimed at investigating the properties of fresh and hardened mortars produced using residues to replace cement. The residues used were fully characterized to determine their specific mass, unitary mass, particle size distribution and morphology, and composition. The performance of the mortars was compared to that of reference compositions, prepared without residues. A total of 18 compositions were prepared, 16 using residues and 2 reference ones. The mortars were prepared using Portland CP II F 32 cement, CH I hydrated lime, river sand and tap water. The compositions of the mortars were 1:1:6 and 1:0.5:4.5 (vol%), and water to cement ratios of 1.87 and 1.45 were used, respectively. The mortars in the fresh state were evaluated by consistency index, water retention, density of mass and incorporated air content tests. In their hardened state, the mortars were evaluated by apparent mass density, modulus of elasticity, flexural tensile strength, compressive strength and water absorption by capillarity. The mortars were also analyzed by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction and fluorescence. Finally, they were classified according to NBR 13281 standards. The mortars prepared using residues partially replacing the cement exhibited lower modulus of elasticity compared to the reference compositions, thus improving the performance in their intended use. On the downside, the water absorption by capillarity was affected by the presence of residues and both the tensile and compressive strength were reduced. However, from the overall standpoint, the replacement of cement by calcareous quarry or porcelain tile polishing residues did not result in significant changes in the properties of the mortars. Therefore, compositions containing these residues can be used in the construction industry

Síntese e impregnação de peneiras moleculares Fe MCM-41 derivada de sílica da casca do arroz

The mesoporous molecular sieves of the MCM-41 and FeMCM-41 type are considered promissory as support for metals used as catalysts in oil-based materials refine processes and as adsorbents for environmental protection proposes. In this work MCM-41 and FeMCM41 were synthesized using rice husk ash - RHA as alternative to the conventional silica source. Hydrothermal synthesis was the method chosen to prepare the materials. Pre-defined synthesis parameters were 100°C for 168 hours, later the precursor was calcinated at 550°C for 2 hours under nitrogen and air flow. The sieves containing different proportions of iron were produced by two routes: introduction of iron salt direct synthesis; and a modification post synthesis consisting in iron salt 1 % and 5% impregnation in the material followed by thermal decomposition. The molecular sieves were characterized by X ray diffraction XRD, Fourier transform infrared spectroscopy FT-IR, X ray fluorescence spectroscopy XFR, scanning electronic microscopy SEM, specific surface area using the BET method, Termogravimetry TG. The kinetic model of Flynn Wall was used with the aim of determining the apparent activation energy of the surfactant remove (CTMABr) in the MCM- 41 porous. The analysis made possible the morphology characterization, identifying the presence of hexagonal structure typical for mesoporous materials, as well as observation of the MCM41 and iron of characteristic bands.

Caracterização de atapulgita visando aplicação para reforço de materiais poloméricos

The clay mineral attapulgite is a group of hormitas, which has its structures formed by microchannels, which give superior technological properties classified the industrial clays, clays of this group has a very versatile range of applications, ranging from the drilling fluid for wells oil has applications in the pharmaceutical industry. Such properties can be improved by activating acid and / or thermal activation. The attapulgite when activated can improve by up to 5-8 times some of its properties. The clay was characterized by X-ray diffraction, fluorescence, thermogravimetric analysis, differential thermal analysis, scanning electron microscopy and transmission electron microscopy before and after chemical activation. It can be seen through the results the efficiency of chemical treatment, which modified the clay without damaging its structure, as well as production of polymer matrix composites with particles dispersed atapugita

Análise mecânica e microestrutural das ligas de cobalto-cromo de uso odontológico após sucessivas refundições

The cobalt-chromium alloy is extensively used in the Odontology for the confection of metallic scaffolding in partial removable denture. During the last few years, it has been reported an increasing number of premature imperfections, with a few months of prosthesis use. The manufacture of these components is made in prosthetic laboratories and normally involves recasting, using parts of casting alloy and parts of virgin alloy. Therefore, the objective of the present study was to analyze the mechanical properties of a commercial cobalt-chromium alloy of odontological use after successive recasting, searching information to guide the dental prosthesis laboratories in the correct manipulation of the cobalt-chromium alloy in the process of casting and the possible limits of recasting in the mechanical properties of this material. Seven sample groups were confectioned, each one containing five test bodies, divided in the following way: G1: casting only with virgin alloy; G2: casting with 50% of the alloy of the G1 + 50% of virgin alloy; G3: casting with 50% of the alloy of the G2 + 50% of virgin alloy; G4: casting with 50% of the alloy of the G3 + 50% of virgin alloy; G5: 50% of alloy of the G4 + 50% of virgin alloy; G6: 50% of alloy of the G5 + 50% of virgin alloy and finally the G7, only with recasting alloy. The modifications in the mechanical behavior of the alloy were evaluated. Moreover, it was carried the micro structural characterization of the material by optic and electronic scanning microscopy, and X ray diffraction.and fluorescence looking into the correlatation of the mechanical alterations with structural modifications of the material caused by successive recasting process. Generally the results showed alterations in the fracture energy of the alloy after successive recasting, resulting mainly of the increasing presence of pores and large voids, characteristic of the casting material. Thus, the interpretation of the results showed that the material did not reveal significant differences with respect to the tensile strength or elastic limit, as a function of successive recasting. The elastic modulus increased from the third recasting cycle on, indicating that the material can be recast only twice. The fracture energy of the material decreased, as the number of recasting cycles increased. With respect to the microhardness, the statistical analyses showedno significant differences. Electronic scanning microscopy revealed the presence of imperfections and defects, resulting of the recasting process. X ray diffraction and fluorescence did not show alterations in the composition of the alloy or the formation of crystalline phases between the analyzed groups. The optical micrographs showed an increasing number of voids and porosity as the material was recast. Therefore, the general conclusion of this study is that the successive recasting of of Co-Cr alloys affects the mechanical properties of the material, consequently leading to the failure of the prosthetic work. Based on the results, the best recommendadition is that the use of the material should be limited to two recasting cycles