Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Transmissão de energia sem contacto com aplicação em veículos eléctricos

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and Fruits

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.

Life Cycle Assessment of the Hybrid Vehicle – Toyota Prius

Die Luftverschmutzung, die globale Erwärmung sowie die Verknappung der endlichen Ressourcen sind die größten Bedenken der vergangenen Jahrzehnte. Die Nachfrage nach jeglicher Mobilität steigt rapide. Dementsprechend bemüht ist die Automobilindustrie Lösungen für Mobilität unter dem Aspekt der Nachhaltigkeit und dem Umweltschutz anzubieten. Die Elektrifizierung hat sich hierbei als der beste Weg herausgestellt, um die Umweltprobleme sowie die Abhängigkeit von fossilen Brennstoffen zu lösen. Diese Arbeit soll einen Einblick über die Umweltauswirkungen des Hybridfahrzeuges Toyota Prius geben. Hierbei findet eine Gliederung in vier verschiedene Lebensphasen statt. Im Anschluss bietet die Sachbilanz die Möglichkeit die Umweltauswirkungen mit verschiedenen Antriebsmöglichkeiten und Brennstoffen zu vergleichen. Das Modell hat gezeigt, dass der Toyota Prius während der Nutzung einen hohen Einfluss auf das Treibhauspotenzial aufweist. Durch die Nutzung anderer Brennstoffe, wie beispielsweise Ethanol oder Methanol lassen sich die Auswirkungen am Treibhauspotenzial sowie der Verbrauch an abiotischen Ressourcen reduzieren. Vergleicht man die Elektromobilität mit der konventionellen, so ist festzustellen, dass diese Art der Mobilität die derzeit beste Möglichkeit zur Reduzierung der Umweltbelastungen bietet. Die Auswirkungen der Elektromobilität sind im hohen Maße abhängig von der Art des verwendeten Strommixes.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Does Cleome droserifolia have anti-schistosomiasis mansoni activity?

The present study was undertaken to assess the effect of the crude extract of Cleome droserifolia (CD) leaves on experimentally infected mice with Schistosoma mansoni. Two groups of mice, showing a patent infection of S. mansoni, one of them was daily treated with an alcoholic extract of CD leaves (0.31 g kg-1 body weight, i.p.) for 21 days. The schistosomicidal activity of the CD extract was evaluated, three weeks post-treatment, on some parasitological and histopathological aspects including worm load, oogram pattern, faecal eggs releasing and granuloma formation. In addition, serum thyroid hormones levels (tri-iodothyronine; T3 and tetra-iodo-thyronin; T4), serum total protein contents and hepatic reduced glutathione (GSH) were evaluated. Treatment using CD extract resulted in a weak reduction in worm burden (32.46%) and affected the viability of both mature and immature eggs as indicated by the increase in the percentage of dead eggs and the decrease in the percentage of live ones. In addition, a week post-treatment, eggs elimination was observed in the stool of the infected-treated group which was low compared to the infected group. There was a suppressive effect of the extract on granuloma formation that could be due to the antioxidant effect of the extract. These data are confirmed by increasing hepatic GSH, serum total proteins and thyroid hormone levels in the infected-treated group as compared to the infected group. Treatment significantly enhanced b globulin fractions of the protein. Based on these assumptions, CD extract has beneficial effects on thyroid hormones status and anti-schistosomiasis activity. The beneficial effects of CD extract could be related to its direct effects on the parasite, and secondary to its effect on the antioxidant capacity of the host. The present study could emphasize the precise mechanism (s) of CD extract protection.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

Toxicity of saponin isolated from Gymnema sylvestre R. Br. (Asclepiadaceae) against Culex tritaeniorhynchus Giles (Diptera: Culicidae) Japanese encephalitis vector mosquito in India

To determine the larvicidal activity of various extracts of Gymnema sylvestre against the Japanese Encephalitis vector, Culex tritaeniorynchus in Tamilnadu, India. To identify the active principle present in the promising fraction obtained in Chlorofom:Methanol extract of Fraction 2. The G. sylvestre leaf extracts were tested, employing WHO procedure against fourth instar larvae of C. tritaeniorhynchus and the larval mortalities were recorded at various concentrations (6.25, 12.5, 25.0, 50 and 100 µg/mL); the 24h LC50 values of the G. Sylvestre leaf extracts were determined following Probit analysis. It was noteworthy that treatment level 100 µg/mL exhibited highest mortality rates for the three different crude extracts and was significantly different from the mean mortalities recorded for the other concentrations. The LC50 values of 34.756 µg/mL (24.475-51.41), 31.351 µg/mL (20.634-47.043) and 28.577 µg/mL (25.159-32.308) were calculated for acetone, chloroform and methanol extract with the chi-square values of 10.301, 31.351 and 4.093 respectively. The present investigation proved that G. Sylvestre could be possibly utilized as an important component in the Vector Control Program.

Análise da atual unidade de desasfaltação pelo Propano para o tratamento de crudes não parafínicos

Este trabalho teve como principal objetivo o estudo da simulação da unidade de destilação atmosférica e a vácuo da fábrica de óleos base da Petrogal, permitindo verificar que, uma vez conseguido um modelo num programa de simulação que traduz o comportamento real de um dado processo, é possível sem riscos operacionais avaliar o efeito da alteração das condições normais de funcionamento desse processo. O trabalho foi orientado pelo Eng.º Carlos Reis, no que diz respeito à empresa, e pelo Prof. Dr. Luís Silva, pelo ISEP. O trabalho foi dividido em duas partes, sendo a primeira direcionada à obtenção do novo crude a partir de dois crudes pesados e caracterização dessa mistura. Já a segunda parte reside na refinação deste novo crude através da plataforma Aspen Plus para obtenção do resíduo de vácuo, para posterior processamento na coluna de discos rotativos usando um método de separação por extração liquido-liquido. Estudaram-se as propriedades físicas dos fluidos e verificou-se que na destilação atmosférica as curvas de destilação encontram-se muito próximas comparativamente com os resultados obtidos pela empresa, enquanto na destilação a vácuo os valores encontram-se mais afastados, apresentando uma variação de 30ºC a 100ºC em relação aos valores obtidos pelo Petro-Sim. Com a finalidade de cumprir um dos objetivos em falta, irão ser realizadas futuramente simulações no extrator de discos rotativos para otimização do processo para crudes pesados, sendo os resultados facultados, posteriormente, à Petrogal. Durante a realização do trabalho foi atingido um modelo real representativo da unidade de destilação atmosférica e a vácuo da Petrogal, podendo obter-se resultados para qualquer tipo de crude, fazendo apenas variar o assay da corrente de alimentação.

Relatório de estágio prática profissional e investigação educacional

Relatório de Estágio apresentado na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ensino de Física e de Química

SEROLOGICAL SURVEY OF Ehrlichia SPECIES IN DOGS, HORSES AND HUMANS: ZOONOTIC SCENERY IN A RURAL SETTLEMENT FROM SOUTHERN BRAZIL

SUMMARY The aims of this study were to determine the seroprevalence of Ehrlichia spp. and risk factors for exposure in a restricted population of dogs, horses, and humans highly exposed to tick bites in a Brazilian rural settlement using a commercial ELISA rapid test and two indirect immunofluorescent assays (IFA) with E. canis and E. chaffeensis crude antigens. Serum samples from 132 dogs, 16 horses and 100 humans were used. Fifty-six out of 132 (42.4%) dogs were seropositive for E. canis. Dogs > one year were more likely to be seropositive for E. canis than dogs ≤ one year (p = 0.0051). Ten/16 (62.5%) and 8/16 (50%) horses were seropositive by the commercial ELISA and IFA, respectively. Five out of 100 (5%) humans were seropositive for E. canis and E. chaffeensis. Rhipicephalus sanguineus (n = 291, 97.98%) on dogs and Amblyomma cajennense (n = 25, 96.15%) on horses were the most common ticks found. In conclusion, anti-Ehrlichia spp. antibodies were found in horses; however, the lack of a molecular characterization precludes any conclusion regarding the agent involved. Additionally, the higher seroprevalence of E. canis in dogs and the evidence of anti-Ehrlichia spp. antibodies in humans suggest that human cases of ehrlichiosis in Brazil might be caused by E. canis, or other closely related species.

OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST

SUMMARYSchistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively).