Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-08T05:23:39Z (GMT). No. of bitstreams: 1 Jacinto_RogeriodeCastilho_D.pdf: 1193443 bytes, checksum: cc178c0781498658e7784945f13b0757 (MD5) Previous issue date: 2007 / Resumo: Os objetivos deste estudo foram analisar a microbiota de canais radiculares com necrose e com lesão periapical de dentes sintomáticos e assintomáticos; quantificar a presença de endotoxinas; correlacionar a presença de bactérias específicas e a quantidade de endotoxinas com os sinais e sintomas de origem endodôntica; e investigar a suscetibilidade antimicrobiana de bactérias anaeróbias estritas isoladas dos canais radiculares contra 8 antibióticos, usando o E-test. Amostras microbiológicas foram coletadas de 90 canais radiculares com polpa necrosada e processadas por meio de técnicas microbiológicas. Outras 50 amostras foram obtidas de canais radiculares necrosados, sintomáticos e assintomáticos para realização do teste cromogênico para quantificação das endotoxinas. Análise estatística foi feita pelos testes x2 de Person ou de Fisher. Um total de 400 isolados clínicos foi encontrado, os quais pertenciam a 69 diferentes espécies e 22 diferentes gêneros. Oitenta por cento das bactérias eram anaeróbias estritas e F. nucleatum foi a espécie predominante. Canais radiculares de dentes sintomáticos apresentaram uma predominância de anaeróbios estritos e um número maior de espécies por canal radicular em relação aos dentes assintomáticos. Foi observada uma relação entre grupos microbianos específicos, principalmente anaeróbios Gram-negativos e a presença de dor espontânea ou dor prévia, dor à percussão, dor à palpação e edema. Endotoxinas foram encontradas em altas concentrações em canais radiculares de dentes sintomáticos e houve uma correlação positiva entre os sinais e sintomas e a concentração de endotoxinas. Amoxicilina, amoxicilina associada ao ácido clavulânico e cefaclor foram efetivos contra todas as cepas testadas. Os resultados sugerem que bactérias específicas e endotoxinas estão associadas aos sinais e sintomas de dentes com canais infectados e lesão periapical e que, a maioria das espécies anaeróbias testadas foi suscetível aos antibióticos estudados / Abstract: The aim of this study was to analyse the microflora isolated from infected root canals of symptomatic or asymptomatic teeth, to quantify the presence of endotoxins; to correlate the presence of specific bacteria and the amount of endotoxins with endodontic symptomatology; and to investigate the antibiotic susceptibility of anaerobic bacteria isolated from infected teeth with periapical lesions against 8 antibiotics through the E-test. Microbial samples were taken from 90 root canals of teeth with necrotic dental pulp, and analysed using rigorous culture procedures. Other 50 samples were collected from infected symptomatic or asymptomatic root canals in order to be analysed by a chromogenic test for the endotoxin quantification. Statistical analysis used a Pearson x2 test or a one-sided Fisher's Exact test, as appropriate. A total of 400 cultivable isolates were recovered from 69 different microbial species and 22 different genera. Eight per cent of the bacteria were were strict anaerobes and F. nucleatum was the most frequently isolated species. Root canals from symptomatic teeth harboured more obligate anaerobes and a larger number of bacterial species than the asymptomatic teeth. Relationships were found between specific microorganisms, especially Gram-negative anaerobes and the presence of pain or history of pain, tenderness to percussion, pain to palpation and swelling. High concentrations of endotoxins were found in root canals of symptomatic teeth and there was a positive correlation between endodontic signs and symptoms and the concentration of endotoxins in infected root canals. Amoxicillin, amoxicillin + clavulanate and cephaclor were effective against all the strains tested. Our results suggested that specific bacteria and endotoxins are associated with endodontic symptoms of infected teeth and that the majority of the anaerobic species tested was susceptible to all antibiotics studied. / Doutorado / Endodontia / Doutor em Clínica Odontológica

This study examined the effects of blood extraction on the survival of horseshoe crabs and performed a preliminary investigation into amebocyte maintenance in vitro. Hemolymph volume of L. polyphemus was estimated over a representative size range of adults. Hemolymph volume expressed as a percentage of wet body weight was 25 ± 2.2% (mean ± S.D.) for males and 25 ± 5.1% for females. Mortality associated with blood extraction was evaluated for horseshoe crabs bled 0, 10, 20, 30, and 40% of their estimated hemolymph volume (unstressed group, N = 200). Mortality associated with the same bleeding levels was evaluated in horseshoe crabs that underwent simulated transport and handling procedures of the biomedical industry's bleeding process (stressed group, N = 195). Mortality rates of the unbled crabs were not significantly different between the stressed group and unstressed group. Of the bled animals, there was a higher (8.3%) mortality rate in the stressed group, than that (0%) in the unstressed group (P < 0.0001). Within the stressed group, mortality was significantly associated with bleeding (P = 0.0088). Horseshoe crab serum and a variety of standard insect cell culture media were evaluated for their effects on amebocyte morphology and viability after 7 days of maintenance in vitro. Horseshoe crab serum-supplemented cultures had significantly higher cell viability than serum-free cultures (N = 6; P = 0.0147). Significant differences in amebocyte viability were identified among the six insect cell culture media tested (N = 36; P < 0.0001), with the highest amebocyte viability of 77.2 ± 5.1% (mean ± S.D.) in Grace's Insect Medium without serum. Information gained from this study provides guidance on altering biomedical bleeding protocols to decrease horseshoe crab stress and mortality, and advances information on amebocyte culture medium selection, both of which contribute to decreasing the biomedical industry's impact on the horseshoe crab population. / Master of Science

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-28T12:10:57Z No. of bitstreams: 1 ricardo-cristiano-de-souza-brum.pdf: 1743762 bytes, checksum: 0d2663a8b1457e7b609cb7288212d4da (MD5) / Made available in DSpace on 2012-11-28T12:10:57Z (GMT). No. of bitstreams: 1 ricardo-cristiano-de-souza-brum.pdf: 1743762 bytes, checksum: 0d2663a8b1457e7b609cb7288212d4da (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Nos últimos anos, as farmacopéias e principais agências regulatórias para produtos farmacêuticos e biofarmacêuticos exigem cada vez mais em suas monografias a aplicação do método para detecção de endotoxinas bacterianas pelo lisado de amebócitos de Limulus(LAL) no controle de pirogênio de produtos terminados parenterais. O objetivo do presente estudo foi padronizar e validar o ensaio de LAL para o biofármaco alfainterferona 2b humana recombinante, produzido em E. coli. Foram empregados os métodos Gel-Clot e Cinético-Cromogênico (semiquantitativo e quantitativo, respectivamente). Para o método Cinético-Cromogênico, a máxima diluição válida (MDV) foi calculada para cada apresentação com base na concentração do produto e a sensibilidade do teste (3 MUI, MDV: 1:3.888 e 10 MUI, MVD: 1:12.962). Diluições seriadas abaixo da MDV foram avaliadas emtriplicata para a verificação dos interferentes, sendo eleitas as diluições de 1:80 e1:100 que exibiram a melhor recuperação no controle positivo do produto. Para oensaio Gel-Clot na apresentação de 3 MUI (MDV: 1:17) foi estipulada a diluição de 1:8para a validação dos testes de rotina. Durante a validação dos ensaios, foi utilizada uma curva-padrão de endotoxina nas concentrações de 0,005 – 50 EU/ml e avaliados o seucoeficiente de correlação (R) e o desvio-padrão relativo. Os critérios de desempenho do método cinético (linearidade, especificidade, exatidão, repetibilidade, reprodutibilidade) foram atendidos de acordo com os parâmetros farmacopéicos e regulatórios (ANVISA e FDA), garantindo desta forma a robustez necessária para que o método de LAL possa ser incluído nos ensaios de rotina do Laboratório de Controle de Qualidade. / In recent years, the Pharmacopean, technical reports and international guides for pharmaceuticals products requires each time more intheir monographies the application of Limulus Ambocytes Lysateendotoxins assay (LAL) for release and pyrogen control in parenteral finished products. The objective of thisstudy was the standardization and validation of the LAL test for the human recombinant biopharmaceutical interferon alpha 2b, produced in E. coli, were used Gel-Clot and Chromogenics Kinetic methods (semi-quantitative and quantitative). In the case of Chromogenics method test, the maximum valid dilution (MDV) was calculated for each presentation based on the concentration of product (3 MUI, MDV: 1:3,888 and 10 MUI, MVD: 1:12,962), serial dilutions under the MDV (1:80) were evaluated in triplicate to detect interferences. For the gel-clot test for the 3 MUI presentation (MDV: 1:17) 1:8 dilution was setfor interferences detection test. For tests’ validation, several dilutions were performedusing standard endotoxin concentrations in 0,005 to 50 EU / ml to confirm the criteria for the methods performance (linearity, specificity, accuracy, reply, reproducibility). The results of validation showed that all pharmacopeia and regulatory parameters (ANVISA) studied, are compatible with the MDV used for each studied presentation of the human recombinant biopharmaceuticals interferon alpha 2b and may be used in quality control.

Empregando a técnica de biologia molecular Checkerboard DNA-DNA Hybridization e o teste Limulus Amebocyte Lysate, os objetivos do presente estudo clínico randomizado in vivo foram avaliar, em bráquetes ortodônticos metálicos: 1) A presença de 16 espécies de microrganismos periodontopatogênicos Gram-negativos pertencentes aos complexos laranja e vermelho, por meio de sondas de DNA; 2) A quantidade de endotoxina bacteriana presente; e 3) A eficácia da utilização do gluconato de clorexidina a 0,12%, sob a forma de bochechos, na redução da contaminação pelas 16 espécies de microrganismos periodontopatogênicos Gram-negativos e na redução da quantidade de endotoxina bacteriana. Participaram do estudo 33 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colocados randomicamente 3 bráquetes metálicos novos nos pré-molares. Os pacientes do Grupo Controle (n=17) fizeram 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=16) fizeram bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard®), da mesma forma que o grupo Controle. Decorridos 30 dias, os 3 bráquetes foram removidos de cada paciente e processados para a detecção dos microrganismos, pela técnica Checkerboard DNA-DNA Hybridization, e para a quantificação da endotoxina bacteriana por meio do teste Limulus Amebocyte Lysate. Os resultados obtidos foram analisados por meio dos testes não-paramétricos de Kruskal-Wallis, Mann-Whitney e pós-teste de Dunn, utilizando os softwares SAS e Graphpad Prism. O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que todos os bráquetes dos pacientes do Grupo Controle encontravam-se densamente contaminados pelos microrganismos avaliados. Nesse grupo, as espécies bacterianas do complexo laranja apresentaram-se em maiores quantidades, em relação às espécies do complexo vermelho (p<0,01). A mediana da quantidade de endotoxina para este grupo foi de 0,6673 EU/ml. Quando comparado ao grupo Controle, observou-se que o número total de microrganismos no grupo Experimental foi estatisticamente menor, com mediana de 29.150.000 no grupo Controle e de 13.130.000 no grupo Experimental (p=0,01). Quando os microrganismos foram avaliados por complexos, foi observada diferença estatisticamente significante entre os grupos Controle e Experimental para o complexo laranja (p=0,04), com contagens menores de bactérias após os bochechos com clorexidina. Por outro lado, observou-se que a quantidade de endotoxina no grupo Experimental foi maior, com mediana de 1,2199 EU/ml (p=0,02). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos periodontopatogênicos Gram-negativos, em pacientes portadores de aparelhos ortodônticos fixos. No entanto, em função do aumento da quantidade de endotoxina bacteriana após o uso dos bochechos com clorexidina, estudos adicionais são necessários com a finalidade de desenvolver procedimentos clínicos ou agentes antimicrobianos que tenham ação sobre a endotoxina presente nos bráquetes metálicos. / Using the biomolecular technique Checkerboard DNA-DNA Hybridization and the Limulus Amebocyte Lysate (LAL) assay, the purposes of the present randomized clinical study were to evaluate in orthodontic metallic brackets: 1) The presence of 16 Gram-negative periodontopathogenic microbial species of the orange and red complexes by using DNA probes; 2) The amount of bacterial endotoxin; and 3) The efficacy of 0.12% chlorhexidine gluconate mouthwashes in reducing the contamination by the evaluated microbial species and the amount of bacterial endotoxin. Thirty-three 11-33-year-old patients undergoing orthodontic treatment with fixed appliances were enrolled in the study and all subjects had 3 new metallic brackets bonded to different premolars in a randomized manner. The patients in the Control group (n=17) were instructed to use a placebo mouthwash twice a week, while those in the Experimental group (n=16) were instructed to use a 0.12% chlorhexidine gluconate mouthwash (Periogard®) in the same way. After 30 days, the 3 brackets were removed from each patient and processed for detection of the microorganisms by the Checkerboard DNADNA hybridization technique, and for quantification of bacterial endotoxin by the LAL assay. The data were analyzed statistically by the non-parametric Mann-Whitney, Kruskal-Wallis and Dunn\'s post tests using SAS and GraphPad Prism softwares. A significance level of 5% was set for all analyses. The brackets of the patients in the Control group were densely contaminated by the evaluated microbial species. In this group, the number of bacterial species of the orange complex was larger compared to the number of bacterial species of the red complex (p<0.01). The median of the amount of bacterial endotoxin for this group was 0.6673 EU/ml. The Experimental group had a significantly smaller number of microorganisms than the Control group (median 13,130,000 versus 29,150,000; p=0.01). When the microorganisms were analyzed by complex, there was statistically significant difference between the Control and Experimental groups for the orange complex (p=0.04) with smaller counts of bacteria after use of chlorhexidine oral rinses. On the other hand, there was a greater amount of bacterial endotoxin in the Experimental (median of 1,2199 EU/ml; p=0.02). In conclusion, 0.12% chlorhexidine oral rinse can be useful in the clinical practice to reduce the levels of Gram-negative periodontopathogenic microorganisms in patients with fixed orthodontic appliances. Considering the increase in the amount of bacterial endotoxin after use of chlorhexidine oral rinses, further research is necessary to develop clinical procedures or antimicrobial agents with action against the endotoxin in the metallic brackets

Empregando a técnica de biologia molecular Checkerboard DNA-DNA Hybridization e o teste Limulus Amebocyte Lysate, os objetivos do presente estudo clínico randomizado in vivo foram avaliar, em bráquetes ortodônticos metálicos: 1) A presença de 16 espécies de microrganismos periodontopatogênicos Gram-negativos pertencentes aos complexos laranja e vermelho, por meio de sondas de DNA; 2) A quantidade de endotoxina bacteriana presente; e 3) A eficácia da utilização do gluconato de clorexidina a 0,12%, sob a forma de bochechos, na redução da contaminação pelas 16 espécies de microrganismos periodontopatogênicos Gram-negativos e na redução da quantidade de endotoxina bacteriana. Participaram do estudo 33 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colocados randomicamente 3 bráquetes metálicos novos nos pré-molares. Os pacientes do Grupo Controle (n=17) fizeram 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=16) fizeram bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard®), da mesma forma que o grupo Controle. Decorridos 30 dias, os 3 bráquetes foram removidos de cada paciente e processados para a detecção dos microrganismos, pela técnica Checkerboard DNA-DNA Hybridization, e para a quantificação da endotoxina bacteriana por meio do teste Limulus Amebocyte Lysate. Os resultados obtidos foram analisados por meio dos testes não-paramétricos de Kruskal-Wallis, Mann-Whitney e pós-teste de Dunn, utilizando os softwares SAS e Graphpad Prism. O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que todos os bráquetes dos pacientes do Grupo Controle encontravam-se densamente contaminados pelos microrganismos avaliados. Nesse grupo, as espécies bacterianas do complexo laranja apresentaram-se em maiores quantidades, em relação às espécies do complexo vermelho (p<0,01). A mediana da quantidade de endotoxina para este grupo foi de 0,6673 EU/ml. Quando comparado ao grupo Controle, observou-se que o número total de microrganismos no grupo Experimental foi estatisticamente menor, com mediana de 29.150.000 no grupo Controle e de 13.130.000 no grupo Experimental (p=0,01). Quando os microrganismos foram avaliados por complexos, foi observada diferença estatisticamente significante entre os grupos Controle e Experimental para o complexo laranja (p=0,04), com contagens menores de bactérias após os bochechos com clorexidina. Por outro lado, observou-se que a quantidade de endotoxina no grupo Experimental foi maior, com mediana de 1,2199 EU/ml (p=0,02). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos periodontopatogênicos Gram-negativos, em pacientes portadores de aparelhos ortodônticos fixos. No entanto, em função do aumento da quantidade de endotoxina bacteriana após o uso dos bochechos com clorexidina, estudos adicionais são necessários com a finalidade de desenvolver procedimentos clínicos ou agentes antimicrobianos que tenham ação sobre a endotoxina presente nos bráquetes metálicos. / Using the biomolecular technique Checkerboard DNA-DNA Hybridization and the Limulus Amebocyte Lysate (LAL) assay, the purposes of the present randomized clinical study were to evaluate in orthodontic metallic brackets: 1) The presence of 16 Gram-negative periodontopathogenic microbial species of the orange and red complexes by using DNA probes; 2) The amount of bacterial endotoxin; and 3) The efficacy of 0.12% chlorhexidine gluconate mouthwashes in reducing the contamination by the evaluated microbial species and the amount of bacterial endotoxin. Thirty-three 11-33-year-old patients undergoing orthodontic treatment with fixed appliances were enrolled in the study and all subjects had 3 new metallic brackets bonded to different premolars in a randomized manner. The patients in the Control group (n=17) were instructed to use a placebo mouthwash twice a week, while those in the Experimental group (n=16) were instructed to use a 0.12% chlorhexidine gluconate mouthwash (Periogard®) in the same way. After 30 days, the 3 brackets were removed from each patient and processed for detection of the microorganisms by the Checkerboard DNADNA hybridization technique, and for quantification of bacterial endotoxin by the LAL assay. The data were analyzed statistically by the non-parametric Mann-Whitney, Kruskal-Wallis and Dunn\'s post tests using SAS and GraphPad Prism softwares. A significance level of 5% was set for all analyses. The brackets of the patients in the Control group were densely contaminated by the evaluated microbial species. In this group, the number of bacterial species of the orange complex was larger compared to the number of bacterial species of the red complex (p<0.01). The median of the amount of bacterial endotoxin for this group was 0.6673 EU/ml. The Experimental group had a significantly smaller number of microorganisms than the Control group (median 13,130,000 versus 29,150,000; p=0.01). When the microorganisms were analyzed by complex, there was statistically significant difference between the Control and Experimental groups for the orange complex (p=0.04) with smaller counts of bacteria after use of chlorhexidine oral rinses. On the other hand, there was a greater amount of bacterial endotoxin in the Experimental (median of 1,2199 EU/ml; p=0.02). In conclusion, 0.12% chlorhexidine oral rinse can be useful in the clinical practice to reduce the levels of Gram-negative periodontopathogenic microorganisms in patients with fixed orthodontic appliances. Considering the increase in the amount of bacterial endotoxin after use of chlorhexidine oral rinses, further research is necessary to develop clinical procedures or antimicrobial agents with action against the endotoxin in the metallic brackets

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Oceanografia Física, Química e Geológica, Instituto de Oceanografia, 2007. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2013-02-18T15:57:58Z No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-12T16:59:29Z (GMT) No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) / Made available in DSpace on 2013-06-12T16:59:29Z (GMT). No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) Previous issue date: 2007 / Florações de cianobactérias têm sido freqüentemente encontradas nas águas do estuário da Lagoa dos Patos (RS). Um das principais cianobactérias de ocorrência local é o gênero Microcystis. As células de Microcystis têm tolerância a baixas salinidades, porém com o aumento abrupto da salinidade, as células podem sofrer lise. Muitas vezes, em função da hidrodinâmica local, essas florações de cianobactérias podem atingir a região da Praia do Cassino, possibilitando o contato com banhistas. Devido à natureza Gram-negativa da composição da parede celular das cianobactérias, esse contato com as células pode resultar em na ocorrência de casos de irritação epicutânea e reações alérgicas. O agente causador é o lipídeo A (endotoxina), encontrado no lipopolissacarídeo (LPS) da membrana externa das cianobactérias. Com o objetivo de detectar concentrações ambientais de LPS das florações de cianobactérias foi utilizado o método cromogênico cinético de ponto final do teste do Lisado do Amebócito de Limulus polyphemus (teste LAL). O teste LAL foi realizado em amostras de água de superfície coletadas nas regiões de São Lourenço do Sul, Praia do Laranjal (Pelotas), Museu Oceanográfico da FURG, Yacht Club do Rio Grande e Praia do Cassino (Rio Grande), entre os meses de dezembro de 2005 e março de 2006. Nestas amostras também foram estimados a abundância celular dos principais grupos do fitoplâncton, a concentração de clorofila-a, e medida a salinidade local no momento da coleta. Para avaliar a possível interferência dos sais nos resultados do teste LAL, também foram realizados cultivos da cianobactéria Microcystis crescendo em diferentes salinidades, onde também foram estimados a abundância celular, e as concentrações de clorofila-a, LPS e microcistinas. Os ajustes metodológicos realizados no teste LAL durante o trabalho resultaram em uma sensibilidade para sua aplicação nas amostras ambientais. As maiores concentrações de endotoxinas detectadas nas amostras ambientais (109,5 EU.mL-1, 71,8 EU.mL-1 e 93,7 EU.mL-1) se correlacionaram positivamente com as maiores abundâncias celulares (aproximadamente 600.000 células.mL-1, 400.000 células.mL-1 e 300.000 células.mL-1, respectivamente), todas com a presença da cianobactéria Microcystis. / Cyanobacterial blooms have been frequently observed in the waters of the Patos Lagoon estuary (RS). One of the main cyanobacterium of local occurrence is the genus Microcystis. The Microcystis cells present some tolerance to low salinity, but with the rapid salt increase the cells may suffer breakdown. Usually due to the local hydrodynamics these blooms may reach the recreational waters of the Cassino Beach, exposing swimming bathers to its contact. Due to the Gram-negative nature of the cyanobacterial cell membrane, this contact with the cells may result in case occurrences of epicutaneous irritation and allergic reactions. The causing agent is the lipid A (endotoxin), found on the lipopolysaccharide (LPS) of the external membrane of cyanobacteria. With the objective to detect environmental concentrations of LPS from cyanobacterial blooms, it was used the kinetic chromogenic endpoint method of the Limulus polyphemus Amebocyte Lisate test (LAL test). The LAL test was performed in surface water samples collected from the regions of São Lourenço do Sul, Laranjal Beach (Pelotas), FURG’s Oceanographic Museum, Yacht Club Rio Grande, and Cassino Beach (Rio Grande) betwen December 2005 and March 2006. In these samples it was also estimated the cellular abundance of the main phytoplanktonic groups, the chlorophyll-a concentration, and measured the local salinity. To evaluate the possible interference of salinity in the LAL test results, it was also cultivated the cyanobacterium Microcystis growing in different salinities. From these cultures it was estimate the cellular abundance, and chlorophyll-a, LPS and microcystin concentrations.. The methodological adjustments performed in the LAL test during this work resulted in a sensitivity for its application in environmental samples. The highest endotoxin concentrations detected in the environmental samples (109.5 EU.mL-1, 71.8 EU.mL-1 and 93.7 EU.mL-1) have positively correlated with the highest cell abundances (approximately 600,000 cells.mL-1, 400,000 cells.mL-1 and 300,000 cells.mL-1, respectively), all of them with the presence of the cyanobacteria Microcystis.

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.