Tesi sul tema "Cyanobacteria Toxicology"

Bibliography: leaves 121-139. The aim of this study was to determine the extent to which protein synthesis inhibition, lowered glutathione (GSH) levels and toxin metabolism contribute to the toxicity of cyclindrospermopsin. Both hepatocyte cultures and reticulocyte lysates were utilized as in vitro tools of investigation. The findings imply that the inhibition of protein synthesis by direct action of the toxin cannot be considered a primary cause of hepatocyte cell death over an acute time frame. Cytochrome P450-derived metabolites may play a crucial role in cytotoxicity, and the toxicity process does not appear to involve oxidative damage.

Cyanobacteria are known to produce a variety of toxic compounds. β-N-methylamino-L-alanine (BMAA) is one of the neurotoxins produced by most cyanobacteria. BMAA has been implicated in amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS / PDC) and was suggested to contribute to this pathology after biomagnification and slow release of BMAA from a protein associated form. The uptake and accumulation of BMAA by the aquatic macrophyte Ceratophyllum demersum has recently been shown, but the consumption of aquatic macrophytes by humans is not typical. The uptake by, and accumulation in, crop plants (Nasturtium officinale and Daucus carota) was therefore investigated so as to establish the existence of any risk to humans from the consumption of plants irrigated with water from dams with high cyanobacterial biomass and therefore high BMAA levels. After the exposure to the BMAA through the growth medium, BMAA had no effect on growth and development of N. officinale and D. carota. The uptake and bioaccumulation of BMAA was observed in N. officinale and D. carota, and was found to be concentration-dependent. Both free and bound cellular BMAA was detected following BMAA exposure through the growth medium. The photosynthetic apparatus of N. officinale was not significantly damaged. The uptake and accumulation of BMAA in edible terrestrial plants may constitute another route of human exposure to BMAA; it may now be prudent to avoid spray irrigation of edible plants with waters from dams with high cyanobacterial biomass and therefore high BMAA levels. After uptake by plants, the cyanotoxins may induce oxidative stress. A recent study showed that BMAA has a significant inhibitory effect on the oxidative stress enzymes in C. demersum. Therefore, the toxicological effects on selected plants were investigated by a range of biochemical enzyme assays in order to establish the plant stress response to exogenous BMAA. The inhibition of antioxidant enzymes upon exposure of N. officinale to BMAA through the growth medium was observed. The inhibition of antioxidant defence enzymes by BMAA correlated with the BMAA bioaccumulation in N. officinale. Further investigations are needed to analyze the uptake, accumulation, and ecotoxicology of BMAA in other crop plants, and to examine the fate of BMAA in these plants particularly its distribution and metabolism.

As cianobactérias são micro-organismos reconhecidos por seu potencial em produzir cianotoxinas que afetam não só o ecossistema e a outros organismos dos ambientes aquáticos, mas também aos seres humanos, agindo em diversos órgãos e tecidos. Cerca de 600 metabólitos secundários produzidos por cianobactérias já foram descritos na literatura, sendo que muitos deles possuem potencial biológico. Os peptídeos de baixo peso molecular, produzidos por cianobactérias chamados cianopeptídeos, dos quais se podem citar as anabaenopeptinas, aeruginosinas, microviridinas, cianopeptolinas e microgininas, são compostos provindos do metabolismo secundário de cianobactérias e são descritos como inibidores de proteases e fosfatases em alguns sistemas biológicos. Sendo assim, o objetivo do estudo foi identificar a ocorrência de cianopeptídeos em cianobactérias brasileiras e testar seu efeito de inibição da atividade sobre enzimas proteases. Os objetos de estudo foram: uma linhagem da espécie Sphaerospermopsis torques-reginae, duas de Cylindrospermopsis raciborskii, duas de Microcystis sp, uma de Oscillatoria e uma de Pseudanabena sp. A partir dos resultados obtidos pode-se afirmar que do total de linhagens analisadas, ao menos 4 destas parecem produzir os cianopeptídeos de interesse, quando avaliados por cromatografia líquida de alta eficiência com detector de arranjo de diodos (HPLC-DAD). Em seguida, culturas de duas linhagens de C. raciborskii (uma produtora e outra não produtora de saxitoxina) foram amostradas a cada 3 dias, para a avaliação do crescimento celular, produção de cianopeptídeos e de saxitoxina e suas variantes. Não foi possível confirmar a produção de cianopeptídeos nas duas linhagens desta espécie. Por outro lado, foi evidenciado um aumento da produção de saxitoxinas quando cultivada num meio sem nitrogênio em comparação com a condição controle. Quando analisada a linhagem de Microcystis sp. (LTPNA 08), produtora de microcistinas, foi possível confirmar por cromatografia líquida acoplada a espectrometria de massas (LC-MS) a produção de dois cianopeptídeos, sendo estes, duas microgininas. Desta forma, desenvolveu-se um método cromatográfico para a separação desses compostos e purificação por cromatografia líquida semi-preparativa, onde foi possível a obtenção de frações enriquecidas de microgininas e microcistinas-RR e LR, com cerca de 70%, 86% e 97% de pureza. Por fim, realizaram-se ensaios avaliando a atividade da enzima conversora de angiotensina (ECA) e aminopeptidase M (AMP M) com as frações isoladas de microgininas e microcistina-LR. A atividade da ECA foi ± 50% inibida pelas frações testadas de cianopeptídeos, microcistina-LR isolada e microcistins-lR comercial. A atividade da AMP M foi 100% e 24,5% inibida quando incubada com 20 µM da fração de microgininas e microcistina-LR, respectivamente. Desta forma, as microgininas isoladas de cianobactérias brasileiras, mostraram-se como importantes inibidores da ECA e AMP M, podendo, no futuro, serem utilizadas como compostos para o tratamento de patologias cardiovasculares e renais.
Cyanobacteria are micro-organisms recognized for their potential to produce cyanotoxins that affect not only the ecosystem and other organisms of aquatic environments, but also humans, acting in various organs and tissues. Around 600 secondary metabolites produced by cyanobacteria have been described in the literature; many of them have biological potential. Low molecular weight peptides produced by cyanobacteria are called cyanopeptides, among them we can cite the anabaenopeptins, aeruginosins, microviridins, cyanopeptolins and microginins, these compounds are derived from secondary metabolisms of cyanobacteria and apparently cause inhibition of proteases and phosphatases in some biological systems. Therefore, this study targeted the identification of the occurrence of cyanopeptides in Brazilian cyanobacterias and testing its effect on the inhibition of proteases activity. The targets of study were: a strain of species Sphaerospermopsis torques-reginae, two strains of Cylindrospermopsis raciborskii, two strains of Microcystis sp. and, one strain of Pseudanabena and Oscillatoria sp. From the results obtained in this study it can be stated that at least four of the total of strains analyzed appear to produce cyanopeptides of interest, when analyzed by high-performance liquid chromatography whit phodo diodo array detector (HPLC-PDA). The cultures of two strains of C. raciborskii (a producer of saxitoxin and a non-producer) were sampled every 3 days for assessment of cell growth, production of cyanopeptides and saxitoxins. It was not possible to confirm the production of cyanopeptides in strains of this species. Nevertheless, an increase in production of saxitoxins was shown when cultivated in an environment without nitrogen, as compared to the control condition. When the strain of Microcystis sp. (LTPNA 08), a producer of microcystins, was analyzed, the production of two cyanopeptides was confirmed by using liquid chromatography-mass spectrometry (LC-MS). After confirmation, a method using HPLC-PDA was used to do the separation and purification of these compounds by semi-preparative chromatography, in which it was possible to obtain an enriched fraction of microginins, microcystin-RR and microcystin-LR with approximately 70%, 86% and 97% purity, respectively. Lastly, inhibition experiments were run with angiotensin-converting enzyme (ACE) and aminopeptidase M (AMP M) with the isolated fractions. The microginins fractions, MC-LR commercial and MC-LR isolated, showed an inhibition of ± 50% of the angiotensin-converting enzyme. The activity of AMP M was 100% and 24.5% inhibited when incubated with microginins and microcystin-LR fractions in a concentration of 20 µM, respectively. Thus, isolated microginins from Brazilian cyanobacteria have exhibited properties as potential therapeutic agents in development of inhibitors of ACE and AMP M, which can be a benefit of using these molecules in the treatment of cardiovascular and renal pathologies.

O crescimento desordenado das cidades tem trazido à tona problemas de saneamento e degradação dos recursos naturais, entre eles a água. O despejo de efluentes domésticos e industriais têm ocasionado a eutrofização dos mananciais, culminando com a proliferação dos fitoplânctons. Esta proliferação tem chegado ao ponto, em certos momentos, de acarretar episódios de floração de algas. Entre os organismos fitoplanctônicos que se desenvolvem no ambiente, estão as cianobactérias, com vários gêneros capazes de produzir diversas cianotoxinas, tais como as microcistinas, anatoxinas, cilindrospermopsinas (CY), saxitoxinas (PSPs), entre outras. Com o aumento da freqüência dos episódios de floração de algas, a probabilidade da ocorrência destas toxinas também aumenta. Como conseqüência disto e devido às exigências legais, os corpos dágua devem ser monitorados para garantir a qualidade da água para consumo humano. Com vistas ao monitoramento da presença das cianotoxinas CY e PSPs, foram realizadas investigações em alguns corpos dágua do Estado de São Paulo. Das investigações realizadas, a neosaxitoxina foi identificada pela primeira vez no Reservatório Billings e os congêneres, saxitoxina, goniautoxina 2, goniautoxina 3, foram identificados de forma inédita em amostras de água deste reservatório. Com relação à CY, foi desenvolvido um método analítico, parcialmente validado. Entretanto, esta cianotoxina não foi localizada nas amostras de água ou cianobactérias das águas superficiais dos corpos dágua estudados. Este estudo mostra a importância do monitoramento da qualidade das águas dos mananciais quanto à presença de cianotoxinas, especialmente daqueles corpos dágua com a finalidade do consumo humano.
Cities growth usually occur in an unorganized manner. This tendence can generate a variety of sanitary problems, including the degradation of natural resources, such as water bodies. As a consequence, domestic and industrial efluents cause eutrofication of water reservoir, increasing the natural level of phytoplancton, what may form algal bloom. Among the phytoplanktonic organisms that grow in this modified environment it is found the cyanobacteria. Some of them can produce different types of cyanotoxins such as microcystin, anatoxin, cylindrospermopsin (CY) and saxitoxin (PSPs). The probability of production of these cyanotoxins increase according to frequent occurrence of algal blooms episodes. Consequently, water bodies monitoring becomes important to assure water quality. The aim of this project was to develop a specific method to identify the presence of cyanotoxins CY and to investigate PSPs in water bodies in São Paulo State. The results confirmed the presence of neosaxitoxin (NEO), a toxin of PSPs family. It was the first time that Neo was indentified in Billings Reservoir along with other PSPs types: saxitoxin, gonyautoxin 2, gonyautoxin 3. Although the study also included CY monitoring, CY was not identified in the tested samples. The present study confirmed the importance of continuous searching and monitoring of water bodies to grant quality to water used for human consumption.

A demanda crescente de água doce de boa qualidade são problemas atuais e mundiais, além do descaso com os dejetos lançados nos ambientes aquáticos que comprometem a qualidade dos recursos hídricos. Um dos parâmetros que atesta a potabilidade da água é a presença de cianobactérias e cianotoxinas. Cianobactérias são microrganismos procariontes aeróbicos fotoautróficos que sintetizam as cianotoxinas. Estes compostos podem ser classificados de acordo com seus mecanismos de ação em hepatotóxicos, neurotóxicos e dermatotóxicos. Por sua diversidade, representam diferentes riscos não só ao ecossistema e a outros organismos dos ambientes aquáticos, como também aos seres humanos. Esse projeto visou o isolamento e cultivo de cepas de cianobactérias produtoras de toxinas para a investigação da biossíntese desses compostos. Com este intuito, foram realizadas coletas de água em três reservatórios no estado de São Paulo e um no Paraná. Cepas de cianobactérais foram isoladas, identificadas e analisadas quanto à produção de toxinas. Uma cepa de Microcystis aeruginosa (LTPNA 02) produtora de microcistinas (MC-LR, MC-RR, MC-YR, MC-LF, MC-LW e desm-MC-LR e desm- MC-RR) foi escolhida para ser estudada frente diferentes condições de cultivo e ter o seu crescimento, produção de toxinas e expressão gênica estudados. Foram utilizados os meios de cultura já referidos na literatura: ASM-1 (N:P=1, 10 e 20), MLA (N:P=10), Bold 3N (N:P=16) e BG-11 (N:P=10 e 100). Para acompanhar o crescimento, dois métodos foram utilizados: contagem de células e espectrofotometria. As toxinas foram quantificadas por LC-MS - QTrap. A análise da expressão gênica foi realizada por reação de PCR em tempo real pelo método de quantificação relativa ΔΔCt. Foi observada diferença no crescimento da cepa estudada nos diferentes meios de cultivo empregados. A contagem das células permitiu a identificação das fases logarítmica e total de crescimento. Durante a fase logarítmica, três experimentos demonstraram diferenças estatísticas quando comparadas ao controle (p<0,05). Ao se avaliar o crescimento total, quatro experimentos foram menores (p<0,01). As leituras das absorvâncias e a contagem de células demonstraram alta correlação Para ambas as leituras em 680 nm e 750 nm o coeficiente de correlação (r) esteve entre 0,93 e 0,99. A quantificação das microcistinas (MC) foi realizada por LC-MS - QTrap. Foram quantificadas as variantes MC-LR, MR-RR e MC-YR. Apesar da relação toxina/célula ser distinto para cada experimento, não representou grande variação naqueles realizados com meio ASM-1 (N:P 1; 10 e 20), meio MLA (N:P=10) e BG11(N:P=10). O experimento realizado em Bold3N (N:P=16,6) apresentou menor concentração de toxina/célula e as variantes MC-LR e MC-YR não foram detectadas. Por outro lado, o experimento realizado em BG-11 (N:P=100) apresentou a maior relação toxina por célula. Estes resultados sugerem que o excesso de nitrato seja um fator estressante para o desenvolvimento e crescimento da cepa de M. aeruginosa avaliada e ao mesmo tempo um fator estimulante para a produção das toxinas analisadas. Os experimentos que avaliaram a expressão dos genes 16S e mcyB em relação ao gene da ficocianina (controle endógeno) foram realizados em meio ASM-1 (N:P=10 e 100) e BG 11 (N:P= 10 e 100). Os parâmetros anteriores, como crescimento e produção de toxinas também foram avaliados. Novamente foram encontradas diferenças entre as fases de crescimento e produção de toxina, porém a expressão dos genes avaliados não demonstrou variação significativa entre os experimentos. Porém ambos os genes avaliados demonstraram menor expressão nos experimentos condizidos em (N:P=100).
There is a great concern these days about potable and good quality water due to the increase of the population needs and also to the arising problems with contamination caused by anthropogenic sources. The presence of cyanobacteria and cyanotoxins are some parameters that attest water potability. Cyanobacteria are prokaryotic aerobic photoautotrophic microorganisms that may synthesize cyanotoxins. These compounds can be classified as hepatotoxic, neurotoxic and dermatotoxic according to their action mechanisms. Because of their diversity, they may represent different risks, not only to their ecosystem and other aquatic living organisms, but also to human beings. The aim of this project was the isolation and cultivation of cyanotoxin-producing cyanobacteria for further investigation on the biosynthesis of these compounds. Water samples from three different reservoirs in São Paulo state and one in Paraná state were collected in order to isolate cyanobacteria strains and accomplish their identification and to evaluate the toxin production. The Microcystis aeruginosa (LTPNA 02) microcystin producer strain (MCLR, MC-RR, MC-YR, MC-LF, MC-LW, desm-MC-LR and desm-MC-RR) was chosen to be grown in different cultivation conditions and later analyzed for its growth rate, toxin production and gene expression. All culture media used in this research were chosen according to the literature: ASM-1 (N:P=1, 10 and 20), MLA (N:P=10), Bold 3N (N:P=16) and BG-11 (N:P=10 and 100). To evaluate growth rate, two techniques were used: cell counting and absorbance determination in two different wavelengths (680 nm and 750 nm). Toxins were quantified by LC-MS in a hybrid triple-quadrupole instrument (Qtrap). Gene expression was assessed by real time PCR, using the ΔΔCt relative quantification method. Cell counting allowed total growth and logarithmic phase identification. During the last, three experiments showed statistical difference from control group (p<0,05). Four experiments resulted in a lower total growth rate (p<0,05). A high correlation between cell counting and absorbance levels was found for both wavelengths tested. Correlation coefficients (r) were from 0,93 to 0,99. Three microcystin variants (MC-LR, MR-RR e MC-YR) were quantified by LC-MS. The toxin content per cell was calculated and showed no statistc variation among those experiments performed on ASM-1 (N:P 1; 10 and 20), MLA (N:P=10) and BG-11 (N:P=10). The lowest toxin/cell concentration was found for Bold3N (N:P=16,6) medium, where MC-LR and MC-YR production was not detected. On the other hand, the experiment with BG-11 (N:P=100) medium showed the highest toxin/cell content. These results suggest that high levels of nitrate in the culture medium may be a stressing factor for the development and growth of the M. aeruginosa tested strain, as well as a disturbing factor for microcystin production. Gene expression experiments regarding 16S and mycB genes using the phycocyanin gene as endogen control were performed on ASM-1 (N:P=10 and 100) and BG 11 (N:P= 10 and 100) media, along with the evaluation of growth rate and toxin production. Differences between growth rates and toxin production were once more observed, however gene expression did not show a significant variation among experiments.

Florações de cianobactérias são facilmente encontradas, devido ao crescente aporte de nutrientes nos corpos de águas naturais e artificiais, ocasionado pelos acelerados processos de eutrofização frutos da ocupação urbana e rural sem a observação de critérios mínimos. Microcystis aeruginosa é uma espécie de cianobactéria potencialmente produtora de cianotoxinas, comumente associada a casos de intoxicação em escala mundial. Novas tecnologias para o tratamento de água têm sido implementadas para cumprimento dos padrões de potabilidade exigidos pela legislação. O presente trabalho buscou analisar a produção científica mundial relacionada ao tratamento de água com presença de M.aeruginosa e MCLR, buscando identificar o estado da arte, além de embasar a discussão dos métodos propostos. O presente estudo está dividido em três artigos, no primeiro realizou-se uma análise bibliométrica das pesquisas mundiais relacionadas à cianobactérias, cianotoxinas e o tratamento de água, a partir da base de dados Scopus. No segundo artigo buscou-se avaliar a aplicabilidade dos AOPs UV-C e UV-C/H2O2 na degradação de Microcystis aeruginosa BB005 e MC-LR, e a análise dos efeitos da adição de nanopartículas de Ag, com base em um produto comercial composto por peróxido de hidrogênio (H2O2) e nanopartículas de prata (NAg). No terceiro artigo buscou-se avaliar a qualidade da água produzida a partir de ensaios de toxicidade aguda com Daphnia magna. Os resultados indicam que a fotólise e o processo UV-C/H2O2 apresentaram resultados satisfatórios, sendo uma alternativa eficiente. Porém, os resultados dos ensaios de ecotoxicidade inferem que estes tratamentos utilizados com a finalidade de degradar M. aeruginosa e MC-LR, possuem potencial de geração de subprodutos de degradação tóxicos: os ensaios com D. magna demonstraram toxicidade mesmo quando a água submetida a fotólise foi diluída quatro vezes. Com relação ao processo UV-C/H2O2 (sem e com adição de NAg), a amostra foi tóxica quando não diluída. Já quando empregada as NAg combinadas a radiação UV-C, esta apresentou toxicidade extremamente alta, afetando a mobilidade de todos os organismos teste em todas as diluições (até 16 x).
Cyanobacterial blooms are easily found, due to the increasing nutrient supply in natural and artificial bodies of water, caused by the accelerated processes of eutrophication, fruits of urban and rural occupation without observing minimum criteria. Microcystis aeruginosa is a specie of cyanobacteria that are potentially cyanotoxin-producing, commonly associated with cases of worldwide intoxication. New technologies for water treatment have been implemented to meet the standards of potability required by legislation. The present study looked for analyze the world scientific production related to the treatment of water with presence of M. aeruginosa and MC-LR, seeking to identify the state of the art, besides supporting the discussion of the proposed methods. The present study is divided into three articles, the first one was a bibliometric analysis of the world-wide research related to cyanobacteria, cyanotoxins and water treatment, from the Scopus database. In the second article evaluated the aplicability of UV-C e UV-C/H2O2 AOPs on degradation of Microcystis aeruginosa BB005 and MC-LR, and the analysis of effects Ag nanoparticles addition, based on a commercial product composed of hydrogen peroxide (H2O2) and silver nanoparticles (NAg). In the trird article evaluated the water quality produced, from acute toxicity tests with Daphnia magna. The results indicate that photolysis and the UV-C/H2O2 process presents satisfactory results, being an efficient alternative. However, the results of the ecotoxicity assays infer that these treatments used for the purpose of degrading M. aeruginosa and MCLR, have potential to generate toxic degradation byproducts: the D. magna assays demonstrated toxicity even when the water submitted to photolysis was diluted four times. Regarding the UV-C/H2O2 process (without and with NAg addition), the sample was toxic when undiluted. When NAg was used in combination with UV-C radiation, it showed extremely high toxicity, affecting the mobility of all test organisms at all dilutions (until 16x).

CNPq
Florações de cianobactérias em reservatórios de abastecimento de água têm ocorrido com uma frequência cada vez maior, causando diversos problemas de ordem operacional nos sistemas de tratamento de água em decorrência da elevada densidade de células, além de preocupações quanto à eficiência do tratamento na remoção de metabólitos como cianotoxinas e compostos odoríferos. Este trabalho teve por objetivo avaliar a aplicabilidade da Moringa oleifera Lam pura e associada ao policloreto de alumínio (PACl) na remoção de células de Microcystis aeruginosa, microcistinas, 2-MIB e geosmina por meio de flotação por ar dissolvido e filtração rápida, utilizando carvão ativado granular. Primeiramente, os sais NaCl e CaCl2 foram avaliados para a extração do coagulante de M. oleifera. As amostras consistiram em água sintética adicionada de ácido húmico e células de M. aeruginosa para valores iniciais de 25 uT. O coagulante obtido com 1M CaCl2 de M. oleifera apresentou maior eficiência de remoção de cor, turbidez e número de células, sendo, para ele, indicada como ideal a dose de 50 mg L-1. É indicado que o CaCl2 não permite uma maior eficiência de extração do coagulante, mas sim que participe na formação dos flocos. A partir desses resultados, considerou-se a substituição de 10 a 50% do coagulante salino por PACl. O conjunto de coagulantes em proporções de 70:30 e 60:40 de M. oleifera e PACl permitiram uma melhoria na eficiência de remoção de células e redução do carbono orgânico residual. Finalmente, para essas proporções, foi avaliada a contribuição do uso de carvão ativado granular (CAG) como camada intermediária de filtro de areia visando à remoção de microcistinas, 2-MIB e geosmina. As amostras foram adicionadas de 50 ng L-1 de 2-MIB e geosmina, e 25 μg L-1 de microcistina-LR equivalente, antes dos ensaios. O uso do filtro com camada intermediária de CAG para o conjunto de coagulantes na proporção 70:30 (M.oleifera:PACl) resultou em eficiências globais acima de 95% para a remoção de cor, turbidez, células de M. aeruginosa, microcistinas e geosmina, e de 51 a 75% de remoção de 2-MIB e carbono orgânico dissolvido. Deste modo, o uso de M. oleifera como clarificante de águas com a substituição de 30% por PACl pode reduzir gastos com reagentes por parte de alguns países que hoje importam seu material para clarificação da água, e a adição de CAG no filtro de areia poderia reduzir custos e espaço com a instalação de mais de uma etapa para a remoção de metabólitos. Assim, este conjunto é indicado como uma alternativa de tratamento convencional de água, devido à sua capacidade de remoção de células e metabólitos, além da manutenção de cor, turbidez e microcistinas abaixo dos níveis estipulados para água de consumo.
Nutrient inputs leads to more frequent algal blooms in water supply reservoir which causes operational problems in water treatment plants due to high density of cells, aside from complications induced by its capacity of production of cyanotoxins and taste and odour compounds. The present study had as purpose an evaluation of the applicability of Moringa oleifera Lam as a coagulant with and without polyaluminium chloride (PACl) in the removal of Microcystis aeruginosa cells, microcystins, 2-MIB and geosmin using dissolved air flotation and filtration, using granulated activated carbon (GAC). First, NaCl and CaCl2 salts were studied for extraction of the coagulant. Samples were obtained by the addition of humic acid and M. aeruginosa cells in synthetic water in order to obtain 25 NTU. Coagulant obtained with 1M CaCl2 showed a better performance for color, turbidity and cells removal, being indicated 50 mg L-1 dosage. CaCl2 would not extract better the active component of M. oleifera seeds, but participate on flocs formation. Based on this, PACl addition was evaluated and added in the ranges of 10 to 50% substitution of the saline coagulant. 70:30 and 60:40 proportions of M.oleifera:PACl were indicated in order to to remove turbidity, color and cells. Finally, the use of GAC as an intermediate layer in rapid sand filtration bed was evaluated as a function of microcystins, 2-mib and geosmine retention capacity. Cited proportions were followed by filtration, added of 2-MIB and geosmin 50 ng L-1 as well as 25 μg L-1 of microcystin-LR equivalent before tests. A 70:30 (M.oleifera:PACl) proportion followed by rapid sand filtration combined with GAC led to removal efficiencies above 95% for color, turbidity, M. aeruginosa cells, geosmin and microcystins, and 51 to 75% efficiencies for 2-MIB and dissolved organic carbon. Hence, M. oleifera usage as water coagulant with 30% of PACl can reduce costs for some countries, and the addition of a GAC layer on a sand filter can reduce cost and space in water treatment plants. This process is indicated as an alternative conventional treatment for the removal of cyanobacteria cells and metabolites, besides its capacity to maintain turbidity, color and microcystins below the stipulated levels for water consumption.

Many cyanobacteria are reported to produce the nonprotein amino acid β-N-methylamino-L-alanine (BMAA). Cyanobacteria are extensively distributed in terrestrial and aquatic environments and recently BMAA was detected in temperate aquatic ecosystems, e.g. the Baltic Sea. Little is known about developmental effects of the mixed glutamate receptor agonist BMAA. Brain development requires an optimal level of glutamate receptor activity as the glutamatergic system modulates many vital neurodevelopmental processes. The aim of this thesis was to investigate the developmental neurotoxicity of BMAA, and its interaction with the pigment melanin. Autoradiography was utilized to determine the tissue distribution of 3H-labelled BMAA in experimental animals. Behavioral studies and histological techniques were used to study short and long-term changes in the brain following neonatal exposure to BMAA. Long-term changes in protein expression in the brain was also investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). A notable targeting of 3H-BMAA to discrete brain regions e.g. hippocampus and striatum in mouse fetuses and neonates was determined by autoradiography. BMAA treatment of neonatal rats on postnatal days 9–10 induced acute but transient ataxia and hyperactivity. Postnatal exposure to BMAA also gave rise to reduced spatial learning and memory abilities in adulthood. Neonatal rat pups treated with BMAA at 600 mg/kg showed early neuronal cell death in the hippocampus, retrosplenial and cingulate cortices. In adulthood the CA1 region of the hippocampus displayed neuronal loss and astrogliosis. Lower doses of BMAA (50 and 200 mg/kg) caused impairments in learning and memory function without any acute or long-term morphological changes in the brain. The MALDI IMS studies, however, revealed changes in protein expression in the hippocampus and striatum suggesting more subtle effects on neurodevelopmental processes. The studies also showed that BMAA was bound and incorporated in melanin and neuromelanin, suggesting that pigmented tissues such as in the substantia nigra and eye may be sequestering BMAA. In conclusion, the findings in this thesis show that BMAA is a developmental neurotoxin in rodents. The risks posed by BMAA as a potential human neurotoxin merits further consideration, particularly if the proposed biomagnifications in the food chain are confirmed.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
As cianobactérias e algas são organismos encontrados, abundantemente, em ambientes aquáticos, onde atuam como produtores primários transferindo energia para os diferentes níveis tróficos. A ocorrência de substâncias tóxicas ou xenobióticas tem sido uma ameça constante nos ecossistemas aquáticos. Tais substâncias podem causar efeitos tóxicos sobre diferentes grupos de cianobactérias e algas afetando em nível celular, de população e comunidade, causando prejuízos a cadeia trófica, nestes ambientes. O arsênio (As) é um elemento tóxico freqüentemente associado com depósitos de ouro. Através da mineração, o As é trazido à superfície pelo processo de drenagem ácida sendo, posteriormente, lixiviado e contaminando os corpos d água. Estudos evidenciam que cianobactérias e algas podem acumular As intracelularmente, sendo capazes de biotransformar as formas tóxicas em formas não tóxicas. A utilização destes processos permite a utilização destes organismos como biorremediadores na descontaminação de ambientes aquáticos contaminados pelo As. Com o objetivo de caracterizar os gêneros Geitlerinema UFV-E01 e Stigeoclonium UFV- E02, cultivados na presença de As, foram conduzidos, em laboratório, estudos sobre o crescimento, a absorção e a toxicidade do As. Para a caracterização foram utilizados como parâmetros à alteração morfológica, a produção de biomassa, para ambos os gêneros. A detecção do gene smtA que codifica a proteína metalotioneína (técnica de PCR), foi feita apenas em Geitlerinema UFV-E01. Os resultados mostraram que a absorção de As, em Geitlerinema UFV-E01, foi maior em células cultivadas em meio BG-11 B, com baixa concentração de fosfato (6,5 μmol.mL -1 ). A absorção de As foi proporcional às concentrações crescentes do elemento no meio de cultivo, atingindo o pico máximo de absorção em 24 horas de exposição (665,25 μg As.g -1 ms para células cultivadas em meio BG-11 A e 1.290,32 μg de As.g -1 ms, para células cultivadas em meio BG-11 B), sendo observado uma queda em 48 horas de exposição (500,00 μg As.g -1 ms para células cultivadas em meio BG-11 A, com 17,5 μmol.mL -1 de fosfato, e 810,00 μg de As.g -1 ms para células cultivadas em meio BG-11 B). As células de Geitlerinema UFV-E01 não apresentaram alterações morfológicas e não houve redução na produção de biomassa. Entretanto, foi observada a presença de grânulos densos no citoplasma das células. Os resultados obtidos, por meio da técnica de PCR, mostraram que Geitlerinema UFV-E01 não possui o gene smtA que codifica a proteína metalotioneína, sugerindo que a cianobactéria não utiliza esta estratégia nos processos de detoxificação celular. A absorção de As pelas células de Stigeoclonium UFV-E02, não foram influenciadas pelas concentrações de fosfato no meio BG-11 líquido. A absorção máxima de As ocorreu em 24 horas de exposição (1.300 μg As.g - ms) e foi mantida constante em 48 horas de exposição. Entretanto, em Stigeoclonium UFV- E02, houve redução na produção de biomassa e, também, alterações morfológicas nos filamentos caracterizados pelo aparecimento de células estreitas e longas, com cloroplastos deformados e de tamanho reduzido quando comparado com células sadias (controle).
Cianobacteria and algae are important primary producers in aquatic ecosystems and their energy is transferred throughout the different levels of the environmental trophic chain. The presence of xenobiotic or toxic elements has been a constant threat to aquatic ecosystems. These elements can cause considerable negative effects on microorganisms, since it can affect their different organization levels and consequently impairing the whole trophic chain. The arsenic (As) is a potential toxic element frequently associated with gold deposits. It can be brought to the surface by the gold mining activity, alongside the acid drainage and latter lixiviated, causing water bodies contamination. Previous study has shown that cianobacteria and algae can accumulated toxic As within the cells and sometimes transforming it into a non toxic form. Such trait can be useful for bioremediation purpose, by using these organisms on As decontamination of aquatic environments. In order to assess the bioremediator potential of Geitlerinema UFV-E01 (Cyanobacteria) and Stigeoclonium UFV-02 genera, absorption and toxicity tests were carried out on selected lineages, cultivated in presence of As, under laboratory conditions. The morphological alterations and biomass production were used as evaluation parameters to portray the two genera in relation to As presence. The identification of the smtA gene, which codifies the metallotioneina, was performed on the Geitlerinema UFV-E01 genus by PCR technique. The results indicated that the As absorption by Geitlerinema UFV-E01 cells was greater when they were cultivated in low phosphorus content (6,5 μmomL -1 ) BG-11 medium. The As absorption was proportional to the increase on the element content in culture medium, reaching the maximum after 24 hours of exposure either in the BG-11A medium (665,25 μg As.g -1 ms in BG-11 A and 1.290,32 μg As.g -1 ms in medium BG-11 B). After 48 hours of exposure to As, it was observed a decrease in the amount of element in the cells on both mediums tested, (500,00 μg As.g -1 ms in medium BG- 11 A with 17,5 μmol.mL -1 of fosfato, and 810,00 μg de As.g -1 ms in medium BG-11 B).The G. cells did not shown any signs of morphological alteration or biomass production decay in any of the culture medium tested. Nevertheless, dense corpuscles were observed in the cytoplasm of the cells cultivated. The PCR results revealed that the Geitlerinema UFV-E01 genus does not have the SmtA gene, suggesting that this cianobacteria can be using other mechanism for As detoxification. As detoxification by S. cells was not influenced by the phosphate concentration in the BG-11 medium. The maximum As absorption took place after 24 hours of exposure (1,300μg As.g -1 ms) and stayed constant for 48 hours. It was observed that Stigeoclonium UFV-02 genus had a decrease in the production of biomass and morphological alterations. The filaments shown longer and striated cells, containing smaller and deformed chloroplasts when compared to the control material.
Dissertação importada do Alexandria

Les efflorescences de cyanobactéries sont susceptibles d’avoir des effets néfastes sur les organismes des écosystèmes aquatiques, ainsi que sur les populations environnantes, notamment à travers la production de nombreuses molécules potentiellement toxiques (appelées cyanotoxines). Jusqu'à présent, une des cyanotoxines les plus étudiées est la microcystine (MC). Cette thèse a pour objectif d’évaluer la toxicité potentielle sur la reproduction de la MC-LR et de l'extrait d'une souche de Microcystis productrice de MCs en étudiant leurs effets toxiques sur le foie et les gonades de poissons medaka adultes exposés de manière aiguë ou chronique.Une étude complète du foie des poissons médaka deux sexes a été menés par ailleurs, attestant d'un fort dimorphisme sexuel aussi bien au niveau cellulaire que moléculaire et souligne les importantes spécificités métaboliques du foie entre les deux sexes, notamment pour le maintien de la compétence de reproduction chez les poissons medaka adultes femelles.Dans l'étude des effets induits par une exposition aiguë, les poissons medaka adultes ont été exposés par gavage à 10 μg.g-1 bw de MC-LR pure pendant 1 heure. L'examen histologique et l'immunolocalisation des MCs du foie de poisson traité par la MC-LR ont révélé des lésions hépatiques sévères ainsi qu'une distribution intense de la MC-LR dans le foie, localisée particulièrement dans le cytoplasme et dans le noyau des hépatocytes. Dans la gonade des poissons traités, la MC-LR a été détectée dans les tissus conjonctifs de l'ovaire et des testicules. De plus, l’observation par microscopie électronique couplé à la technique d’immunogold a révélé, pour la première fois, que la MC-LR était également détectable dans le chorion, le cytoplasme et le vitellus des ovocytes matures.Au cours des études des effets induits par l’exposition chronique, les poissons medaka adultes ont été exposés durant 28 jours par balnéation à 1 et 5 μg.L-1 de MC-LR et à un extrait de la souche de Microcystis aeruginosa (PCC 7820) productrice de microcystines (5 μg.L-1 équivalent MC-LR). Ces résultats ont révélé que la MC-LR et l'extrait de Microcystis induisent des effets délétères sur différents paramètres de reproduction, tels la fécondité et le taux d’éclosion des embryons. La cause principale de ces perturbations de la reproduction semblent principalement résulté d’un dysfonction hépatique globale induite par les traitements aux MCs (hépatotoxiques, notoires), plutôt qu’à des effets directs sur les gonades. Dans l'ensemble, les résultats de cette thèse démontrent que même si les microcystines pourraient avoir un impact direct, mais modéré, sur la fonction gonadique en induisant une cytotoxicité dans les cellules somatiques gonadiques et les cellules reproductrices, elle semble avoir principalement avoir un impact indirect sur la fonction reproductrice en perturbant la fonction hépatique générale. Ces données améliorent notre compréhension des processus liés à la toxicité potentielle des cyanotoxines pour la reproduction chez un poisson modèle, et fait d’une manière générale progresser questionnement quant à la protection des populations exposées à ces cyanotoxines
Cyanobacterial blooms threaten human health as well as other living organisms of the aquatic environment, particularly due to the production of natural toxic components (called cyanotoxins). So far, one of the most studied cyanotoxins is the microcystin (MC). This thesis evaluated the potential reproductive toxicity of MC-LR and the extract of one Microcystis strain (MC-producing) by investigating their toxic effects on the liver and gonad of adult medaka fish with one acute and one chronic study.An investigation of the metabolic specificities of the liver in two genders of medaka fish was performed prior to the MC-containing exposure, which attests to a strong sexual dimorphism of medaka liver, and highlights the importance of metabolic adjustments of the liver for maintaining the reproductive competency in adult medaka fish.In the acute study, adult medaka fish were administered with 10 μg.g-1 bw of pure MC-LR for 1 hour by gavage. The histological examination and immunolocalization of the MC-treated fish liver revealed a severe liver lesion along with an intense distribution of MC-LR in the liver, being particularly localized in the cytoplasm and nucleus of hepatocytes. In the gonad of MC-treated fish, MC-LR was shown to be present in the connective tissue of ovary and testis. Additionally, immunogold electron microscopy, for the first time, revealed that MC-LR was also localized in the chorion, cytoplasm and yolk vesicles of oocytes.Overall, the results of this thesis demonstrates that MC might directly impact gonadal function by inducing cytotoxicity in gonadal somatic cells and reproductive cells, and it could also impact the reproductive function indirectly by disturbing the general liver function. This improves our understanding of the potential reproductive toxicity of cyanotoxins in model fish, and advances our current knowledge on the protection of aquatic organism populations as well as human health from cyanotoxin issues

Thesis (PhD)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Several genera of cyanobacteria produce a range of toxins. The increased rate of eutrophication of surface fresh waters due to anthropogenic inputs has resulted in more frequent and severe cyanobacterial bloom events. Such bloom events make impoundments unsuitable for recreational use and increase the cost of production of potable water due to the necessity for removal of toxins released from cells during the purification process. Microcystis aeruginosa is the major freshwater bloom-forming toxic cyanobacterium. Concentrations of the hepatotoxin, microcystin, are highly variable in blooms. Published literature on environmental conditions leading to increased microcystin production was often contradictory and in many cases did not consider all relevant parameters. However, environmental nitrogen and phosphorus, temperature and light, and growth rate were implicated in regulation of toxin content. The purpose of this work was therefore to investigate environmental factors (specifically nitrogen and phosphorus) and cellular activities (specifically carbon fixation and nitrogen uptake rates and growth rate) involved in the modulation of microcystin production in M. aeruginosa in order to clarify the role of these parameters, and in an attempt to identify regulatory mechanisms for microcystin production. Environmental nitrogen, phosphorus and growth rate were shown to co-modulate microcystin production in M. aeruginosa. Adequate phosphorus is required for photosynthetic carbon fixation. Phosphorus uptake by M. aeruginosa is strongly correlated with carbon fixation rate. Although microcystin content increased with increasing nitrogen:phosphorus ratios in culture medium, under phosphorus limitation microcystin content was lower irrespective of nitrogen concentrations. This observation and the requirements for fixed carbon for nitrogen assimilation therefore prompted investigation of the effects of cellular carbon fixation and nitrogen uptake in the modulation of microcystin production. Microcystin production was found to be enhanced when nitrogen uptake rate relative to carbon fixation rate was higher than that required for balanced growth. The cellular nitrogen:carbon ratio above which microcystin concentrations increased substantially, corresponded to the Redfield ratio for balanced growth. Investigation of potential regulatory mechanisms involving the cyanobacterial nitrogen regulator, NtcA, yielded putative NtcA binding sites indicative of repression in the microcystin synthetase gene cluster. In culture, the polypeptide synthetase module gene, mcyA, and ntcA were inversely expressed as a function of carbon-fixation:nitrogen-uptake potential. However, no increase or decrease in microcystin production could be linked to either glutamine, glutamate or a-ketoglutarate, metabolites that are involved in regulation of ntcA. The role of NtcA in regulation of microcystin production could therefore not be confirmed. In conclusion, these data suggest that microcystin production is metabolically regulated by cellular C:N balance and specific growth rate. The primary importance of nitrogen and carbon was demonstrated by a simple model where only nitrogen uptake, carbon fixation and growth rate were used to predict microcystin levels. The model also explains results previously described in literature. Similarly, an artificial neural network model was used to show that the carbon fixation dependence on phosphorus allows accurate prediction of microcystin levels based on growth rate and environmental nitrogen and phosphorus.
AFRIKAANSE OPSOMMING: Verskeie genera van sianobakterieë produseer 'n verskeidenheid van toksiene. Die toename in die tempo van eutrofikasie van varswater oppervlaktes as gevolg van antropogeniese insette veroorsaak al hoe meer en al hoe erger sianobakteriële infestasies. Dit veroorsaak probleme vir ontspanninggebruik van hierdie waters en verhoog die koste van produksie van drinkbare water as gevolg van die noodsaak om die toksiene wat deur die selle gedurende die suiweringsproses vrygelaat word te verwyder. Microcystis aeruginosa is die belangrikste varswater bloeisel-vormende toksiese sianobakterium. Die konsentrasie van die hepatotoksien mikrosistien is hoogs varieerbaar in sulke bloeisels. Gepubliseerde literatuur oor die omgewingskondisies wat lei na verhoogde mikrosistienproduksie is dikwels weersprekend en neem in vele gevalle nie al die relevante parameters in ag nie. Desnieteenstaande word omgewingstikstof, fosfor, temperatuur en lig, asook groeisnelheid, geïmpliseer in die regulering van toksieninhoud. Die doel van hierdie navorsing was dus om omgewingsfaktore (spesifiek stikstof en fosfor) en sellulêre aktiwiteite (spesifiek koolstoffiskering en die snelheid van stikstofopname en van groei) betrokke by die modulering van mikrosistienproduksie in M. aeruginosa te ondersoek in 'n poging om die rol van hierdie parameters te verstaan en om regulatoriese meganismes vir mikrosistienproduksie te identifiseer. In hierdie studie is aangetoon dat omgewingstikstof en fosfor sowel as groeisnelheid mikrosistienproduksie in M. aeruginosa ko-moduleer. Genoegsame fosfor word benodig vir fotosintetiese koolstoffiksering. Fosforopname deur M. aeruginosa korreleer sterk met die snelheid van koolstoffiksering. Alhoewel mikrosistieninhoud toegeneem het met 'n toename in die stikstof:fosfor verhouding in die kultuurmedium, was die mikrosistieninhoud onder kondisies van fosforlimitering laer ongeag die stikstofkonsentrasie. Hierdie waarneming, tesame met die noodsaak van gefikseerde koolstof vir stikstofassimilering, het gelei na 'n studie van die effekte van sellulêre koolstoffiksering and stikstofopname op die modulering van mikrosistienproduksie. Dit is gevind dat mikrosistienproduksie verhoog was wanneer die snelheid van stikstofopname relatief tot die snelheid van koolstoffiksering hoër was as die waarde wat benodig word vir gebalanseerde groei. Die sellulêre stikstof:koolstof verhouding waarbo mikrosistienkonsentrasies beduidend verhoog is stem ooreen met die Redfield verhouding vir gebalanseerde groei. 'n Ondersoek na potensiële reguleringsmeganismes waarby die sianobakteriële stikstofreguleerder NtcA betrokke is het gelei na die ontdekking van moontlike NtcA bindingseteis; dit kan dui op die repressie van die mikrosistiensintetase geengroepering. Onder kultuurkondisies is gevind dat die geen vir die polipeptiedsintetase module, mcyA, en ntcA omgekeerd uitgedruk word as 'n funksie van koolstofopname:stikstofopname potensiale. Geen toename of afname in mikrosistienproduksie kon egter gekoppel word aan óf glutamien, óf glutamaat, óf a-ketoglutaraat nie, metaboliete wat betrokke is by die regulering van ntcA. Die rol van NtcA in die regulering van mikrosistienproduksie kon dus nie bevestig word nie. Die gevolgtrekking is dus gemaak dat mikrosistienproduksie metabolies gereguleer word deur die C:N balans en die spesifieke groeisnelheid. Die primêre belang van stikstof en koolstof is gedemonstreer deur 'n eenvoudige model waarin slegs stikstofopname, koolstoffiksering en groeisnelheid gebruik word om mikrosistienvlakke te voorspel. Die model verklaar ook resultate wat tevore in die literatuur beskryf is. Soortgelyk is 'n artifisiële neurale netwerkmodel gebruik om te toon dat die afhanklikheid van koolstoffiksering van fosfor akkurate voorspelling van mikrosistienvlakke gebaseer of groeisnelheid en omgewingstikstof en fosfor moontlik maak.

Les proliférations de cyanobactéries dans les milieux aquatiques continentaux représentent un problème environnemental, économique et sanitaire, notamment à cause de la capacité de certains genres à produire des toxines. En étudiant les variations spatio-temporelles de la diversité génétique et du potentiel toxique de populations de la cyanobactérie hépatotoxique Microcystis dans différents types d'écosystèmes aquatiques (retenues, étangs, fleuve) situés dans une même aire géographique, nous avons montré une structuration génétique des populations ainsi que d'importantes différences du potentiel toxique de ces proliférations. L'étude des variations de la structure en taille des colonies de Microcystis et des quotas cellulaires en microcystines en lien avec la stabilité de la colonne d'eau ont suggéré l'importance des colonies de petite taille dans le maintien des populations ainsi que dans la production de microcystines
Proliferations of cyanobacteria in freshwater ecosystems are a source of growing concern because they lead to ecological disturbances and the toxins they produce constitute health risks for animals and human beings. By studying the spatiotemporal variations in genetic diversity and toxic potential of several populations of the hepatotoxic cyanobacterium Microcystis in different kind of freshwater ecosystems located in the same geographical area, we evidenced a genetic structuration of the populations, and great differences in the proportion of potentially microcystinproducing genotypes in these populations. The temporal variations in the population size structure and in the microcystin cellular quotas related to the stability of the water column suggested the importance of small Microcystis colonies in sustaining the populations in unfavourable conditions for growth and also in microcystin production

L‟eutrophisation croissante des écosystèmes aquatiques favorise le développement des cyanobactéries, parmi lesquelles Microcystis est la plus représentée dans les régions tempérées. La capacité de Microcystis à produire une puissante hépatotoxine, la microcystine, est à l‟origine de diverses perturbations écologiques, et de nombreuses nuisances sanitaires. La compréhension des facteurs déterminant la toxicité des efflorescences de Microcystis constitue, de fait, un enjeu majeur des recherches actuelles. Dans ce contexte, l‟objectif premier de ce travail de thèse était d‟étudier la variabilité temporelle et l‟implication potentielle de la toxicité de Microcystis à l‟échelle de son cycle de développement annuel. Pour cela, il était nécessaire de considérer, en particulier, les parties les moins connues du cycle de développement : la phase de survie benthique, et les transitions entre les phases benthique et planctonique, via les processus de recrutement et de sédimentation. Nous avons alors étudié le potentiel toxique des populations de Microcystis grâce à des approches complémentaires menées à différentes échelles spatio-temporelles, en considérant à la fois les gènes impliqués dans la synthèse de microcystines, leur transcription et les concentrations en microcystines. Cette étude s‟est appuyée, en parallèle, sur la caractérisation de la structure génétique des populations de Microcystis dans les compartiments benthique et planctonique. La prise en compte systématique de la phase de vie benthique a tout d‟abord permis d‟améliorer nos connaissances sur cette phase du cycle de développement de Microcystis. Ainsi, Microcystis peut survivre plusieurs années en profondeur dans les sédiments, sans que les populations ne perdent leur potentiel toxique, ou que leur structure génétique soit altérée. En revanche, en surface des sédiments, le potentiel toxique et la structure génétique des populations sont variables, de manière similaire à ce qui peut être observé dans la colonne d‟eau. Enfin, ces travaux ont également mis en évidence l‟influence des phases de transition entre l‟eau et les sédiments dans la variabilité du potentiel toxique et de la structure génétique des populations de Microcystis. Les processus de recrutement benthique et de sédimentation occasionnent, en effet, une sélection génétique, qui, bien que paraissant indépendante du potentiel toxique des génotypes, peut grandement affecter le potentiel toxique des sous-populations benthiques et planctoniques de Microcystis
The increasing eutrophication of aquatic ecosystems promotes the development of cyanobacteria, among which Microcystis is the most widespread in temperate regions. The ability of this cyanobacterium to produce a potent hepatotoxin, called the microcystin, represent a serious threat for both natural life and human health. Thus, understanding the factors determining the toxicity of Microcystis blooms is a major challenge of actual research. In this context, the main goal of this work was to study the temporal variability and the potential implication of Microcystis toxicity, at the scale of its annual life cycle. For that, it was necessary to consider more particularly, the least known parts of the cycle : the benthic survival phase, and the transition between the benthic and the planktonic phases, through the benthic recruitment and the sedimentation processes. Then, we studied the toxic potential of Microcystis populations through complementary approaches conducted at different spatio-temporal scales, by considering the genes controlling the synthesis of the microcystin, their transcription and the concentrations of microcystin. In parallel, the genetic structure of Microcystis populations was characterized in both benthic and planktonic compartments. By considering systematically the benthic life stage, we were first able to improve our knowledge on this phase of Microcystis development cycle. Thus, Microcystis is able to survive several years in deep sediments, without the population‟s toxic potential or genetic structure being degraded. On the other hand, at the sediment surface, the toxic potential and the genetic structure of the populations vary, in a similar range to what observed in the water column. Furthermore, this work also shed the light on the influence of benthic-pelagic transitions in the variability of the genetic structure and the toxic potential of the populations of Microcystis. Indeed, a genetic selection occurs during the benthic recruitment and the sedimentation processes. Although such a selection does not seem to rely on the toxic potential of the genotypes, it can greatly modify the toxic potential of both benthic and planktonic sub-populations of Microcystis

La prolifération grandissante des épisodes de fleurs d’eau de cyanobactéries toxiques dans les plans d'eau potable et dans les usines de traitement d'eau potable est une préoccupation mondiale. L’utilisation de sondes in vivo, permettant la détection de la phycocyanine des cyanobactéries par fluorescence, est une technologie innovante, de plus en plus couramment utilisée. Néanmoins, pour favoriser son implémentation à grande échelle, elle doit être validée scientifiquement. De nombreuses sources d’interférences dans ces sondes sont à l’origine de biais de lecture. Les objectifs de cette recherche sont :1. La caractérisation des espèces dominantes et la succession des espèces de cyanobactéries dans deux grands lacs utilisés pour la production de l’eau potable2. L’analyse de la variabilité des espèces de cyanobactéries et d’autres groupes de phytoplancton en fonction de la température et des nutriments3. La validation du suivi des cyanobactéries par sonde fluorométriques aux sources d’eau potable en corrigeant le signal pour les interférences d’autres groupes de phytoplanctonLes résultats de ce travail de recherche ont montré qu’il existe de nombreses sources d’interférences aux sondes à fluoresence YSI, mais qu’il était possible de développer un facteur de correction afin d’éviter de surestimer les cyanobactéries. Suite à la validation de la sonde, celle-ci a été utilisée pour comprendre la dynamique des cyanobactéries à la baie Missisquoi afin de caractériser les espèces dominantes et de mieux comprendre la succession phytoplanctonique de la baie afin d’aider les opérateurs de l’usine de traitement d’eau potable à planifier les traitements en fonction de la qualité biologique de la baie. Parmi les résultats les plus intéressants, notons l’apparition précoce, de plus en plus marquée à travers les années, des blooms de Microcystis sp. et le développement de bloom co-dominés par des Chroococcales et des Nostocales. Le développement de fleurs d’eau dominées par Aphanizomenon sp. ou Dolichospermum sp. est généralement précédé d’une période où le milieu est limité en N ce qui favorise le développement de ces espèces fixatrices d’azote atmosphérique
The increase of toxic cyanobacterial blooms in source waters that can lead to breakthrough into drinking water treatments plants is a worldwide concern. The use of in situ probes allows for the detection of cyanobacterial phycocyanin through fluorescence. It is an innovative technology becoming more widely used. However, to facilitate the implementation of this technology, it must be validated. Several sources of interferences can lead to biases in their application. The objectives of this research are to :1. characterize the dominant species and cyanobacterial succession in two large lakes used for drinking water production2. analyse the variability of cyanobacterial species as well as other groups of phytoplankton as a function of temperature and nutrients3. validate cyanobacterial monitoring by fluorometric probe in drinking water sources by correcting the signal for other groups of phytoplanktonThe results of this research have shown that there are many sources of interference in fluorescence probes, but that a correction factor can be used to prevent the overestimation of cyanobacteria. Following the validation of the probe, it was used to improve our understanding of the dynamics of phytoplankton succession in Missisquoi Bay in order to characterize the dominant species and succession to improve the operation of drinking water treatment at Missisquoi Bay. Among the interesting findings was the earlier apparition of cyanobacteria throughout the years, Micocystis sp. blooms and blooms co-dominated by Chroococcales and Nostocales. The development of cyanobacterial blooms dominated by Aphanizomenon sp. or Dolichospermum sp. was generally preceded by a period where the water body was limited in nitrogen, which favours the development of these species capable of fixing nitrogen

Le milieu marin représente une source d'inspiration pour les chimistes, grâce à l'incroyable diversité structurale des composés isolés des organismes et micro-organismes marins. Parmi eux, la laxaphycine B, un lipopeptide cyclique isolée de la cyanobactérie Anabaena torulosa est comme la plupart des peptides marins produit selon une voie de biosynthèse non ribosomale « NRPS /PKS » et présente une forte activité cytotoxique sur différentes lignées cellulaires cancéreuses. Une des problématiques de ce lipopeptide est l'absence d'information sur son mécanisme d'action. Afin d'identifier des cibles potentielles, mais surtout d'entreprendre des études des relations structure-activités, la confirmation de sa structure est nécessaire. C'est dans cette optique que nous avons entrepris la synthèse de la laxaphycine B en synthèse peptidique en phase solide (SPPS). Dans un premier temps, nous avons entrepris la synthèse des quatre aminoacides non ribosomaux. Dans un second temps, nos efforts se sont concentrés sur le développement d'une stratégie de synthèse pour l'obtention de ce lipopeptide. En dernier lieu, nous avons étudié la structure secondaire possible de la laxaphycine B, afin d'apporter une explication sur son mécanisme d'action
Marine environment is a source of inspiration for chemists, thanks to the incredible structural diversity isolated from marine organisms and microorganism's compounds. Among them, laxaphycine B, a cyclic lipopeptide isolated from Anabena torulosa cyanobacteria, as like most marine peptides produced by a non-ribosomal biosynthesis pathway "NRPS/PKS”. Furthemore, this peptide has shown a strong cytotoxic activity on various cancer cell lines. One of the problems of this lipopeptide, is the lack of information on its mechanism of action. To identify potential targets and also to study in structure activity relationships, confirmation of its structure is necessary. It is in this context that we undertook laxaphycine B's synthesis using SPPS. In a first step, the four non-ribosomal aminoacids have been synthesized. In a second step, our efforts have focused on the development of a synthesis strategy to obtain laxaphycine B. Lastly, we studied the laxaphycin B's secondary structure to understand its mechanism of action

As cianobactérias apresentam distribuição variada e podem ocorrer desde as regiões frias do ártico até os trópicos, em corpos dágua doce e no ambiente marinho. Algumas espécies de cianobactérias produzem compostos com conhecida toxidade, podendo causar efeitos deletérios em seres humanos e animais. Entre estes compostos estão os com atividade neurotóxica devido aos mais variados mecanismos de ação. O objetivo geral deste projeto é a obtenção por via sintética de algumas neurotoxinas e variantes, entre elas a β-N-metil-L-alanina (L-BMAA), anatoxina-a e anatoxina-a(s), bem como algumas aplicações. Para a L-BMAA, foi buscada a determinação de rotas sintéticas viáveis para a obtenção deste aminoácido modificado sob a forma racêmica e enantiomericamente pura, além de um possível produto cíclico que pode ser gerado naturalmente a partir da L-BMAA. Algumas rotas estratégicas foram determinadas com êxito para a síntese destes compostos. Entre as aplicações dos produtos obtidos podem ser citados: (i) a determinação de um método analítico para a determinação deste aminoácido por RMN de 1H; (ii) um método analítico por LC-MS utilizando D3-L-BMAA como padrão interno e (iii) a indicação de um possível mecanismo de neurotoxidade ligado a neurodegeneração via um intermediário produzido naturalmente do equilíbrio entre L-BMAA e íons bicarbonato. Duas rotas sintéticas estratégicas foram traçadas para a anatoxina-a. Na primeira, que não foi bem sucedida, partiu-se de um biciclo já formado e em sua etapa chave deveria ocorrer à expansão de um anel tropânico que conduziria ao biciclo 1:2:4. Na segunda, tentou-se a síntese a partir de uma molécula extremamente simples, o 1,5- ciclooctadieno, e após cinco passos reacionais obteve-se a síntese total da anatoxina-a sob a forma racêmica. A etapa mais importante para essa rota foi a ciclização utilizando-se paládio divalente. Entre as possíveis aplicações dos produtos obtidos sugere-se: (i) o desenvolvimento de um método analítico por LC-MS utilizando D3-anatoxina-a como padrão interno e (ii) ensaios biológicos com os análogos para se determinar potencial agonista, antagonista ou agonista parcial de receptores nicotínicos para esses compostos. A anatoxina-a(s) apresentou um grande desafio para a sua síntese total. Por se tratar de um organofosforado com uma parte alquílica heterocíclica, buscou-se primeiramente a sua síntese parcial. Assim, foi possivel a síntese da guanidina cíclica sob a forma racêmica e quiral, além da obtenção de alguns compostos análogos inéditos. Para a síntese da N-hidroxiguanidina cíclica traçou-se duas diferentes rotas, uma enantioseletiva, partindo do aminoácido asparagina, e outra racêmica, com a utilização de álcool benzílico como material de partida. No entanto, apesar da produção de intermediários e análogos, não foi possível a obtenção do produto final. Entre as aplicações dos produtos sintéticos obtidos estão: (i) o desenvolvimento e validação de um método analítico para a quantificação indireta de anatoxina-a(s) utilizando a N-hidroxiguanidina cíclica como padrão e (ii) a utilização destes intermediários sintéticos em ensaios para se determinar os mecanismos de ação tóxicos, especialmente como inibidores de colinesterases.
Cyanobacteria are aquatic and photosynthetic organisms that can be present in cold areas, such as the Arctic, as well as in tropical waters, in both marine and freshwater environment. They are important species for aquatic life and terrestrial ecosystems since they are on the base of the food web. However, some cyanobacterial species can produce toxic secondary metabolites that have deleterious effects for animals and human. These toxic metabolites are also called cyanotoxins and show different mechanisms of action ranging from hepatotoxic to neurotoxic effects. The aim of this study is the organic synthesis of some common neurotoxins, including some analogues, such as β-N-methyl-L-alanine (L-BMAA), anatoxin-a and anatoxin-a(s). Moreover, focus was given for some possible application of these compounds, especially as analytical standards for water monitoring. Some synthetic routes were carried out in order to produce L-BMAA in both racemic and enantiomerically pure forms. Also, it was synthesized a possible cyclic analogue that may be formed in vivo from L-BMAA. The racemate and the pure L-BMAA were successfully obtained. Some possible application of these compounds were suggested: (i) the development of an analytical method based on 1H NMR analyses for the determination of L-BMAA in environmental and biological samples; (ii) the development of an analytical method based on LC-MS for the determination of L-BMAA, using D3-L-BMAA as an internal standard, in different matrices and (iii) the indication of a cyclic derivative formed in vivo that can be involved in the neurotoxicity of L-BMAA. Two strategies were planned in order to produce anatoxin-a. The first one was not successful and was started by using the bicyclic precursor. The crucial step was the expansion of tropanic ring to produce the 1:2:4 byciclic anatoxin-a precursor. The second strategy was initiated with the 1,5 cyclicoctadiene and after five reaction steps anatoxin-a was finally synthesized in its racemic form. The most important step for this route was the use of palladium. Similar applications were suggested for anatoxin-a: (i) the development of an analytical method based on LC-MS for the determination of anatoxin-a using D3-anatoxin-a as an internal standard in different matrices and (ii) the screening of anatoxin-a and analogues in bioassays based on nicotinic receptors in order to determine their agonistic and antagonistic mechanism of action. The total synthesis of anatoxin-a(s) is still a challenge. This cyanotoxin is a natural organophosphate with an alkylic heterocyclic chain. Therefore, the first step was the production of the cyclic guanidine in both racemic and enantiomerically pure forms. Some cyclic guanidine derivatives were prepared via asparagine and/or benzilic alcohol. However, anatoxin-a(s) was not achieved. The use of the cyclic guanidine can be recommended for: (i) the development of an analytical method based on LC-MS for the indirect determination of anatoxin-a(s) using its alkylic chain as a standard after sample alkalinization and (ii) the screening of anatoxin-a(s) derivatives in cholinesterase assays to determine their toxicity and mechanisms of action.

La recherche a été initiée par l'observation d'efflorescences de Planktothrix agardhii, une cyanobactérie dulçaquicole potentiellement toxique, dans deux étangs saumâtres, les étangs de l'Olivier et de Bolmon, avec dans ce dernier le déclin de l'espèce concomitamment à une augmentation de salinité. L'objectif de l'étude consistait à évaluer l'influence de la salinité du milieu sur la performance, l'hégémonie et la production de toxine de Planktothrix agardhii au sein de la communauté phytoplanctonique.La réalisation de suivis pluriannuels in situ couplés à des expérimentations en milieu contrôlé a permis de démontrer (i) la capacité d'acclimatation et d'adaptation à la salinité de Planktothrix agardhii, laquelle garantit sa suprématie et sa production toxinique en milieu saumâtre ; et (ii) la modification structurale et fonctionnelle de la communauté phytoplanctonique suite à une augmentation de salinité supérieure au seuil d'halotolérance de Planktothrix agardhii. La recherche témoigne ainsi de la versatilité des cyanobactéries qui renforce leur aptitude à être de bons compétiteurs, laissant présager leur persistance, la continuité de leurs nuisances, et leur expansion dans le futur
The research was launched by the observation of Planktothrix agardhii blooms, a potentially toxic freshwater cyanobacterium, in two brackish ponds, the Olivier and Bolmon ponds, with in the latter, the concomitantly collapse of P. agardhii with an increase in salinity. The goal of the study was to assess the salinity influence on the performance, the dominance and the toxin production of P. agardhii within the phytoplankton community.The achievement of a long-term monitoring in situ combined with batch cultures experiments has demonstrated (i) the ability of P. agardhii to acclimate and adapt to salinity, which ensure its supremacy and its toxin production in brackish areas, and (ii) the structural and functional changes of the phytoplankton community with the exceeding of the salt-tolerance threshold of P. agardhii .The research reflects the cyanobacteria versatility that enhances their suitability for being good performers, suggesting their persistence along with their nuisances, and their expansion in the future

Bibliography: leaves 235-265.
xvi, 265 leaves : ill. ; 30 cm.
Examines the tumour promoting effects of the microcystins through a long-term study in which cyanobacterial extract containing a range of microcystins was given in drinking water to mice previously treated with the tumour initiator N-nitroso-N-methyluren by gavage ; and through examining the effects of pure microcystin-LR in cultured primary hepatocytes from immature mice.
Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1998?

M.Sc. (Environmental Management)
In South Africa, there are practically no freshwater lakes. Therefore, exploitable water supplies are confined to rivers, artificial lakes behind dams, and groundwater. The many demands for water, and the erratic flow of most South African rivers, have led to the creation of artificial lakes and dams, i.e. impoundments on all the major rivers, in order to stabilise flow and therefore guarantee annual water supply. Cyanobacterial bloom formation in freshwater sources, such as rivers, lakes, dams and reservoirs are known to occur throughout the world. In South Africa, the occurrence of cyanobacteria has also been recorded with the best known being the bloom of the hyper-eutrophic Hartbeespoort Dam. In South Africa specifically, cyanobacteria are mostly seasonally driven. Species that are known to cause bloom formation are Microcystis sp., Anabaena sp., Oscillatoria sp. and Cylindrospermopsis sp. These species are known to produce cyanotoxins that cause health problems in animals and humans, but also produce taste and odour problems in drinking water, if not treated effectively. In most cases where cyanobacteria blooms have been known to occur, it also enters source water for drinking water purification plants. Because source water containing cyanobacteria and the effect it has on the consumer, environment and animals, it is thus important to identify the dominant algae species. Cyanotoxin drinking water guidelines must be developed and implemented and a management plan for the Water Treatment Plant must be produced, to ensure that the risk of human exposure to the cyanotoxins are minimised. The present study focuses on the Vaalkop Dam from which raw water is abstracted and treated by the Magalies Water Vaalkop Water Treatment Plant (MWVWTP) to produce potable water. The source water abstracted from the Vaalkop Dam can contain high numbers of cyanobacteria as well as cyanotoxins that must be removed by the MWVWTP during potable water purification to ensure compliance to water quality standards. The overall objective of the study is to investigate the occurrence of cyanobacteria and cyanotoxins in the Vaalkop Dam at the point where the source water is abstracted for drinking water purification.

As cyanobacteria (also known as blue green algae) produce a range of cyclic peptides which are highly toxic, capillary electrophoresis and associated techniques have been investigated to assess their applicability for toxin monitoring in the water bodies of kwaZulu Natal, South Africa. Capillary electrophoresis (CE) is a technique in which charged molecules can be efficiently separated in a buffer solution within a capillary tube under the influence of a strong electric field. Two CE modes, namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were initially evaluated using a laboratory-built CE instrument. The former mode lacked selectivity due to the similar charge to size ratio of the algal toxins. However, with the latter mode, incorporation of a surfactant (sodium dodecyl sulphate) into the buffer, produced sufficient resolution between components. Parameters including surfactant concentration, buffer ionic strength, buffer pH and operating voltage were systematically optimized to separate the four algal toxins under investigation (microcystin YR, microcystin LR, microcystin RR and nodularin). The optimum separation conditions were: 30 mM borax, 9 mM sodium dodecyl sulphate, pH 9.18, 30 kV applied voltage, 10 s hydrodynamic injection, 70 cm x 50 Ilm Ld. bare fused silica capillary (LEFF 40 cm) and UV detection at 238 nm. Under these conditions, typical detection limits were in the low ng/IlL range (14.13 ng/IlL for microcystin LR to 29.85 ng/ILL for nodularin). The MECC method was evaluated in terms of migration time precision, efficiency and resolution, peak area and normalised peak area precision. Standard deviation values for retention times acquired using replicate electrokinetic injections ranged from 0.018 to 0.054 and 0.069 to 0.148 for hydrodynamic injections. Normalised peak area precision for replicate hydrodynamic injections were in the range 84 to 97 % RSD, while improved % RSD values of 11.5 to 18.7 were achieved for electrokinetic injections. Due to poor precision resulting from the lack of automation on the laboratory built CE system, poor correlation between increasing concentration and a corresponding change in normalised peak areas were achieved. The MECC method developed was applied to the analysis of an algal scum extract to illustrate the technique. A general problem with CE is that it suffers from poor detection sensitivity. Hence in this study, alternative injection modes, sample concentration strategies and alternative detection techniques were investigated in an attempt to improve detection limits for algal toxins. Using optimized electrokinetic injection conditions, detection limits were five to ten times better than those obtained with hydrodynamic injections. On-line sample concentration methods were partially successful. Field amplified back and forth MECC in which analyte injected in the entire column volume and subsequently focused in a narrow band by manipulating the electric field, resulted in an enormous sensitivity enhancement that ranged from 197 times for microcystin RR to 777 times for microcystin YR when compared to hydrodynamic injections. Field amplified sample stacking (FASI) was ineffective for toxin preconcentration, while electro-extraction produced detection limits ranging from 0.27 ng/J.tL for microcystin YR to 1.08 ng/J.tL for microcystin RR. Solid phase extraction, in which analytes are first trapped and concentrated on HPLC material in a cartridge and then eluted in a more concentrated form for injection, was found to be practical only in the offline mode. A concentration detection limit of less than 0.002 ng/J.tL was obtained. Attempts with on-line solid phase extraction failed due to problems associated with coupling the cartridge with the separation capillary. Finally, laser induced fluorescence (LIF) detection was investigated as an alternative to UV detection. Unfortunately, the algal toxins were not amenable to LIF detection because tagging with the fluorescent moiety, fluorescein isothiocyanate (FITC), was prevented by the stereochemistry of these cyclic peptides. A comparative study between HPLC and MECC revealed that the former displayed poor efficiency peaks and long analysis times for toxin analysis. However HPLC was superior in terms of retention time precision (0.12 to 0.64 % RSD) and area precision (1.78 to 2.86 % RSD). Mass detection limits for MECC (0.0142 to 0.0603 ng) were far superior to those achieved by HPLC (0.55 to 1.025 ng). In addition to HPLC and MECC, a preliminary investigation of micro-high performance liquid chromatography (J.tHPLC) and capillary electrochromatography (CEC) for the analysis of algal toxins was made using 50 J.tm Ld. capillary columns packed in-house, with reverse phase HPLC packing material.
Thesis (M.Sc.)-University of Natal, 1997.