Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-25T16:49:25Z No. of bitstreams: 1 raphaelamoreiraferreira.pdf: 591822 bytes, checksum: ada6d23a894a6ca1835f8b88fcef5bc9 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-26T11:54:14Z (GMT) No. of bitstreams: 1 raphaelamoreiraferreira.pdf: 591822 bytes, checksum: ada6d23a894a6ca1835f8b88fcef5bc9 (MD5) / Made available in DSpace on 2017-05-26T11:54:14Z (GMT). No. of bitstreams: 1 raphaelamoreiraferreira.pdf: 591822 bytes, checksum: ada6d23a894a6ca1835f8b88fcef5bc9 (MD5) Previous issue date: 2007-10-11 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cylindrospermopsis raciborskii é uma cianobactéria filamentosa fixadora de nitrogênio que tem se tornado alvo de diversos estudos devido a sua toxicidade potencial e sua elevada adaptabilidade ecofisiológica. Seu sucesso adaptativo é atribuído, dentre outras coisas, a sua tolerância à baixa disponibilidade de luz e sua elevada afinidade por fósforo, além da capacidade de estocar esse nutriente. Devido a recente ocorrência de florações de C. raciborskii em todo o mundo, faz-se necessário estudar a ecologia e fisiologia dessa espécie visando o controle das florações. O presente trabalho buscou avaliar os efeitos da limitação de fósforo nas taxas de produtividade e eficiência fotossintética, na morfologia e no crescimento de populações tóxicas e não tóxicas de C. raciborskii. As cepas utilizadas apresentaram baixa eficiência fotossintética, sendo esta ainda menor após período de limitação de fósforo, o que foi atribuído ao baixo conteúdo de clorofila a. No entanto, a produtividade específica foi elevada, mesmo após a limitação por fósforo. Tal fato é certamente essencial para o desencadeamento de florações. A limitação de fósforo imposta as cepas provocou diminuição na relação superfície:volume das células, crescimento lento, aparecimento de heterocitos e acinetos e aparecimento de filamentos com células de diferentes tamanhos evidenciando a importância desse nutriente na determinação da morfologia de C. raciborskii. As alterações morfológicas relacionadas à diminuição do tamanho celular também estão relacionadas à baixa eficiência fotossintética devida a alterações na arquitetura celular. Os resultados encontrados sugerem cautela na identificação exclusivamente morfológica da espécie bem como demonstram que a interrupção do aporte de fósforo em ecossistemas aquáticos sujeitos a florações de C. raciborskii, enquanto medida isolada, não é suficiente para prevenir a ocorrência desses eventos. / Cylindrospermopsis raciborskii is a filamentous cyanobacteria able to fix N2 and has been object of many studies because of its potential toxicity and wide ecophysiologic adaptability. Its success is ascribed to, among other things, its shade adaptability, high affinity for phosphorus besides being able to store this nutrient. Since there has been many recent blooms of C. raciborskii around the world it become indispensable to know the ecology and physiology of these species. These work intent to evaluate the effects of phosphorus limitation on photosynthetic efficiency, morphology and growth of C. raciborskii. The species showed a low photosynthetic efficiency (α) under natural conditions and an even lower α after P limitation, which were attributed to its low chlorophyll a content. However, C. raciborskii had a high photosynthetic capacity (Pmb), before and after P limitation period. These ability is essential for the development of blooms. P limitation also reduced the growth and induced extreme morphological changes, such as: low surface:volume ratio, formation of akinetes, trichomes with different cell sizes proving the relevance of these nutrient on morphology of the species. These morphological changes had also influenced the low α found in these work. Our results suggest prudence on the taxonomic identification of C. raciborskii based on morphological characteristics and they demonstrate that the discontinuity on P load to aquatic ecosystems subject to C. raciborskii blooms does not assure that the blooms will not happen.

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-12-08T00:14:00Z No. of bitstreams: 1 LaissaMacedoTorres_DISSERT.pdf: 1199182 bytes, checksum: 8e8e139999c522ac6a56c6e99656daaa (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2015-12-10T22:51:02Z (GMT) No. of bitstreams: 1 LaissaMacedoTorres_DISSERT.pdf: 1199182 bytes, checksum: 8e8e139999c522ac6a56c6e99656daaa (MD5) / Made available in DSpace on 2015-12-10T22:51:02Z (GMT). No. of bitstreams: 1 LaissaMacedoTorres_DISSERT.pdf: 1199182 bytes, checksum: 8e8e139999c522ac6a56c6e99656daaa (MD5) Previous issue date: 2014-07-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Reservat?rios s?o os principais recursos de ?gua superficial no semi?rido do Brasil. A grande maioria destas reservas de ?gua est? comprometida pela eutrofiza??o. A seca severa ocorrida em 2012 contribuiu para perdas consider?veis no volume de ?gua, influ?nciando a disponiblidade de recursos (nutriente e luz) para o fitopl?ncton. O objetivo do trabalho foi verificar o efeito da seca na din?mica de grupos funcionais fitoplanct?nicos e verificar os fatores direcionadores de cada grupo. O efeito da seca favoreceu a homogeneidade de vari?vies bi?ticas e abi?ticas nos reservat?rios, principalmente pela falta de fluxo dos rios intermitentes. Os grupos funcionais de cianobact?rias formadoras de flora??es (S1, M e SN) dominaram ao longo do ano de 2012, nos tr?s reservat?rios estudados (Dourado, Gargalheiras e Passagem das Tra?ras). Os grupos estiveram relacionados ? altas concentra??es de s?lidos vol?teis, f?sforo total (PT) e am?nia (NH3), al?m de dominar tanto em ambientes rasos como em ambientes mais profundos. Cylindrospermopsis raciborskii (grupo SN) foi a esp?cie mais representativa nos tr?s reservat?rios, apresentando as maiores biomassas. O grupo M (Sphaerocavum brasiliense), ao contr?rio do descrito na literatura, registrou seu melhor desempenho em ambientes rasos, com mistura da coluna d??gua. A abordagem dos grupos funcionais de Reynolds et al. (2002) representou uma ferramenta importante na descri??o dos sistemas eutr?ficos do semi?rido, mas n?o refletiu uma resposta clara dos fatores direcionadores de cada grupo. Devido ? longa domin?ncia de cianobact?rias nos reservat?rios do semi?rido, sugerimos que a abordagem de grupos taxon?micos indica mais claramente quais caracter?sticas ambientais contribuem para o melhor desemprenho desse grupo. Ambientes ricos em nutrientes, alto tempo de reten??o da ?gua, alta disponibilidade de luz durante quase todo o ano e estabilidade na coluna d??gua por falta de fluxo s?o caracter?sticas dos reservat?rios do semi?rido que favorecem a domin?ncia dos grupos de cianobact?rias formadoras de flora??es. Sendo assim, nossos resultados mostram que os reservat?rios do semi?rido s?o vulner?veis ? domin?ncia de cianobact?rias formadoras de flora??es, especialmente em per?odos de eventos extremos, como seca severa, refletindo na perda da qualidade de ?gua nos mananciais da regi?o. / Reservoirs are the main sources of surface water in Brazil?s semiarid region. The majority of these water supplies, however, are compromised by eutrophication. A severe drought in 2012 contributed to significant losses in water volume, influencing the availability of resources (nutrients and light) for phytoplankton. The aim of this study is to understand the dynamics of the functional groups of phytoplankton and the factors that affect them during a severe drought in the semiarid reservoirs of the northeast. We therefore studied the Dourado, Gargalheiras and Passagem das Tra?ras reservoirs in Rio Grande do Norte from January 2012 to January 2013. The effect of drought favoured homogeneity within the reservoir, in relation to biotic and abiotic variables, notably the absence of water supply given the lack of flow from its tributaries (intermittent river). The phytoplankton functional groups of bloomforming cyanobacteria (SN, S1 and M) dominated throughout the year 2012, in both the shallow and deep areas of the three reservoirs studied. The groups were related to high concentrations of volatile solids, total phosphorus and ammonia, and high turbidity. Cylindrospermopsis raciborskii (SN group) was the species with the greatest biomass in the three reservoirs. M group (Sphaerocavum brasiliense) performed better in shallow waters with more available phosphorus. Our data showed that high concentrations of nutrients and low availability of light, besides the stability of the water column due to lack of flow and the system?s high residence time, favoured the dominance of bloom-forming cyanobacteria groups, especially those tolerant to shadow

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-06T20:31:23Z No. of bitstreams: 1 FabianaOliveiraDeAraujoSilva_TESE.pdf: 783632 bytes, checksum: d5a28b9c131a2a8187ccb70b95821d14 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-08T17:34:29Z (GMT) No. of bitstreams: 1 FabianaOliveiraDeAraujoSilva_TESE.pdf: 783632 bytes, checksum: d5a28b9c131a2a8187ccb70b95821d14 (MD5) / Made available in DSpace on 2016-07-08T17:34:29Z (GMT). No. of bitstreams: 1 FabianaOliveiraDeAraujoSilva_TESE.pdf: 783632 bytes, checksum: d5a28b9c131a2a8187ccb70b95821d14 (MD5) Previous issue date: 2015-02-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / A eutrofiza??o ? a causa mais comum de deteriora??o da qualidade da ?gua no mundo. Este processo se d? pela entrada excessiva de nutrientes, nitrog?nio e f?sforo, nos corpos aqu?ticos causando flora??es de algas e cianobact?rias. Em lagos rasos esses efeitos s?o mais complicados devido a uma maior intera??o do corpo aqu?tico com o entorno, com o ar e o sedimento. Existem v?rias t?cnicas de restaura??o de lagos eutrofizados, com uma vasta gama de resultados bem sucedidos, mas no Brasil h? apenas um ?nico caso de restaura??o bem sucedida: o lago Parano? em Bras?lia. A regi?o semi?rida brasileira possui milhares de lagos artificiais, regionalmente chamados de a?udes, em sua maioria rasos e eutr?ficos. A eutrofiza??o desses corpos aqu?ticos ? documentada e o fitopl?ncton desses ambientes ? frequentemente dominado por cianobact?rias potencialmente t?xicas. O principal objetivo desta tese de doutorado ? testar diferentes t?cnicas de restaura??o da qualidade da ?gua que possam ser facilmente aplicadas em lagos rasos do semi?rido brasileiro. Resultados de um experimento em laborat?rio sugerem que a aplica??o de argila adsorvente de f?sforo associada a um coagulante ? base de alum?nio ? uma t?cnica efetiva na remo??o do f?sforo reativo sol?vel e na diminui??o da taxa de crescimento da Cylindrospermopsis raciborskii, cianobact?ria potencialmente t?xica que domina nos reservat?rios do semi?rido brasileiro, mas que esse efeito ? dependente da biomassa no momento da aplica??o da t?cnica. Os resultados de um experimento de campo realizado em mesocosmos num lago raso eutrofizado demonstraram que a aplica??o de coagulante ? base de alumn?nio em conjunto com a da remo??o de peixes bent?voros ? mais eficiente na remo??o de f?sforo total e clorofila-a da coluna de ?gua do que a aplica??o isolada de apenas uma dessas t?cnicas. Por fim, testes de laborat?rio demostraram que o coagulante ? base de alum?nio apresentou um bom desempenho em remover turbidez e f?sforo total em testes de bancada com ?gua de seis reservat?rios do semi?rido, sendo a efici?ncia reduzida com o aumento da biomassa de clorofila e pH. Os resultados deste estudo mostram que ? poss?vel melhorar a qualidade da ?gua de reservat?rios eutrofizados no semi?rido brasileiro atrav?s do controle da carga interna de nutrientes seja pela precipita??o e inativa??o do f?sforo no sedimento, como tamb?m pela inibi??o da libera??o do f?sforo no sedimento por peixes bioturbadores, e que os resultados s?o aditivos quando as t?cnicas s?o aplicadas em conjunto. / Eutrophication is the most common cause of water quality degradation in the world. This process occurs by excessive nutrients inputs, nitrogen and phosphorus, to the aquatic systems resulting in algal and cyanobacterial blooms. In shallow lakes these effects are pronounced due to the higher interaction of the lake with watershed, air and sediment. There are innumerous restoration techniques of eutrophied lakes with a range of successful results but there is only one case of successful lake restoration in Brazil: Parano? Lake in Bras?lia city. The Brazilian semiarid region has many artificial lakes, named a?udes, which are mostly eutrophic and shallow lakes. The eutrophication in these lakes is reported and the phytoplankton community is dominated by potentially toxic cyanobacteria species, mainly Cylindrospermopsis raciborskii. The aim of this thesis is to test techniques for water quality management which can be easily applied in Brazilian semiarid lakes. Results from a laboratory experiment suggest that the addition of a phosphorus sorbent clay associated with an aluminium based coagulant is an effective technique in removing soluble reactive phosphorus and reducing C. raciborskii growth rate ? cyanobacteria potentially toxic dominant in reservoirs of Brazilian semiarid ? but this effect is dependent on the biomass in the application moment. Results from a field experiment in mesocosm in a eutrophied lake showed that the addition of aluminium based coagulant and removal of benthivorous fish is more efficient in removing total phosphorus and chlorophyll-a from water column than the isolated application of one of the techniques. Lastly, laboratory tests showed that aluminium based coagulant exhibited good performance in removing turbidity and total phosphorus from water of six reservoirs but the efficiency was reduced by algal biomass and pH. The results of this study showed that the improvement in water quality of eutrophied reservoirs in semiarid region is possible through internal loading control by phosphorus precipitation and inactivation in sediments or inhibition of phosphorus release by benthivorous fishes, and also that these results show are additives in water quality improvement.

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.