O gene de RNAr 16S tem sido amplamente utilizado na inferência filogenética de organismos procariotos, entretanto, sequências desse gene de cianobactérias isoladas de manguezais brasileiros são inexistentes. Neste estudo, 42 sequências inéditas do gene de RNAr 16S de cianobactérias isoladas de manguezais brasileiros foram geradas. Na análise BLAST as sequências apresentaram similaridades variando de 91 a 99% com outras sequências conhecidas de cianobactérias depositadas no GenBank. Na análise filogenética do gene de RNAr 16S várias sequências do gênero Chlorogloea ficaram agrupadas com sequências do gênero Synechococcus e algumas sequências de Nostoc ficaram mais próximas de sequências de Nodularia. As sequências de Leptolyngbya agruparam-se separadas das típicas de Leptolyngbya. Esses resultados corroboram com outros estudos, os quais mostraram que o filo Cyanobacteria necessita de revisão taxonômica. Na avaliação do potencial de produção de substâncias bioativas de 44 isolados de manguezais utilizando a técnica de PCR e dois conjuntos de oligonucleotídeos iniciadores degenerados específicos para sequências gênicas codificantes de peptídeo sintetase não ribossômica (NRPS) e policetídeo sintase (PKS) modular, 10 linhagens apresentaram resultado positivo para presença de NRPS e todos os isolados apresentaram resultado positivo para PKS. Na tentativa de atribuir função a esses genes e posteriormente explorar as potencialidades das linhagens, um produto da PCR de NRPS e 19 de PKS foram sequenciados, as sequências obtidas foram traduzidas para aminoácidos e utilizadas na construção de árvores filogenéticas. A análise filogenética da sequência de aminoácido do domínio de adenilação da NRPS indicou uma possível síntese de sideróforo pela linhagem Phormidium CENA135. Extratos extracelulares dessa linhagem analisados por Q-TOF/MS e RMN indicaram a produção de um sideróforo anfifílico com massa molecular e estrutura similar ao sideróforo synechobactina A. As sequências de aminoácidos do domínio da cetossintase de PKS das 19 linhagens mostraram relações filogenéticas com várias PKSs de linhagens conhecidas por produzirem substâncias, tais como sideróforo, algicida, microginina, oscilaginina B, hectoclorina e scytonemina. No teste da atividade antimicrobiana contra micro-organismos patogênicos dos extratos intra e extracelulares das 44 linhagens, 17 delas inibiram o crescimento de vários dos micro-organismos testados. Os extratos ativos foram analisados por Q-TOF/MS e 34 substâncias putativas conhecidas foram identificadas, tais como fungicidas, inibidores de proteases, antimaláricos e outras de funções desconhecidas. O teste imunológico ELISA específico para detecção da hepatotoxina microcistina (MC) apresentou resultado positivo para 16 das 23 linhagens testadas. Fragmentos de sequências do gene mcyA envolvido na síntese de MC foram amplificados por PCR em cinco linhagens (Synechococcus CENA136, Phormidium CENA135, Chlorogloea CENA142, Chlorogloea CENA146 e Nostoc CENA160), inclusive em dois gêneros sem registro de produção dessa toxina (Synechococcus e Chlorogloea). O teste ELISA específico para detecção da neurotoxina saxitoxina (SXT) apresentou resultado positivo para três linhagens. Entretanto, a análise em Q-TOF/MS e FT-ICR/MS de 15 linhagens, inclusive das que apresentaram resultado positivo no ELISA, não confirmaram a produção de saxitoxinas. / The 16S rRNA gene has been widely used in phylogenetic inference of prokaryotic organisms, however, sequences of this gene of cyanobacteria isolated from Brazilian mangroves are absent. In this study, 42 newly sequences of 16S rRNA gene of cyanobacteria isolated from Brazilian mangroves were generated. BLAST analysis of the sequences showed similarities ranging from 91-99% with other known cyanobacterial sequences deposited in GenBank. Phylogenetic analysis of several 16S rRNA sequences from the genus Chlorogloea were clustered with sequences from the genus Synechococcus and some sequences of Nostoc were closer to sequences of Nodularia. The Leptolyngbya sequences clustered separated from those of typical Leptolyngbya. These results corroborate with other studies which showed that the phylum Cyanobacteria need taxonomic revision. In assessing the potential of bioactive compounds production of 44 mangrove isolates using PCR technique and two sets of degenerated primers specific for gene sequences encoding non-ribosomal peptide synthetase (NRPS) and modular polyketide synthase (PKS), 10 strains showed positive results for the presence of NRPS and all isolates were positive for PKS. In an attempt to assign function to these genes and further explore the potential of the strains, a PCR product of NRPS and 19 PKS were sequenced and the sequences obtained were translated into amino acids and used to construct phylogenetic trees. Phylogenetic analysis of amino acid sequence of the NRPS adenylation domain indicated a possible synthesis of siderophore by the Phormidium strain CENA135. Extracellular extracts of this strain analyzed by Q-TOF/MS and NMR indicated the production of an amphiphilic siderophore with molecular mass and structure similar to the siderophore synechobactin A. The amino acid sequences of the PKS ketosynthase domain of 19 strains showed phylogenetic relationships with several PKSs of strains known to produce compounds such as siderophore, algicide, microginin, oscillaginin B, hectochlorin and scytonemin. In the antimicrobial activity test against pathogenic microorganisms of the intra- and extracellular extracts of 44 strains, 17 of them inhibited the growth of various microorganisms tested. The active extracts were analyzed by Q-TOF/MS and 34 putative known substances were identified such as fungicides, protease inhibitors, antimalarials, and others of unknown function. The immunological test ELISA specific for detection of the hepatotoxin microcystin (MC) was positive for 16 of the 23 strains tested. Sequence fragments mcyA gene involved in the synthesis of MC were amplified by PCR in five strains (Synechococcus CENA136, Phormidium CENA135, Chlorogloea CENA142, Chlorogloea CENA146 and Nostoc CENA160), including two genera with no record of production of this toxin (Synechococcus and Chlorogloea). Specific ELISA test for detection of the neurotoxin saxitoxin (SXT) showed positive results for three strains. However, analysis by Q-TOF/MS and FT-ICR/MS of 15 strains, including those that showed positive ELISA results, did not confirm the saxitoxin production.

Cianobactérias são microrganismos que desempenham um papel ecológico fundamental na natureza. O processo de eutrofização em ambientes aquáticos, tais como lagos e reservatórios, constitui um dos principais fatores relacionados à ocorrência de florações de cianobactérias nestes locais. Quatro lagoas presentes no Estado de São Paulo, nas cidades de Campinas, Limeira e Piracicaba foram estudadas visando descobrir a diversidade de cianobactérias presentes, seu potencial para a produção de cianotoxinas e os efeitos destas em organismos aquáticos bioindicadores, como Hydra attenuata. As amostras foram coletadas no mês de setembro de 2010 e divididas em 3 partes: a primeira foi utilizada para extração de DNA de cianobactérias para análises em DGGE, construção de bibliotecas genômicas e amplificação de genes produtores de toxinas; a segunda para a extração de cianotoxinas e análises em LCMS/ MS; e a terceira parte foi usada em ensaios ecotoxicológicos utilizando organismos aquáticos bioindicadores. A análise de DGGE mostrou que o padrão de bandas entre as diferentes amostras ambientais de água pareceu não apresentar grandes variações entre si e mostrou a predominância de poucas UTOs em todas as amostras, o que sugere que nessas localizações algumas espécies são predominantes sobre as outras. A construção de bibliotecas genômica gerou 233 clones, distribuídos da seguinte maneira: 57 clones da lagoa de Limeira, 60 clones da Lagoa do Taquaral, 59 clones da lagoa ESALQ1 e 57 clones da lagoa ESALQ2. Um total de 9 gêneros de cianobactérias foi observado nas amostras das lagoas, os quais são Anabaena, Brasilonema, Cylindrospermopsis, Limnococcus, Microcystis, Nostoc, Pseudanabaena, Synechococcus e Woronichinia. Neste estudo, as lagoas ESALQ2, Taquaral e Limeira apresentaram mais do que 80% da comunidade de cianobactérias avaliadas como sendo do gênero Microcystis. Este fato comprovou que no período analisado estavam ocorrendo florações de Microcystis em 3 das 4 lagoas analisadas. Pela amplificação de genes codificadores de cianotoxinas e cianopeptídeos foi possível detectar a presença de aeruginosina, cianopeptolina, microcistina e saxitoxina nas amostras ambientais. Estes dados foram comprovados através das análises em LC-MS/MS onde foi possível detectar a presença de aeruginosina, cianopeptolina e microcistina. Somente a presença de saxitoxina não foi confirmada por esta análise. Os ensaios toxicológicos com H. attenuata mostraram que todos os extratos foram tóxicos para este organismo teste, com exceção do extrato da lagoa ESALQ2, que causou apenas efeitos sub-letais nestes organismos. / Cyanobacteria are microorganisms that play a crucial ecological role in nature. The process of eutrophication in aquatic environments such as lakes and reservoirs, is one of the main factors related to the occurrence of cyanobacterial blooms in these locations. Four ponds located in the state of São Paulo at Campinas, Limeira and Piracicaba cities were studied in order to explore the diversity of cyanobacteria, their potential for cyanotoxins production and their effects on aquatic organisms as biological indicators, such as Hydra attenuata. The samples were collected in September 2010 and divided into 3 parts: the first one was used for cyanobacterial genomic DNA extraction, DGGE analysis, genomic library construction and amplification of toxinproducing genes; the second was used for the cyanotoxins extraction and LC-MS/MS analysis; and the third part was used in ecotoxicological tests using aquatic bioindicators. DGGE analysis showed that the banding pattern between the different water environmental samples did not seem to vary widely among themselves and showed the dominance of a few UTOs in all samples, suggesting that some species in these locations are predominant over the others. The construction of genomic libraries generated 233 clones, distributed as follow: 57 clones from Limeira lake, 60 clones from Taquaral Lake, 59 clones from ESALQ1 lake and 57 clones from ESALQ2 lake. A total of nine genera of cyanobacteria were observed in samples of the ponds, which are Anabaena, Brasilonema, Cylindrospermopsis, Limnococcus, Microcystis, Nostoc, Pseudanabaena, Synechococcus and Woronichinia.In this study, the lake ESALQ2, Taquaral and Limeira had more than 80% of the community of cyanobacteria assessed as belonging to the genus Microcystis. This fact proved that in the analyzed period the occurring blooms were of Microcystis in three of the four lakes studied. By amplification of genes coding for cyanotoxins and cyanopeptides it was possible to detect the presence of aeruginosin, cyanopeptolin, saxitoxin and microcystin in environmental samples. These data were confirmed by LC-MS/MS analysis and it was possible to detect the production of aeruginosin, cyanopeptolin and microcystin. Only the presence of saxitoxin was not confirmed by this analysis. The toxicological tests with H. attenuata demonstrated that all extracts were toxic to this organism, except the extract of ESALQ2 lake, which caused only sub-lethal effects in these organisms.

O contato com as cerdas da lagarta de Premolis semirufa (Pararama) desperta sintomas da inflamação aguda e nos indivíduos poliacidentados pode causar deformidades comuns às sinovites crônicas (pararamose). No presente estudo foi mostrado que o extrato das cerdas da lagarta apresenta intensa atividade proteolítica, sendo capaz de ativar o Sistema Complemento, promover hidrólise de C3, C4 e C5 e a geração de anafilatoxinas. Análises cromatográficas do extrato permitiram o isolamento de uma serinoprotease de 82 kDa capaz de promover tais atividades. Em modelo murino, foi verificado que o extrato é capaz de induzir altos títulos de anticorpos, pronunciada reação inflamatória, ativação de linfócitos T e APCs, bem como produção de citocinas pró-inflamatórias. Os dados obtidos demonstram a existência, no extrato das cerdas da pararama, de várias enzimas que podem atuar em conjunto na geração e desenvolvimento das manifestações clínicas da pararamose. / The contact with the Premolis semirufas caterpillar bristles (Pararama) causes symptoms of the acute inflammation and, in the individuals after multiple accidents, joint deformities common to chronic synovitis (pararamose) can occur. In the current study it was shown that caterpillar bristles extract has intense proteolytic activity, being able to activate the Complement System, induce cleavage of C3, C4 and C5, and the generation of anaphylatoxins. Chromatographic analysis of the extract allowed the isolation of a serine protease with Mr of 82 kDa able to promote such activities. In murine model, it was demonstrated that the extract is able to induce high antibody titers, a pronounced inflammatory reaction, activation of T lymphocytes and APCs, as well as the generation of proinflammatory cytokines. The data obtained demonstrate the existence, in the pararama bristles extract, of numerous enzymes that can act together in the generation and development of clinical manifestations of pararamose.

O Acidente Vascular Cerebral (AVC) é um quadro de alta incidência e uma das principais causas de incapacidade no mundo. Uma de suas complicações frequentes e de grande impacto na funcionalidade é a Síndrome do Ombro Doloroso Hemiplégico (SODH). O presente trabalho consiste em revisar a literatura a respeito da eficácia do tratamento da SODH com toxina botulínica tipo A (TXB-A). Foram pesquisados os bancos de dados eletrônicos PubMed e ISI web of Science, no período de 1996 a 2014. Incluímos os estudos tipo ensaio clínico duplo-cego, randomizado e aleatorizado que utilizaram a TXB-A como tratamento da SODH. Os artigos deveriam obter pontuação 3 ou superior pelos critérios de Jadad. Seis estudos foram encontrados e dois foram considerados de baixa qualidade. Os quatro artigos restantes possuíam 114 participantes no total. Três autores avaliaram os artigos de forma independente quanto aos critérios de inclusão. A extração de dados incluiu as características dos pacientes, dose e marca comercial da TXB-A utilizada, músculos e efeitos na avaliação da dor. Houve grande variação de dose, técnicas de aplicação e músculos tratados, bem como das intervenções após a aplicação. Três estudos demonstraram efeitos positivos do tratamento da dor e função do membro superior. A meta-análise não pode ser realizada, pois a informação quantitativa necessária foi indisponível. Os resultados da literatura são discordantes e apesar dos indícios serem favoráveis, não há resultados estatisticamente significantes para corroborar tal prática. Maior investigação e pesquisa são necessários para definir a indicação da TXB-A como tratamento da SODH. Mesmo assim, esta abordagem pode ser considerada particularmente quando a espasticidade é relevante. A intervenção pode ser realizada como uma alternativa ou ser utilizada em combinação com outros tratamentos. Para melhores resultados, mais de um músculo deve ser alvo, preferencialmente os músculos peitoral maior e subescapular. / The Stroke presents a high incidence and is a major cause of disability the in the world. One of it`s frequent complications and a cause of great loss of quality of life is the post-stroke shoulder pain (PSSP). The purpose of this study is to explore the effectiveness of botulinum neurotoxin type A (BoNT-A) in the treatment of PSSP. The electronic databases PubMed and ISI web of Science were searched from 1996 to 2014. We included double-blind randomized controlled trials that used BoNT-A for the treatment of PSSP in spastic hemiplegic patients. Articles should be scored 3 or more with the Jadad criteria. Six studies fulfilled the first search and two were considered low quality. The four remaining studies comprised 114 participants. Three authors independently evaluated articles eligible for inclusions. Data extracted included patient characteristics, doses and types of commercialized BoNT-A used, muscles injected, and effects on pain evaluation. There was a large variation in doses, injection techniques and treated muscles, as well as post-injection interventions. Three studies demonstrated positive effects of BoNT-A in the treatment of pain or upper limb functioning. Meta-analysis was not performed because of unavailable quantitative data. Literature results are discordant and despite evidence be favorable, there are no statistically significant results to support this practice. Further investigation and research is needed to define the indication of BoNT-A on PSSP. Nevertheless, this approach can be seen particularly when the spasticity is relevant. The intervention may be performed as an alternative or used in combination with other treatments. For best results, more than one muscle should be targeted, preferably the pectoralis major and subscapularis muscles.

Les systèmes toxine-antitoxine (TA) sont composés d’une toxine intracellulaire qui cible un processus cellulaire essentiel et qui est neutralisée par une antitoxine. Ces systèmes sont très abondant chez les bactéries et sont impliqués dans la réponse aux stress, la formation de biofilm, le phénomène de persistance, etc.<p>Mon projet de thèse a porté sur l’étude de la diversité des systèmes TA à deux niveaux. Dans un premier temps, plusieurs toxines de la famille RelE provenant de différentes espèces bactériennes et associées à des antitoxines non-canoniques ont été étudiées. Dans la seconde partie de ma thèse, nous avons caractérisé l’activation spécifique de deux systèmes TA d’Escherichia coli au niveau de la régulation transcriptionnelle du système et de l’activation de la toxine. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les systèmes toxine-antitoxine sont divisés en trois classes suivant la nature et le mode d’action de l’antitoxine. Ils sont fortement représentés au sein du règne bactérien et se trouvent sur des éléments génétiques mobiles qu’ils stabilisent dans la population bactérienne, mais aussi sur les chromosomes bactériens où leur fonction n’a pas encore été établie avec certitude. Au cours de ce travail, nous avons étudié les systèmes toxine-antitoxine bactériens de classe II, qui sont généralement composés de deux gènes organisés en opéron. Le premier gène code pour une antitoxine qui antagonise l’activité de la toxine, le produit du second gène. L’antitoxine, en complexe avec la toxine, est également capable de réguler l’expression de l’opéron en se fixant au promoteur de l’opéron. Lors du commencement de cette étude, les systèmes toxine-antitoxine étaient divisés en 10 familles sur base des similarités de séquences partagées par les toxines. A chaque famille de toxine était associée une famille d’antitoxine. <p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les systèmes toxine-antitoxine (TA) sont composés de deux gènes organisés en opéron retrouvés chez la quasi-totalité des bactéries ainsi que chez les archées. Ils ont été découverts sur des plasmides, où ils induisent une tuerie post-segregationnelle (PSK). En effet, l’antitoxine est instable car elle est dégradée par une protéase ATP dépendante. Lors de la réplication, si un plasmide portant un système TA n’est pas transmis à la cellule fille, celle-ci recevra tout de même une partie du cytoplasme de la cellule mère qui contenait des protéines de toxine et d’antitoxine. Cette dernière étant instable, et sans synthèse de novo, la toxine va se retrouver libre d’affecter sa cible cellulaire. Un système TA crée donc une addiction au plasmide qui le porte, stabilisant celui-ci.<p>Les systèmes TA se retrouvent également, dans un nombre de copies variable, au sein du chromosome. Dans ce cas, plusieurs hypothèses existent quant à leur fonction, comme la mort cellulaire programmée, la réponse au stress, la stabilisation de régions génomiques non essentielles, ou l’anti-addiction au plasmide.<p>L’origine et l’évolution des systèmes TA restent inconnues, alors qu’ils présentent des aspects intrigants. En effet, les toxines CcdB et MazF ont des séquences et des activités toxiques très différentes, malgré une structure proche ;les toxines RelE et ParE présentent des séquences proches, mais des fonctions différentes. À l’heure actuelle, les deux hypothèses concernant l’origine évolutive des systèmes TA sont soit qu’ils seraient tous issus d’un ancêtre commun, soit qu’ils auraient été réinventés plusieurs fois au cours de l’évolution, à partir d’un nombre limité de gènes.<p>Au cours de cette thèse, nous avons abordé la question de l’origine et l’évolution des systèmes TA de plusieurs manières :par la reconstruction de phylogénies et de séquences ancestrales, et par l’étude d’un système chromosomique particulier au sein de nombreuses souches d’Escherichia coli. Enfin, nous nous avons décidé d’analyser le contexte génomique des systèmes TA chromosomiques. Ces travaux ont notamment permis de mieux comprendre l’évolution des systèmes TA, et de conforter l’hypothèse selon laquelle ils seraient des éléments égoïstes, évoluant au sein des génomes sans gain d’aptitude pour l’hôte.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique. / The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests.

Kidney failure results in fluid and toxin accumulation within body fluid compartments, contributing to the excess mortality seen in this condition. Such uremic toxins have been measured in plasma, with levels assumed to reflect extraplasmatic concentrations such as in interstitial fluid (ISF). ISF is separated from plasma by nanometre-order microvascular pores; toxins may not circulate “freely” between the two compartments. This work set out to characterise the ISF in uremic subjects, with the hypothesis that there may be differences with plasma. Any such difference may be clinically relevant, owing to the much larger size of the ISF compartment, its proximity to cell metabolic processes, and its expansion in renal impairment.We used a modified microdialysis technique to successfully sample subcorneal ISF of some the uremic toxins (urea, creatinine, urate, phosphate). Reverse iontophoresis (RI) was also used as a non-invasive technique to sample epidermal ISF of urea. Hollow microneedles were developed and their ability to extract ISF tested in CKD patients and controls. The mechanical properties (pressure, volume, permeability) and biochemical composition (proteomic and metabolomic profiles) of the interstitial compartment were also investigated.Microdialysis and RI performed very well as interstitial uremic toxin sampling techniques. Small differences were seen in steady states between ISF and plasma urea, creatinine, phosphate and urate, with slightly lower ISF levels. Dialysis seemed to enhance this difference, with a lag in the clearance of ISF toxins seen in some patients, most remarkable with phosphate. Metabolomic analysis identified several uremic toxins in ISF, whilst proteomics found some significant differences between the two compartments, with toxins like beta-2 microglobulin occurring in ISF only. Microneedle arrays successfully extracted ISF in 68.8% of patients with oedema. Successful extraction of ISF with microneedles occurred mainly in oedematous patients, who were found to have raised interstitial pressures (ISP) and volumes. ISP correlated significantly with body fluid volumes and seemed time-dependent, lower in more chronic oedema. ISP and volumes also correlated with the oedema depitting time (after thumb pressure), a potential novel parameter that probably relates to tissue hydraulic conductivity and hence volume status and fluid mobility within the interstitium.This study demonstrates that interstitial fluid may need to be considered as a separate active compartment in patients with renal dysfunction, with a different “uremic" composition and unique pathophysiological characteristics that cannot be explained by blood compartment based measurements alone. There is a need for more studies, to further characterise this compartment and elucidate its importance.

O estudo das florações de cianobactérias potencialmente tóxicas é de fundamental importância, principalmente se tratando de um reservatório de grande valor econômico e social, devido a seus usos múltiplos e localização próxima a grandes centros urbanos como é o reservatório de Salto Grande (Americana - SP). Assim, este trabalho visou fornecer subsídios para formulação de prognóstico da ocorrência de cianobactérias e produção de toxinas em ambientes aquáticos com elevado grau de eutrofização. Para isto, a pesquisa foi desenvolvida em três escalas: em macrocosmo (represa), foi determinada a variação da ocorrência das espécies de cianobactérias e das demais classes fitoplanctônicas, e analisado o potencial tóxico das amostras coletadas nos meses chuvosos (janeiro, fevereiro e março/98) e de estiagem (junho, julho, agosto e setembro/98) em duas estações de coleta no reservatório de Salto Grande; em mesocosmo (tanques), verificou-se, durante o período de seca, as variações na ocorrência das classes fitoplanctônicas e das espécies de cianobatérias e produção de toxinas, em função da manipulação da razão N/P através da dosagem de nitrogênio e fósforo na água; em microcosmo (garrafões de vidro, em laboratório), foi testado o efeito da redução de fósforo e conseqüente aumento da razão N/P sobre o crescimento e produção de toxinas em culturas de cepas de Microcystis aeruginosa (Kützing) Kützing, isoladas a partir de amostras de florações desta espécie no reservatório de Salto Grande, durante os meses chuvosos e os secos. No ambiente foi detectada a presença de microcistinas na água de todos os dias de coleta, exceto em 25/02/99. Em geral, as concentrações estiveram abaixo do limite de aceitabilidade (1 &#956g.L-1), com exceção da estação 2 nos dias 27/01/99 (39,53 &#956g.L-1) e 22/03/99 (3,98 &#956g.L-1). Nos experimentos em mesocosmos notou-se um aumento da densidade fitoplanctônica) nas 3 condições distintas. Na condição controle (sem manipulação), ocorreu um sensível aumento da porcentagem de contribuição das cianobactérias e diminuição dos demais grupos ao longo dos 11 dias. Na condição de razão N/P baixa, houve um pequeno aumento na porcentagem de contribuição das cianobactérias e clorofíceas, uma diminuição das criptofíceas e os demais grupos não apresentaram grandes alterações. Sob razões N/P alta, as cianobactérias tiveram um aumento, as clorofíceas mantiveram-se constantes e as criptofíceas diminuíram em relação às porcentagens iniciais. As condições dos tanques mantidos com razão N/P baixa foram mais favoráveis às clorofíceas. As cianobactérias apresentaram um aumento de biomassa nas condições dos tanques mantidos com razão N/P alta. Em microcosmos, a fase exponencial teve início no oitavo dia de cultivo em todos os testes. Ao completar aproximadamente 18 dias de experimento, notou-se uma diminuição no rendimento das culturas em meio ASM-1 com redução de fósforo. Em todas as escalas estudadas constatou-se que a concentração de microcistina esteve relacionada a fatores favoráveis ao desenvolvimento das espécies tóxicas. Entretanto, os fatores determinantes para o crescimento de cianobactérias tóxicas, apresentaram diferentes papéis em casa escala estudada. No macrocosmo, a estabilidade da coluna d\'água foi fundamental para o estabelecimento de maiores densidades de espécies tóxicas. Nos mesocosmos, o enriquecimento foi responsável pelo aumento da densidade de espécies tóxicas. Nos microcosmos, a disponibilidade de fósforo esteve diretamente relacionada à taxa de crescimento de Microcystis aeruginosa e conseqüentemente, ao aumento da concentração de microcistina. / The study of the bloom of potential toxic cyanobacteria is of paramount significance, mainly when a reservoir endowed with great social and economical values due to both its multiple uses and its nearness to big urban centres - such as the Salto Grande reservoir, located near the city of Americana in the inland of the State of São Paulo, Brazil - is concerned. Based on such actuality, this work was aimed at supplying resources to devise forecasting the occurrence of cyanobacteria as well as the production of toxins in aquatic environments in which high eutrophication levels are observed. Towards this target, the research was carried out in the three steps that follow. In macrocosmic level (reservoir), the variations of the occurrence of both the cyanobacteria species and the remaining phytoplankton classes were found out, and then the toxic potentiality of the samples collected during the rainy season (January, February and March 1998) and the dry season (June, July, August and September 1998) in two sampling stations at the Salto Grande reservoir was analysed. In mesocosrnic scale (tanks), the variations in the occurrences of the classes of phytoplankton and of the cyanobacteria species - as well as the variations in production of toxins as a function of the ratio N/P that was dealt with by means of dosing nitrogen and phosphorus in the water - were observed during the dry season. In microcosmic level (glass bottles \"in lab\"), the effect of reducing phosphorus (and consequently increasing the ratio N/P) on the growth and the production of toxins in cultures of Microcystis aeruginosa (Kützing) Kützing strains - isolated from bloom samples of this species at the Salto Grande reservoir, during the wet and the dry seasons - was tested. At the environment, the presence of rnicrocystin in the water of all the samples, with the exception of the 25th of February 1999, was detected. Apart from the station 2 during January 2th 1999 (39.53 µg.L-1) and March 2th 1999 (3.98 µg.L-1), the concentrations stayed below the limit of acceptability (1 µg.L-1). At the experiments in mesocosmic scale, in 3 different conditions, increasing in phytoplankton density was observed. At the control condition (without manipulation), both reasonable augmentation of the percentage of the cyanobacteria contribution and diminishment of the other groups during the 11 days were detected. Under the condition of low N/P ratio, a slight rise of the percentage of the cyanobacteria and chlorophycea contribution and a decrease of the cryptophycea were observed; the other groups did not present much change. Under the condition of high N/P ratio, it was seen that the cyanobacteria increased, the chlorophycea remained unaffected and the cryptophycea decreased in comparison to the initial percentages. The conditions ofthe tanks that had been maintained at low N/P ratio favoured more the chlorophycea. The cyanobacteria presented biomass augmentation under the conditions of the tanks that had been maintained with high N/P ratio. For every test at the microcosmic level, the exponential stage had begun at the 8th development day. After being experimented for nearly 18 days, yield decrease of the cultures at the environment ASM-1 with phosphorus reduction was observed. In every scale that had been studied, it was noted that the microcystin concentration is related to unfavourable factors as far as the development of the toxic species is concerned. However, the determining causes for the growth of toxic cyanobacteria played different roles in each scale studied. At the macrocosmic level, enrichment was the responsible for increasing the density of the toxic species. At the microcosmic level, phosphorus availability had been directly related to Microcystis aeruginosa growth rate and, therefore, to the increase of microcystin concentration.