Los ácidos grasos poliinsaturados de cadena larga (LC-PUFAs) son nutrientes esenciales altamente demandados por las industrias acuícola, nutracéutica y alimentaria. Actualmente, el principal recurso que abastece de LC-PUFAs a la industria es el aceite de pescado. Sin embargo, las diversas problemáticas asociadas a su producción hacen necesaria la búsqueda de recursos alternativos. Ciertas microalgas marinas producen naturalmente estos compuestos por lo que surgen como una fuente alternativa prometedora de LC-PUFAs. Además, su síntesis puede modificarse manipulando las condiciones de crecimiento y, frente a condiciones de baja temperatura, ciertas especies responden sintetizando aún más LC-PUFAs. Sobre la base de estos antecedentes, el objetivo de esta Tesis fue aislar microalgas nativas del estuario de Bahía Blanca y desarrollar estrategias de cultivo que involucren el estrés por baja temperatura. Se plantearon como objetivos específicos aislar potenciales especies productoras de PUFAs e identificarlas morfológica y molecularmente; caracterizar su crecimiento a escala de laboratorio; cultivarlas en fotobiorreactor bajo condiciones óptimas y estresantes en términos de temperatura determinando su efecto sobre parámetros de crecimiento, producción de biomasa y cantidad, calidad y producción lipídica, con especial énfasis en LC-PUFAs, y de otros compuestos de alto valor como esteroles, pigmentos y/o proteínas. De las tres especies de diatomeas aisladas, se seleccionó a Cylindrotheca closterium por presentar los mejores resultados en cuanto crecimiento y producción de biomasa. Se corroboró la identidad de la cepa mediante análisis del gen cloroplástico rcbl. Se realizaron cultivos en fotobiorreactor de C. closterium a 20°C (control), y disminuyendo abruptamente la temperatura a 10oC durante la fase exponencial de crecimiento (EBTEx) y la fase estacionaria de crecimiento (EBTEs). La condición EBTEs, fue la estrategia de cultivo más adecuada para incrementar la producción de biomasa, lípidos y LC-PUFAs esenciales, particularmente en términos de ácidos eicosapentaenoico (EPA) y araquidónico (ARA). A su vez, la presencia de esteroles y aminoácidos esenciales para organismos acuáticos revelaron el valor de la biomasa para su posible uso en acuicultura. Por otra parte, otra especie aislada fue identificada como Pavlova gyrans (Haptophyta) a partir de sus características morfológicas y estudios moleculares en base al gen nuclear 18s rARN. P. gyrans mostró ser robusta para el crecimiento bajo las condiciones empleadas en el laboratorio. Se ensayaron cultivos en fotobiorreactor a una temperatura óptima (20oC) y bajo EBTEs. Ésta última condición demostró ser efectiva para incrementar la producción de biomasa y LC-PUFAs, particularmente los ácidos grasos EPA y docosapentaenoico (DPA). Además, la presencia de fitoesteroles y pigmentos otorgaron aún más valor a la biomasa. Finalmente, se analizó la calidad de los lípidos teniendo en cuenta indicadores de calidad nutricional de los mismos. P. gyrans mostró valores similares a los reportados para diferentes aceites de pescado por lo que puede considerarse una especie para uso alimentario. En conjunto, estos resultados evidencian el potencial de las cepas nativas C. closterium y P. gyrans como una posible fuente alternativa de LC-PUFAs. / Long-chain polyunsaturated fatty acids (LC-PUFA) are essential nutrients highly demanded by aquaculture, nutraceutical and food industries. Currently, the main source that supplies LC-PUFAs to the industry is fish oil. However, various problems associated with its production make it necessary to search for alternative resources. Certain marine microalgae naturally produce these compounds, emerging as promising alternative sources of LC-PUFA. Furthermore, its synthesis can be modify by manipulating their growth conditions and faced with low temperature conditions, certain species respond by synthesizing even more LC-PUFA. Based on this background, the objective of this Thesis was to isolate native microalgae from the Bahía Blanca Estuary and develop culture strategies involving low temperature stress. The specific objectives were to isolate possible PUFA- producing species and identify them morphologically and molecularly; characterize their growth on a laboratory scale; cultivate them in a photobioreactor under optimal and stressful conditions in terms of temperature, determining their effect on growth parameters, biomass production and quantity, quality and lipid production, with special emphasis on LC-PUFA, and other high-value compounds such as sterols, pigments and/or proteins. Of the three isolated diatom species, a Cylindrotheca closterium was selected as it showed the best results in terms of growth and biomass production. The identity of the strain was confirmed by analysis of the rcbl chloroplast gene. C. closterium cultures were performed in a photobioreactor at 20°C (control), and the temperature was abruptly decreased to 10oC during the exponential growth phase (EBTEx) and the stationary growth phase (EBTEs). The EBTEs condition was the most suitable culture strategy to increase the production of biomass, lipids and LC-PUFAs, particularly in terms of eicosapentaenoic (EPA) and arachidonic (ARA) acids. In turn, the presence of sterols and essential amino acids for aquatic organisms revealed the value of the biomass for its possible use in aquaculture. On the other hand, another isolated species was identified as Pavlova gyrans (Haptophyta) based on its morphological characteristics and molecular studies based on the nuclear 18s rRNA gene. P. gyrans was shown to be robust for growth under laboratory conditions. Cultures were also performed in a photobioreactor at optimal temperature (20oC) and under EBTEs. This last condition proved to be effective in increasing the production of biomass and LC-PUFAs, particularly EPA and docosapentaenoic (DPA) fatty acids. In addition, the presence of phytosterols and pigments added even more value to the biomass. Finally, lipids were analyzed taking into account indicators of nutritional quality. P. gyrans showed similar values to those reported for different fish oils, so it can be considered a species for food use. Taken together, these results show the potential of two native strains, C. closterium and P. gyrans, as alternative sources of LC-PUFAs.

No description available.

No description available.

The goal of this thesis is to determine if Sinorhizobium meliloti can be useful as a sentinel soil microorganism for assessing the impacts of contaminant stressors on the metabolome of a microorganism. Not only is a good deal known about this organism, but it is an important organism in agriculture. Moreover, the currently available gene array and a large library of gene fusion can be used as facile pathways to explore genetic and genomic impacts in addition to metabolomic impacts of contaminants, should such studies be deemed worthwhile. In this study, the polar metabolome of the soil microorganism, Sinorhizobium meliloti, has been analyzed by LC-ESI-MS using a HILIC column coupled to a medium mass resolution time-of-flight mass spectrometer. This approach has resulted in the retention (k' > 0.7) of over 300 polar metabolites as detected in both positive ion and negative ion modes. These data do not include ions corresponding to adduct ions, isotopic features or ions resulting from in-source decay processes. The retained peaks showed excellent linear responses and did not suffer from ion suppression, a common problem in flow-injection ESI analysis. This methodology has been applied to the analysis of S. meliloti exposed to fluorene, a common PAH contaminant, and to a coal tar fraction containing low molecular mass PAHs. Multiple cultures of S. meliloti were grown on M9 glucose minimal medium in the absence and presence of fluorene (0.14 mg/L and 1.4 mg/L) and a PAH mixture (total PAH concentrations of 0.14 mg/L and 1.4 mg/L). Analyses of biological replicates were performed in pentuplicate. The retention times of the resulting chromatograms were aligned, peak areas determined and the resulting data processed using PCA and OPLS-DA methods. The retention time reproducibilities of peaks were within ± 10 seconds and the biological variabilities of over 700 components averaged 23% ± 15% (n=25) . The impacts of fluorene exposures and PAH mixture exposures on the S. meliloti metabolomes (polar) caused significant changes in the metabolome. The lower concentration exposures had less of an impact than the higher dosages. Low dosages of both fluorene and the PAH mixture produced a similar metabolic response in S. meliloti, while at higher dosages the responses were more specific to each toxin. The use of SUS plots coupled with S-plots of the OPLS-DA analysis were particularly advantageous for the identification of metabolites of interest. Changes were seen in the levels of adenine, adenosine, glutamate, and aspartate, among others. In the future, the profiles of the non-polar metabolites of each of sample will be analyzed using a previously developed 'shotgun lipidomics' method. / Thesis / Master of Science (MS)

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Les analogues de nucléosides représentent une famille thérapeutique très largement utilisée en cancérologie dans le traitement des hémopathies malignes et autres cancers. Leur implication sur les variations des pools des nucléotides endogènes a été largement décrite et a constitué le point de départ de ce travail. C’est dans le but d’étudier ces variations que nous avons développé une méthode de dosage sensible et spécifique des ribonucléotides et désoxyribonucléotides physiologiques intracellulaires. Dans la première partie de ce mémoire nous avons réalisé une revue de la bibliographie sur les méthodes de dosage décrites dans la littérature puis nous avons détaillé le métabolisme des nucléotides endogènes et de leurs analogues. Dans la deuxième partie, nous présentons la validation de notre méthode de dosage et l’application à l’étude des variations des pools endogènes de ribonucléotides et désoxyribonucléotides dans différents modèles cellulaires : lignées résistantes versus lignées parentales et lignées traitées versus lignées non traitées. Les résultats obtenus sur les cellules montrent que notre méthode de dosage permet d’étudier l’impact des analogues nucleosidiques sur les pools endogènes de nucléotides mais aussi les dosages des dérivés phosphates de ces nucléosides / Nucleoside analogues constitute family of drugs widely used in cancer therapeutic for the treatment of malignancy hemopathies and several type of cancer. Numerous studies described their action on the variation of endogen nucleotide pools. This constitutes the point of starting of the present work. A sensible and specific assay for the quantification of intracellular ribonucleotides and désoxyribonucleotides was developed. In the first part, a review concerning the analytical methods published is presented. We also focused on the metabolism of endogen nucleotides and their analogues. In the second part, our method for the quantification of endogen nucleotides and désoxynucleotides is presented from different cellular models: resistant cell lines versus wild type lines, treated cells with analogues versus untreated cells. Results obtain from the cells lines studied show that our analytical method allows to study the impact of nucleoside analogues on endogenous nucleotides, but also the quantification of the phosphate derivatives of these analogues

As elevadas taxas de colesterol plasmático representam um grande risco à saúde, uma vez que podem causar doenças cardiovasculares. Para o tratamento e prevenção da dislipidemia são utilizados medicamentos reguladores do colesterol, como as estatinas. Embora eficazes e extensamente utilizados, esses fármacos apresentam efeitos adversos se administrados na dosagem errada. Assim, faz-se necessário o desenvolvimento de um método de monitorização terapêutica a fim de se ajustar a concentração desses compostos no sangue. Este trabalho visa o desenvolvimento de um método para análise de pravastatina (PRA), atorvastatina (AT), fluvastatina (FLV) e sinvastatina (SV) em plasma humano usando cromatografia líquida acoplada à espectrometria de massas (LC-MS). Na etapa de preparo de amostras, de forma inédita, utilizou-se a técnica microextração por sorvente empacotado (MEPS) para a análise de plasma humano contendo quatro estatinas. Para a otimização das condições de extração avaliaram-se, por experimentos univariados, parâmetros como fase extratora, composição do solvente de eluição e de lavagem. Outros fatores como volume de amostra, ciclos de amostragem, ciclos de eluição e etapas de eluição foram avaliados empregando-se planejamento experimental multivariado. A extração foi realizada utilizando-se uma fase estacionária C18 Chromabond como sorvente. O método MEPS-LC-MS/MS desenvolvido foi validado baseando-se nas recomendações da agência nacional de vigilância sanitária (ANVISA) e apresentou linearidade, seletividade, precisão, exatidão e recuperação adequadas para as estatinas, excetuando-se para a sinvastatina. A faixa de linearidade obtida foi de 10-200 ng mL-1 (FLV e AT) e 20-200 ng mL-1 (PRA). Os limites de quantificação obtidos foram da ordem de 10 ng mL-1 (AT e FLV) e 20 ng mL-1 (PRA). Desta forma o método desenvolvido poderá ser utilizado para a determinação dos níveis de pravastatina, fluvastatina e atorvastatina em amostras de plasma humano. / Elevated plasma cholesterol level is a risk factor for coronary diseases, which are the most deadly sickness according to the World Health Organization (WHO). In order to fight the hypercholesterolemia in patients, statins are a well-established class of drugs to be prescribed. Even though they are efficient, some side effects can be associated with statin therapy, especially when interactions with other drugs occur. In these cases, monitoring the concentration can optimize the drug dosage to therapeutic effectiveness whilst minimizing the adverse effects. The aim of this work was to develop a method for analysis of pravastatin (PRA), atorvastatin (AT), fluvastatin (FLV) and simvastatin (SV) in human plasma. The experimental means chosen to attain the goal was liquid chromatography-tandem mass spectrometry (LC-MS/MS) and for the sample preparation, microextraction by packed sorbent (MEPS). To optimize the extraction conditions, parameters such as sorbent, elution and washing solution were evaluated. Other parameters such as sampling, elution cycles, sample volume and elution steps were evaluated using multivariate experimental design. The extraction was performed using C18 Chromabond as sorbent. The method was validated based on ANVISA recommendations and featured appropriated linearity, selectivity, accuracy, precision, and recovery, except for simvastatin. The calibration curve in plasma was obtained in the concentration range 10-200 ng mL-1 (FLV and AT) and 20-200 ng mL-1 (PRA) and the limit of quantification (LOQ) was 10 ng mL-1 (FLV and AT) and 20 ng mL-1 (PRA). The method developed proved to be suitable for the analysis of pravastatin, fluvastatin and atorvastatin in human plasma sample, but not simvastatin, and it can contribute to a more efficient usage of the statins in the treatment of hypercholesterolemia.

O quadro evolutivo da agricultura brasileira resulta em benefícios à população exigindo crescentes avanços tecnológicos no setor. Constantemente, novos agrotóxicos são introduzidos estimulando estudos científicos com a finalidade de determinar e avaliar os impactos na população e no meio ambiente. No presente trabalho, a matriz avaliada foi o lodo gerado no processo de tratamento de água para consumo humano, coletado na região do Vale do Ribeira, SP. A técnica empregada foi a cromatografia líquida de fase reversa acoplada à espectrometria de massas triploquadrupolar em tandem com ionização por electrospray. Os compostos foram extraídos previamente da matriz. O desenvolvimento da metodologia exigiu tratamento dos dados para que esses pudessem ser utilizados e transformados em informações confiáveis. Os processos envolvidos foram avaliados usando o conceito da validação de ensaios químicos. Os indicadores avaliados foram seletividade, linearidade, intervalo de trabalho, sensibilidade, exatidão, precisão, limite de detecção, limite de quantificação e robustez. Esses indicadores produziram valores quantitativos e qualitativos que foram estatisticamente evidenciados de forma objetiva. A metodologia desenvolvida e validade é simples. Como resultado, mesmo explorando a sensibilidade da técnica, os compostos estudados não foram encontrados no lodo da ETA de Registro. Isso leva a crer que esses compostos podem estar presentes em concentrações muito baixas, podem sofrer degradação durante o tratamento da água ou não são retidos completamente pela ETA. 7 / The evolving scenario of Brazilian agriculture brings benefits to the population and demands technological advances to this field. Constantly, new pesticides are introduced encouraging scientific studies with the aim of determine and evaluate impacts on the population and on environment. In this work, the evaluated sample was the sludge resulted from water treatment plant located in the Vale do Ribeira, São Paulo, Brazil. The technique used was the reversed phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Compounds were previously liquid extracted from the matrix. The development of the methodology demanded data processing in order to be transformed into reliable information. The processes involved concepts of validation of chemical analysis. The evaluated parameters were selectivity, linearity, range, sensitivity, accuracy, precision, limit of detection, limit of quantification and robustness. The obtained qualitative and quantitative results were statistically treated and presented. The developed and validated methodology is simple. As results, even exploring the sensitivity of the analytical technique, the work compounds were not detected in the sludge of the WTP. One can explain that these compounds can be present in a very low concentration, can be degraded under the conditions of the water treatment process or are not completely retained by the WTP.

O número crescente de descobertas de substâncias bioativas produzidas pelo metabolismo secundário de cianobactérias tem despertado o interesse de grupos de pesquisa no mundo todo com o objetivo comum de descrever e explorar estas moléculas e entender a sua biossíntese. No Brasil, as pesquisas sobre moléculas bioativas produzidas por linhagens de cianobactérias nativas são escassas. Neste trabalho, utilizando iniciadores específicos da PCR e sequenciamento, a presença de genes envolvidos na biossíntese da neurotoxina saxitoxina (STX) foi confirmada em representantes dos gêneros Dolichospermum, Sphaerospermopsis, Cuspidothrix e Cylindrospermopsis, enquanto que genes da citotoxina cilindrospermopsina (CYN) foram detectados somente em representantes de Cylindrospermopsis. Genes envolvidos com a produção dos inibidores enzimáticos, microviridina (MDN) e a cianobactina microciclamida (MCA) foram sequenciados em isolados do gênero Microcystis. Os genomas das linhagens de Cylindrospermopsis raciborskii CENA302 e CENA303 foram sequenciados usando a plataforma HiScan SQ (Ilumina) com biblioteca pareada 2 x 100 pb. O genoma da Sphaerospermopsis torques-reginae ITEP-024 foi sequenciado utilizando a plataforma Ion Torrent (Life Technologies) com tamanhos de fragmentos de até 200 pb. As tentativas de montagem ab initio dos genomas foram realizadas e o agrupamento gênico da saxitoxina (28 kb) da linhagem C. raciborskii CENA302 foi identificado e caracterizado. As análises filogenéticas das sequências de aminoácidos envolvidos com a biossíntese das moléculas bioativas avaliadas demonstraram que os isolados brasileiros de cianobactérias formam clados com elevado valor de reamostragem com sequências homólogas de cianobactérias conhecidas como produtoras dessas moléculas. Neste estudo é relatada pela primeira vez a presença de genes cyr em linhagens da América do Sul de C. raciborskii e a presença simultânea de genes cyr e sxt em uma única linhagem de C. raciborskii. Além disso, este é o primeiro estudo que relata a presença de genes envolvidos na biossíntese de MDN e MCA nas espécies de cianobactérias M. protocystis, M. panniformis e M. wesenbergii. Análises por espectrometria de massas acoplada a cromatografia líquida (LC-MS) e imunoensaio enzimático (ELISA) foram utilizadas a fim de detectar e identificar variantes estruturais das moléculas bioativas das cianobactérias que tiveram os genes biossintéticos sequenciados. A análise de LC-MS mostrou a produção das variantes GTX2, GTX3, STX e dc-STX pela linhagem C. raciborskii CENA302, enquanto que a linhagem C. raciborskii CENA305 apresentou as variantes NEO, C1 e dcGTX3. As quatro novas variantes de MCY, [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR e [D-Phe1]MC-LR, foram encontradas nas espécies M. panniformis SPC702 e M. protocystis SPC697. Este é o primeiro relato da produção de MCY por essas duas espécies de Microcystis. Dezesseis linhagens que ainda não possuíam as sequências do gene de RNAr 16S foram sequenciadas. O resultado da análise filogenética das sequências do gene de RNAr 16S foi coerente com as descrições morfológicas, sendo que todas as linhagens foram caracterizadas em nível de espécie. As informações geradas neste estudo contribuem para o aumento do conhecimento da diversidade metabólica dos isolados brasileiros de cianobactérias e trazem nova visão sobre a evolução dessas moléculas produzidas pelo metabolismo secundário / The growing numbers of discoveries of bioactive substances produced by cyanobacterial secondary metabolism has attracted the interest of research groups around the world with the common goal of describing and exploring these molecules and understanding their biosynthesis. In Brazil, researches on bioactive molecules produced by native cyanobacterial strains are scarce. In this work, using specific PCR primers and sequencing, the presence of genes involved in the biosynthesis of the neurotoxin saxitoxin (STX) was confirmed in representatives of the genera Dolichospermum, Sphaerospermopsis, Cuspidothrix and Cylindrospermopsis, while genes of the cytotoxin cylindrospermopsin (CYN) were detected only in representatives of Cylindrospermopsis. Genes involved in the production of protease inhibitors, microviridin (MDN) and the cianobactin microciclamide (MCA), were sequenced in isolates of the genus Microcystis. The genomes of Cylindrospermopsis raciborskii strains CENA302 and CENA303 were sequenced using the high-throughput platform HiScan SQ (Illumina) as paired-ends 2 x 100 bp. The Sphaerospermopsis torques-reginae ITEP-024 genome was sequenced using the high-throughput platform Ion Torrent (Life Technologies) with fragment sizes up to 200 bp. Attempts of ab initio genomes assembly were performed and the 28 kb saxitoxin gene cluster of C. raciborskii strains CENA302 was identified and characterized. Phylogenetic analyses of amino acid sequences involved in the biosynthesis of the bioactive molecules evaluated showed that the Brazilian cyanobacterial isolates formed clades with high bootstrap values with homologous sequences of known cyanobacterial producers of these molecules. In this study is reported for the first time the presence of cyr genes in South America strains of C. raciborskii and the simultaneous presence of cyr and sxt genes in a single C. raciborskii strain. Furthermore, this is the first study reporting the presence of genes involved in the biosynthesis of MDN and MCA in the cyanobacterial species M. protocystis, M. panniformis e M. wesenbergii. Analyses by mass spectrometry coupled to liquid chromatography (LC-MS) and enzyme immunoassay (ELISA) were used to detect and identify structural variants of bioactive molecules of the cyanobacteria that had the biosynthetic genes sequenced. Analysis of LC-MS showed the production of the variants GTX2, GTX3, STX and dc-STX by the C. raciborskii strain CENA302, whereas the strain C. raciborskii CENA305 presented the variants NEO, C1 and dcGTX3. The new four MCY variants [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR and [D-Phe1]MC-LR were found in the species M. panniformis SPC702 and M. protocystis SPC697. This is the first report of the MCY production by these two species of Microcystis. Sixteen strains that still lacked the 16S rRNA gene sequences were sequenced. The result of the phylogenetic analysis of 16S rRNA gene sequences was consistent with the morphological descriptions, and all strains were characterized to species level. The informations generated in this study contribute to the increase of knowledge on metabolic diversity of Brazilian cyanobacterial strains and bring new insight into the evolution of these molecules produced by secondary metabolism