A identificação e seleção de animais com maior potencial genético de crescimento vem proporcionando um aumento substancial nos índices produtivos. Para tanto, ferramentas do melhoramento genético vem sendo utilizadas, além disso, mudanças nos padrões de crescimento animal, podem alterar o metabolismo animal e consequentemente os metabólitos. Dessa forma, o objetivo desse trabalho, foi analisar o efeito do potencial genético para crescimento avaliado pós-desmama no desempenho e metaboloma do plasma de machos Nelore durante o período de terminação. Foram coletadas amostras de plasma de 74 bovinos Nelore com diferente potencial de crescimento e provindos de dois rebanhos distintos. Ambos os rebanhos foram divididos em animais de alta potencial genético para crescimento e baixo potencial genético para crescimento. Os animais permaneceram alojados em confinamento, recebendo a mesma dieta durante todo o período experimental. Os animais foram pesados periodicamente para avaliar a ganho médio diário e no dia anterior ao abate foi realizada coleta de sangue para analise metabolômica do plasma. O perfil metabolômico das amostras de plasma foi determinado utilizando a Ressonância Magnética Nuclear. Durante a seleção dos animais do rebanho 1 durante a fase de recria, o ganho de peso dos animais do rebanho 1 de alto crescimento, foi 323 g/dia superior (P <0,0001) e o peso vivo ao sobreano foi de 54 kg maior (P = 0,05) comparado aos animais de baixo crescimento, evidenciando diferenças fisiológicas e fenotípicas entre os grupos nesse momento. Também foi observado no rebanho 1 maior peso vivo ao final do período de 35 kg (P = 0,004) para os animais de alto crescimento. Além disso, os resultados obtidos nesse estudo, não mostraram diferenças estatística entre os diferentes potenciais genéticos para crescimento ao final da terminação. Dessa forma, o presente estudo conclui que a diferença fenotípica em função do potencial genético para crescimento ocorreu no período de seleção da mesma, porém essa diferença não foi observada durante a terminação dos animais, da mesma forma, não houve diferença metabolômica no plasma dos animais de acordo com o potencial genético para crescimento, avaliada no período final da terminação. / The identification and animals selection with greater genetic potential of growth has provided a substantial increase in the productive indexes. For many tools of genetic breeding, changes in animal growth patterns have been used, they can modify animal metabolism and consequently, metabolites. Therefore, the objective of the present work was to analyze the effect of the genetic potential for post-weaned growth on the performance and metabolome of Nellore male bull plasma during the finishing period. Plasma samples were collected from 74 Nelore bulls with different growth potential and from two different herds. Both herds were divided into animals of high and low genetic potential for growth. The animals were kept in feedlot, receiving the same diet throughout the experimental period. The animals were periodically weighed to evaluate the mean daily gain and the day before slaughter, blood collection was performed for metabolic analysis of the plasma. The metabolomic profile of the plasma samples was determined using Nuclear Magnetic Resonance. During the selection of animals from herd 1 during the growing phase, the weight gain of the high growth herd 1 animals was 323 g / day higher (P <0.0001) and the live weight to the overane was 54 kg higher (P = 0.05) compared to low growth animals, evidencing physiological and phenotypic differences between the groups at that time. It was also observed in the herd 1 greater live weight at the end of the period of 35 kg (P = 0.004) for the high growth animals. In addition, the results obtained in this study did not show statistical differences between the different genetic potentials for growth at the end of the study. Therefore, the present study concludes that the phenotypic difference as a function of the genetic potential for growth occurred during the selection period, althoug this difference was not observed during the finishing of the animals, in the same way, there was no metabolomic difference in the animals plasma according to the genetic potential for growth, evaluated in the final termination period.

As neolignanas, bem como as lignanas, apresentam diversas atividades biológicas comprovadas, sendo a licarina A uma neolignana diidrobenzofurânica, detentora de atividade leishmanicida. A partir dessa atividade e do interesse na produção de fármacos eficazes no tratamento dessa doença, a licarina A foi selecionada para estudo de metabolismo in vitro a partir de reações biomiméticas utililizando [Fe(TPP)Cl] e Mn(Salen) como catalisadores; sob ação de oxidantes (PhIO e mCPBA) em quatro solventes de polaridades distintas (AcOEt, MeOH, ACN, DCM), na proporção de 1:30:30 (catalisador:substrato:oxidante). Inicialmente, as reações realizadas foram submetidas às análises por CG-EM comprovando um produto de oxidação majoritário m/z 342, denominado metabólito A. Os solventes de menor polaridade (DCM e AcOEt), bem como o catalisador de Jacobsen, foram mais eficientes na geração desse metabólito. O aumento do tempo reacional bem como o aumento da concentração do oxidante não favoreceu a produção de metabólitos. Posteriormente, as análises dessas reações também foram realizadas por CLUE-DAD-EM/EM e sete metabólitos puderam ser identificados a partir do auxílio do estudo de fragmentação da licarina A bem como a partir de análises de RMN e EM dos metabólitos isolados por CLAE em escala semipreparativa. As análises de RMN dessas frações permitiram a determinação estrutural de um metabólito de m/z 343 [M+H]+ (metabólito 3) constituído de uma epoxidação nos carbonos C-7\'/C-8\' situados na cadeia lateral da licarina A. Sendo assim, quatro dos sete metabólitos (2, 3, 6 e 7) formados apresentaram m/z 343 [M+H]+ e espectros de massas tandem muito semelhantes, correspondendo aos possíveis diasteroisômeros do metabólito epoxidado, visto que a licarina A apresenta dois centros quirais. Outros três metabólitos foram produzidos e exibiram íons de m/z 361 [M+H]+, m/z 315 [M+H]+ e m/z 341 [M+H]+, correspondentes respectivamente a um diol vicinal em C-7\'/C-8\', um aldeído benzóico e um outro aldeído gerado a partir da oxidação da metila terminal em C-9\'. Foi realizado ainda o estudo de metabolismo in vitro com microssomas hepáticos de ratos, sendo que esse estudo foi capaz de reproduzir as reações biomiméticas a partir da produção do mesmo metabólito (2) (apresentando mesmo TR e mesmo espectro de UV). Em relação à toxicidade e aos possíveis danos que a licarina A poderia acarretar, um estudo de toxicidade aguda foi realizado sendo esse capaz de auxiliar na classificação da licarina A, sendo a mesma tóxica em doses > 300mg/kg e 2000mg/kg (categoria 4). Análises dos parâmetros bioquímicos realizadas foram capazes de determinar possível toxicidade hepática, a partir das alterações enzimáticas (AST, ALT, LDH, ALP) ocorridas. Contudo, análise microscópica de cortes dos tecidos (fígado, coração e rins) não determinaram nenhum comprometimento tecidual que pudesse evidenciar toxicidade. / Neolignans and lignans show several biological activities. Licarin A is a dihydrobenzofuranic neolignan, which exhibits leishmanicidal activity. For this reason, licarina A was selected to carry out the studies of in vitro metabolism. So biomimetic reactions were done by the use of [Fe(TPP)Cl] and Mn (Salen) catalysts, different oxidants (PhIO and mCPBA), as well solvents with different polarities (EtOAc, MeOH, ACN, and DCM), and the ratio of 1:30:30 (catalyst:substrate:oxidant). Initially, the reactions were analyzed by GC-MS and showed one major oxidation product of m/z 342 (metabolite A). The less polar solvents (DCM and EtOAc) and Jacobsen catalyst (Mn (Salen)) were more efficient for the production of metabolite A. The reaction time and concentration were increased, but they did not represent a higher production of metabolites. Subsequently these reactions were also analyzed by UPLC-DADMS/ MS and seven metabolites could be identified based on the fragmentation pattern proposed for licarin A, as well as from NMR and MS data of the metabolites isolated by HPLC. A metabolite of m/z 343 [M+H]+ (metabolite 3) was identified by NMR and MS/MS data and it consists of epoxidation in the carbons of C-7\'/C-8\' from licarin A. Therefore, four of the seven metabolites (2, 3, 6 and 7) exhibited m/z 343 [M+H]+ in the MS spectra and the MS/MS showed high similarity with the epoxided metabolite (metabolite 3). So these metabolites could be diasteroisomers of the metabolite 3. Three other metabolites were produced and exhibited ions at m/z 361 [M+H]+, m/z 315 [M+H]+ and m/z 341 [M+H]+, corresponding respectively to a vicinal diol in C-7\'/C-8\', benzylic aldehyde and other aldehyde generated from the oxidation of the terminal methyl at C-9\'. The study of in vitro metabolism by rat liver microsomes was also carried out, and this study was able to reproduce the biomimetic reactions by production of the same metabolite (2). The acute toxicity and potential damage of licarin A were evaluated and the results indicated toxicity for this compound at doses >300mg/kg and 2000mg/kg (category 4). Analysis of biochemical parameters were able to determine possible liver toxicity caused by enzymatic changes (AST, ALT, LDH, ALP). However, microscopic analysis of tissues sections (liver, heart and kidneys) did not show any tissue damage that could indicate toxicity.

Foram isolados 37 fungos endofíticos de Viguiera arenaria (VA1 a VA37), sendo 32 oriundos de folhas e 5 de raízes. Os fungos foram classificados por métodos de biologia molecular, sendo Glomerella cingulata a espécie predominante. Os fungos foram cultivados em culturas fermentativas em duas etapas, em pequena escala, para obtenção dos extratos em AcOEt, BuOH e MeOH. Os extratos em AcOEt foram avaliados em ensaios antimicrobianos, citotóxicos frente a células leucemia T humana (JURKAT) e frente às enzimas GAPDH de Trypanosoma cruzi e APRT de Leishmania tarentolae. Diversos extratos apresentaram atividades biológicas significativas. Os perfis químicos dos extratos em AcOEt foram avaliados através de CLAE-UV e RMN 1H. Os fungos VA1 (Glomerella cingulata) e VA17 (Fusarium sp.), selecionados após as triagens química e biológica, foram cultivados em escala ampliada. A partir do extrato AcOEt do fungo VA1 foram isoladas duas substâncias, nectriapirona (I) e tirosol (II). A nectriapirona apresentou atividade citotóxica significativa contra células de leucemia T humana (linhagens JURKAT) e melanoma (linhagens B16F10). Ergosterol (III) foi isolado do extrato micelial metanólico do fungo VA1. A partir do extrato micelial metanólico do fungo VA5 (G. cingulata), cultivado em pequena escala, foi isolado o manitol (IV). Após do cultivo em escala ampliada do fungo VA17 (Fusarium sp.) foram isolados três derivados dicetopiperazinícos, um oriundo do extrato AcOEt, substância V, ainda não relatada na literatura, e dois do extrato micelial MeOH, fusaperazina B (VI) e substância VII, também inédita na literatura. O isolamento das substâncias foi realizado através de técnicas cromatográficas, como CC, CCDP e CLAE. As estruturas químicas foram elucidadas com auxílio de técnicas espectroscópicas (RMN 1H e 13C, HMQC, HMBC, NOE-diff) e espectrométricas (ESI-MS). Experimentos de modelagem molecular foram realizados com a fusaperazina B (VI), que corroboraram com os dados de NOE-diff, indicando que a conformação dobrada do anel dicetopiperazínico é a mais predominante. Foi ainda realizada avaliação do perfil químico dos extratos obtidos em pequena escala dos fungos classificados como G. cingulata, via CLAE e RMN 1H, verificando-se diferenças químicas significativas, o que pode sugerir variabilidade genética entre as linhagens. O tirosol (II) foi detectado na maioria dos extratos de G. cingulata. / A total of 37 endophytic fungi were isolated from Viguiera arenaria (VA1 to VA37), 32 from the leaves and 5 from the roots. Endophytes were classified by means of molecular biology methods, and Glomerella cingulata was the predominant species. The endophytes were cultured in a two step fermentative process in small scale to give the EtOAc, BuOH and MeOH extracts. The EtOAc extracts were submitted to antimicrobial assays, citotoxic assays against human leukemia T cells (JURKAT) and assays against two enzymes, GAPDH from Trypanosoma cruzi and APRT from Leishmania tarentolae. Several extracts showed promising activities in the bioassays. The chemical profiles of the EtOAc extracts were obtained through HPLC and 1H NMR. Endophytes VA1 (Glomerella cingulata) e VA17 (Fusarium sp.) were cultured in large scale after chemical and biological screenings. Nectriapyrone (I) and tirosol (II) were isolated from the EtOAc extract from VA1. Nectriapyrone showed high citotoxicity against leukemia T human cells (JURKAT) and melanoma cells (B16F10). Ergosterol (III) was isolated from the micelial MeOH extract of VA1. Mannitol (IV) was isolated from the micelial MeOH extract from the fungus VA5 (G. cingulata). Extracts from VA17 (Fusarium sp.), obtained in large scale, yielded three diketopiperazine derivatives. A novel derivative was isolated from the EtOAc extract (compound V). Two derivatives were obtained from the micelial MeOH extract, fusaperazine B (VI) and a new diketopiperazine (VII). The isolation of the compounds was carried out using chromatography techniques (column, prep. TLC, and HPLC) and the identification was achieved by spectroscopic (1D and 2D NMR) and spectrometric methods (ESI-MS). Molecular modeling experiments were carried out with fusaperazine B (VI), showing that the diketopiperazine ring is predominantly in the folded conformation, in agreement with NOE-diff experiments. Significant differences were observed in the HPLC profiles and 1H NMR spectra of the extracts from the G. cingulata strains, which might be related to genetic variability among the strains. Tirosol (II) was detected in the majority of G. cingulata extracts

Algumas espécies de cianobactérias podem produzir diferentes tipos de peptídeos bioativos (cianopeptídeos), como aeruginosinas, cianopeptolinas, microgininas, microviridinas, anabaenopeptinas e microcistinas. As microcistinas, que compõem a classe mais conhecida na literatura científica, são hepatotoxinas e inibem fosfatases do tipo 1 e 2A. Já as microgininas, moléculas inibidoras da enzima conversora de angiotensina (ECA) e de outras proteases, são importantes candidatas ao desenvolvimento de fármacos anti-hipertensivos. No entanto, as funções fisiológicas desses compostos ainda não foram completamente elucidadas. O objetivo desse estudo é identificar e isolar cianopeptídeos, especialmente da classe das microgininas, produzidos por cepas de cianobactérias brasileiras. O fracionamento dos extratos das cianobactérias foi biomonitorado por meio de ensaios de inibição da ECA. Para tanto, foram produzidos extratos de culturas de 59 cepas de cianobactérias mantidas em laboratório e, quando pertinente, seguiu-se o pré-fracionamento por extração em fase sólida em coluna de fase reversa. As frações obtidas foram testadas para a inibição da ECA e analisadas por cromatografia líquida acoplada à espectrometria de massas (LC-MS). Nos casos em que houve inibição, o fracionamento foi continuado por cromatografia líquida de alta eficiência semipreparativa para o isolamento dos cianopeptídeos. As espécies analisadas pertencem aos gêneros: Microcystis, Oscillatoria, Radiocystis, Sphaerocavum, Sphaerospermopsis, Cylindrospermopsis, Leptolyngbya, Pleurocapsa, Chrooccocidiopsis, Nostoc, Brasilonema, Desmonostoc, Oculatella e Limnothrix. Além de outras espécies das famílias Pseudanabaenaceae, Nostocaceae e Michochaetaceae que não tiveram suas identificações concluídas. Nas análises por HPLC-DAD não foram encontrados picos com espectros de UV característicos de microgininas nas 59 cepas avaliadas. Esses resultados foram corroborados por análises por LC-MS, nas quais também não foi possível a detecção desses compostos. Como método de triagem, as análises por LC-MS triplo quadrupolo foram conduzidas via precursor ion scan para o monitoramento das perdas m/z 128 e 142, que são fragmentos oriundos do aminoáciodo Ahda, exclusivamente presente em microgininas. Algumas linhagens se revelaram produtoras de variantes do cianopeptídeo microcistina, possuindo absorção UV máxima em 238 nm. Amostras de Israel, doadas pelo Prof. Shmuel Carmeli, da Universidade de Tel Aviv, também foram analisadas por espectrometria de massas no modo precursor ion para a investigação da produção de microgininas. Foram encontrados íons de m/z 128 e 142 em uma das amostras de Israel. Ensaios enzimáticos iniciais da ECA foram realizados com frações das cepas controle LTPNA 08 e 09, sabidamente produtoras de microgininas. Realizou-se a otimização do método inicial de detecção dos cianopeptídeos para que fosse procedido o isolamento dos compostos de interesse da cultura LTPNA 08. As frações obtidas foram analisadas por cromatografia líquida acoplada à espectrometria de massas (LC-MS) e isoladas por um coletor de frações. 4,8 mg de microgininas foram obtidos com o isolamento das moléculas a partir de 3 g de biomassa seca da linhagem. Ensaios enzimáticos de inibição da ECA com as frações isoladas de microgininas e microcistina-LR comercial também foram realiazados. Os resultados mostram que tanto as microgininas quanto a microcistina-LR comercial apresentam potencial de inibição da enzima ECA. As microgininas e a microcistina-LR podem ser consideradas como novas moléculas a serem estudadas para o desenvolvimento de fármacos inibidores da ECA. Continuar a investigação da produção desses compostos é relevante tanto por seu potencial ecotoxicológico quanto por seu potencial farmacológico. / Some cyanobacterial species can produce different types of bioactive peptides (cyanopeptides), such as aeruginosins, cyanopeptolins, microginins, microviridins, anabaenopeptins and microcystins. Microcystins are well known in the scientific literature as hepatotoxins that inhibit phosphatases type 1 and 2A. Microginins are reported as inhibitors of the angiotensin converting enzyme (ACE) and other proteases, being important candidates for the development of anti-hypertensive compounds. Nevertheless, the physiological functions of these compounds have not been fully elucidated. The aim of this study is to isolate and identify cyanopeptides, namely microginins, produced by Brazilian cyanobacterial strains. The fractionation of cyanobacterial extracts was screened by ACE inhibition assays. Extracts of 59 cyanobacterial strains were produced from cultures cultivated under laboratory conditions and, in cases where microginins were found, prefractionation were carried out by solid phase extraction on a reverse phase column. The fractions obtained were tested for inhibition of ACE and analyzed by liquid chromatographymass spectrometry (LC-MS) by using precursor ion scan to monitor the loss of m/z 128 and 142, typical fragments detected from the amino acid Ahda, which is exclusively found in microginins. In cases where fractions demonstrated inhibitory activity, the fractionation was continued by semi-preparative high performance liquid chromatography for the isolation of cyanopeptides. The analyzed species belong to the genere: Microcystis, Oscillatoria, Radiocystis, Sphaerocavum, Sphaerospermopsis, Cylindrospermopsis, Leptolyngbya, Pleurocapsa, Chrooccocidiopsis, Nostoc, Brasilonema, Desmonostoc, Oculatella and Limnothrix. And other species of Pseudanabaenaceae, Nostocaceae and Michochaetaceae families did not have their peptides completely characterized. Analyses of HPLC-DAD did not revealed characteristic features by UV spectra of microginins in the 59 strains. These results were corroborated by analyses of LC-MS triple quadrupole, microginins were not detected as well. Some strains were positive for microcystin variants, showing maximum UV absorption at 238 nm and typical MS/MS spectra. Initial enzyme assays for the inhibition of ACE were performed with fractions of the control strains LTPNA 08 and 09, known as microginins\' producers. Samples fromIsrael, kindly donated by Prof. Shmuel Carmeli, of Tel Aviv University, were analyzed by mass spectrometry in the precursor ion mode for the investigation of microginins. Ions m/z 128 and 142 were founded on one sample of Israel. A HPLC method was optimized for the detection and isolation of cyanopeptides produced by culture LTPNA 08. Fractions isolated by semi-preparative HPLC were analyzed by LC-MS and collected when UV and MS spectra were characteristically similar to microginins . 4.8 mg of microginins were obtained by semi-preparative HPLC from 3 g of dried strains. The inhibition assays of ECA were performed with fractions of microginins and microcystin-LR analytical standard. The results showed that microginins as well as microcystin-LR have the potential of inhibiting ACE. Microginins and microcystin-LR can be considered as target molecules for the study of ACE inhibitors. Further research on the production of these classes of peptides is relevant because of its ecotoxicological and pharmacological potential.

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PCβF/PCαR, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining". A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits  (cpcB) and  (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PCβF/PCαR targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

In most plant species artificial polyploidy generally enhances the vigor of determinate plant parts and may be favorable where vegetative organs and biomass constitute the economic product. Furthermore, artificial polyploidy has been considered a method of increasing production potential of plants secondary metabolites. However, despite considerable research on polyploid plants, very few cases of polyploid medicinal plants have been reported. Artemisia annua L. synthesizes artemisinin, an antimalarial sesquiterpene lactone. Artemisinin can be synthesized, but it is costly compared to the naturally derived product. Hairy root cultures of Artemisia annua L. (clone YUT16) show rapid growth and produce artemisinin. This culture offers a good model system for studying artemisinin production. Others have shown that tetraploid Artemisia annua L. plants produce more artemisinin/mg DW than diploids. These yields were offset, however, by decreases in biomass productivity. Little is known about how polyploidy may affect growth production of hairy roots. Using colchicine, we have produced four stable tetraploid clones of Artemisia annua L. from YUT16 hairy root clone. Compared to the diploid clone, these tetraploid clones showed major differences in growth and development. Nevertheless, artemisinin yields of these tetraploid clones were 2-5 times higher than the diploid and their production seemed to be by the age of the inoculum. This work will prove useful in furthering our understanding of the effects of artificial polyploidy on the growth and secondary metabolite production of hairy roots.

<p>S obzirom&nbsp; da su cijanobakterije (modrozelene alge) identifikovane kao jedna od<br />najperspektivnijih grupa organizama za izolaciju novih i biolo&scaron;ki aktivnih prirodnih<br />produkata, cilj ove teze bio&nbsp; je utvrđivanje biotehnolo&scaron;kog potencijala&nbsp; autohtonih<br />filamentoznih sojeva&nbsp; cijanobakterija izolovanih sa područja Vojvodine koji pripadaju<br />azotofiksirajućim rodovima&nbsp;<em> Nostoc</em>&nbsp; i&nbsp; <em>Anabaena</em>&nbsp; i neazotofiksirajućem rodu&nbsp;&nbsp;&nbsp; <em>Spirulina</em>. Biotehnolo&scaron;ki potencijal testiranih sojeva je određen&nbsp; u smislu produkcije biomase, fikobiliproteinskih pigmenata, masnih kiselina, fenolnih jedinjenja, antioksidanata, antibakterijskih i antikancerogenih agenasa. Dobijeni rezultati su pokazali&nbsp; da su produkcija biomase i sadržaj fikobiliproteinskih pigmenata kod svih testiranih sojeva zavisili od primenjenih uslova kultivacije, pri&nbsp; čemu je&nbsp; kod sojeva roda&nbsp; <em>Spirulina</em> produkcija biomase bila jače stimulisana primenom kontinualnog osvetljenja, a kod azotofiksirajućih sojeva rodova&nbsp;<em> Nostoc&nbsp;</em> i&nbsp;<em> Anabaena</em>&nbsp; organskim izvorima ugljenika (glicerolom i glukozom). Kao soj sa najvećim potencijalom za&nbsp; proizvodnju biomase izdvaja se soj&nbsp; <em>Spirulina&nbsp; S1</em>, a za produkciju fikobiliproteina sojevi&nbsp; <em>Spirulina</em>&nbsp; S1,&nbsp;<em> Nostoc</em> 2S1,&nbsp; <em>Anabaena</em>&nbsp; Č2 i&nbsp; <em>Spirulina</em>&nbsp; S2.&nbsp; Određivanjem sadržaja masnih kiselina GC-FID metodom utvrđeno je da su&nbsp; kod svih sojeva&nbsp; najzastupljenije bile palmitinska, palmitoleinska, oleinska i linolna kiselina, pri čemu su&nbsp; sojevi roda <em>Spirulina</em> produkovali i&nbsp; &gamma;-linolensku kiselinu, dok su svi&nbsp; sojevi rodova&nbsp; <em>Nostoc&nbsp;</em> i&nbsp;<em> Anabaena&nbsp;</em> produkovali&nbsp; -linoleinsku kiselinu.&nbsp; Najzastupljenije fenolne komponente testiranih etanolnih ekstrakata određene HPLC-MS/MS metodom&nbsp; bile su hinska kiselina i katehin, pri čemu je najveći sadržaj fenolnih jedinjenja registrovan kod soja&nbsp; <em>Nostoc</em>&nbsp; 2S7B. Hemijskom karakterizacijom ekstrakata kod testiranih sojeva takođe je utvrđen značaj azotnih uslova kultivacije&nbsp; u cilju povećanja produkcije&nbsp; fenolnih jedinjenja,&nbsp; kao i&nbsp; -linoleinske kiseline. Poređenjem rezultata antioksidantne aktivnosti u kori&scaron;ćenim testovima&nbsp; DPPH&nbsp; i FRAP, kao sojevi sa najvećim&nbsp; antioksidantnim potencijalom izdvajaju se&nbsp; <em>Spirulina</em>&nbsp; S1 i<em> Spirulina</em>&nbsp; S2. Antibakterijska aktivnost metanolnih ekstrakata&nbsp; registrovana je kod sojeva<em> Nostoc</em> 2S7B, <em>Nostoc</em> 2S1, <em>Anabaena</em> Č2, <em>Anabaena</em> Č5, <em>Spirulina</em> S1 i&nbsp;<em> Spirulina</em> S2, koji su ispoljili efekat na Gram-pozitivne i Gram-negativne bakterije, pri čemu su&nbsp; sojevi <em>Anabaena</em>&nbsp; Č2,<em> Nostoc&nbsp;</em> 2S7B&nbsp; i&nbsp; <em>Nostoc</em>&nbsp; 2S1&nbsp; delovali na najvi&scaron;e bakterijskih sojeva.&nbsp; Kod svih testiranih sojeva je primenom MTT testa uočena antikancerogena tj. citotoksična aktivnost dimetil sulfoksidnih (DMSO) ekstrakata prema HepG2 ćelijskoj liniji, među kojima su najveću aktivnost&nbsp; ispoljili sojevi&nbsp;<em> Nostoc</em>&nbsp; LC1B i&nbsp; <em>Nostoc&nbsp; </em>2S7B.&nbsp; Primenom bioeseja&nbsp; <em>Artemia salina, Daphnia magna</em>&nbsp; i&nbsp;&nbsp;<em> Danio rerio&nbsp; </em>konstatovan je mali broj sojeva koji su ispoljili toksičnost na test organizme, dok na ćelijsku liniju&nbsp; RTL-W1&nbsp; testirani sojevi nisu ispoljili citotoksičnost&nbsp;<em> in vitro</em>, &scaron;to sa aspekta potencijalne biotehnolo&scaron;ke primene sojeva ima veliki značaj. Kao najtoksičniji izdvojili su se sojevi<em>&nbsp; Nostoc&nbsp; </em>LC1B&nbsp; i <em>Nostoc </em>S8 koji su ispoljili&nbsp; toksičnost u sva tri bioeseja. Ispitivanjem toksičnosti<em> in vitro</em> u enzimskim esejima konstatovano je da je manji broj sojeva inhibirao aktivnost enzimaprotein fosfataze 1 (PP1) u odnosu na aktivnost enzima&nbsp; acetilholinesteraze&nbsp; (AChE). Primenom Analitičkog hijerarhijskog procesa u grupnom kontekstu, najveću težinu su dobili kriterijumi antikancerogena ativnost, produkcija biomase i sadržaj fikocijanina, navedenim redom. Konačno, u vi&scaron;ekriterijumskom kontekstu najbolje rangiran soj je<br /><em>Spirulina</em> S1, na drugom mestu je soj <em>Spirulina</em> S2, dok je na trećem soj&nbsp;<em> Nostoc&nbsp; </em>LC1B.</p> / <p>Cyanobacteria (blue-green algae) have been identified as one&nbsp; of the most promising groups of organisms for the isolation of new and biologically active natural products, therefore, the aim of this thesis was to determine the biotechnological potential of autochthonous filamentous cyanobacterial strains isolated from&nbsp; Vojvodina region,&nbsp; which belong to the N₂-fixing genera&nbsp; <em>Nostoc</em>&nbsp; and&nbsp; Anabaena&nbsp; and non-N₂-fixing genus&nbsp; <em>Spirulina</em>. Biotechnological potential of tested strains was determined using the production of biomass, phycobiliprotein pigments, fatty acids, phenolic co mpounds, antioxidants, antibacterial and anticancer agents. The obtained results showed that the production of biomass and phycobiliprotein pigments, in all tested strains, depended on the cultivationconditions, whereas biomass production was strongly stimulated by continuous light in<em> Spirulina</em>&nbsp; strains, and by organic carbon sources (glycerol and glucose) in N₂-fixingstrains. The highest potential for biomass production was shown in&nbsp;<em> Spirulina</em>&nbsp; S1 strain.On the other hand, the highest potential&nbsp; for the production of phycobiliproteins wasshown in strains&nbsp; <em>Spirulina</em>&nbsp; S1,&nbsp; <em>Nostoc&nbsp;</em> 2S1,&nbsp; <em>Anabaena</em> C2 and&nbsp; <em>Spirulina</em>&nbsp; S2. By determination of the content of fatty acids using GC-FID method it was found that in allthe tested strains the most common fatty acids were palmit ic, palmitoleic, oleic andlinoleic acid, whereby the strains of the genus&nbsp; <em>Spirulina</em> produced &gamma;-linolenic acid as well,while all strains of the<em> Nostoc&nbsp;</em> and <em>Anabaena&nbsp;</em> genera produced y-linolenic acid. The most frequent phenolic compounds of tested strains determined by using the HPLC-MS/MSmethod were quinic acid and catechin, with the highest content of phenolic compounds registered in Nostoc&nbsp; strain 2S7B. By chemical characterization of the extracts in the tested strains it was also stated a significance of&nbsp; the nitrogen cultivation conditions in order toincrease the production of phenolic compounds, as well as&nbsp; y-linolenic acid. Comparing the results of the antioxidant activity in the DPPH and FRAP tests, it&nbsp; was shown that strains&nbsp; <em>Spirulina</em>&nbsp; S1 and&nbsp; <em>Spirulina&nbsp;</em> S2 had the highest antioxidant potential. The antibacterial activity of the intracellular methanolic extracts was registered in strains <em>Nostoc&nbsp;</em> 2S7B,&nbsp; <em>Nostoc&nbsp;</em> 2S1,&nbsp; <em>Anabaena</em>&nbsp; C2,&nbsp; <em>Anabaena</em>&nbsp; C5,&nbsp; Spirulina&nbsp; S1 and&nbsp; Spirulina&nbsp; S2, that&nbsp; inhibited the growth of&nbsp; Gram-positive and Gram&nbsp; -negative bacteria. Using MTT test, anti-cancer ie. cytotoxic activity of&nbsp; dimethyl sulfoxide&nbsp; (DMSO) extracts to the HepG2 cell line was detected in all tested strains, however, the highest activity was exhibited in strains <em>Nostoc</em>&nbsp; LC1B and<em> Nostoc</em> 2S7B . In bioassays <em>Artemia salina,&nbsp; Daphnia magna</em>&nbsp; and <em>Danio rerio</em>&nbsp; a small number of strains exhibited toxicity to the test organisms, while in case of cell line RTL-W1 tested strains did not show&nbsp; in vitro&nbsp; cytotoxicity, which is of great importance from the aspect of the potential biotechnological application of thestrains.&nbsp;<em> Nostoc</em>&nbsp; LC1B and&nbsp; <em>Nostoc</em>&nbsp; S8 strains induced toxicity in all three bioassays, and therefore considered as the most toxic strains. By testing&nbsp; in vitro&nbsp; toxicity in enzyme assays, it was found that few strains inhibited the activity of&nbsp; protein phosphatase (PP1) enzyme in relation to&nbsp; acetylcholinesterase&nbsp; enzyme&nbsp; (AChE) activity. Using the Analytical hierarchical process in the group context, the highest weight was given to the criteria of anticancer&nbsp; activity, biomass production, and the phycocyanin content, respectively. Finally, in the multi-criteria context, the best-ranked strain is&nbsp;<em> Spirulina</em>&nbsp; S1,&nbsp; <em>Spirulina&nbsp;</em> strain S2 is on the second place, while <em>Nostoc</em> strain LC1B is the third one.</p>

<p>Kao jedna od najstarijih grupa fotoautotrofnih mikroorganizama, cijanobakterije predstavljaju &scaron;iroko rasprostranjene prokariote sa raznovrsnim metaboličkim strategijama u cilju preživljavanja i adaptacije na različite uslove životne sredine. Upravo zbog toga, cijanobakterije su značajne kao producenti različitih biolo&scaron;ki aktivnih metabolita. Međutim, većina studija o cijanobakterijama uglavnom je vezana za cijanobakterije vodenih ekosistema. Predmet istraživanja ovog rada je<br />utvrđivanje diverziteta autohtonih cijanobakterija &scaron;umskih ekosistema i karakterizacija odabranih terestričnih cijanobakterijskih predstavnika planinskih područja Srbije izolovanih tokom trogodi&scaron;njeg monitoringa kroz utvrđivanje njihovih osnovnih ekofiziolo&scaron;kih, biohemijskih i genetičkih karakteristika. Rezultati dobijeni u ovom radu su ukazali na diverzitet zemlji&scaron;nih cijanobakterija &scaron;umskih stani&scaron;ta ispitivanih planinskih područja kao i na njihov metabolički diverzitet, odnosno potencijal produkcije različitih bioaktivnih jedinjenja. Na osnovu taksonomski važnih odlika&nbsp;identifikovano je i okarakterisano 20 cijanobakterijskih sojeva za koje je utvrđeno da pripadaju sledećim rodovima:&nbsp;<em>Nostoc, Anabaena, Tolypothrix, Calothrix</em>, <em>Cylindrospermum, Lyngbya, Oscillatoria, Phormidium</em>. Identifikacija izolovanih cijanobakterija primenom molekularanog markera 16S rRNK u većini slučajeva (90%) je potvrdila preliminarnu identifikaciju rodova na osnovu morfolo&scaron;kih kriterijuma. U pogledu produkcije biomase dobijeni rezultati su pokazali da je&nbsp;produkcija biomase kod odabranih testiranih cijanobakterijskih sojeva zavisila od primenjenih uslova kultivacije. Utvrđeno je da su azot, glukoza i saharoza delovali u pravcu stimulacije produkcije biomase kod velikog broja sojeva. Najveća produkcija biomase detektovana je kod soja&nbsp;<em>Calothrix&nbsp;</em>M2 u prisustvu azota u medijumu. Kod soja&nbsp;<em>Nostoc&nbsp;</em>T18 zabeleženo je najveće povećanje produkcije biomase u prisustvu glukoze i saharoze u medijumu. Takođe, sadržaj fikobiliproteina bio je povećan kod većine testiranih sojeva u prisustvu glukoze i saharoze u medijumu. Ispitivanjem sadržaja ugljenih hidrata (glukoze, fruktoze i ksiloze) konstatovano je prisustvo sva tri monosaharida kod svih sojeva pri čemu je svaki soj imao specifičan ugljeno-hidratni profil. Sadržaj monosaharida kod svih testiranih sojeva opadao je u sledećem redosledu glukoza ˃fruktoza ˃ ksiloza. Izuzetnu sposobnost produkcije heksoza i pentoza ispoljila su tri soja<em>&nbsp;Nostoc&nbsp;</em>M1<em>, Phormidium&nbsp;</em>T11 i&nbsp;<em>Calothrix&nbsp;</em>M2. Antibakterijska aktivnost intracelularnih ekstrakata registrovana je kod 16 testiranih&nbsp;cijanobakterijskih sojeva i zavisila je od kombinacije cijanobakterijski-bakterijski soj i tipa primenjenog ekstrakta. U odnosu na heksanske ekstrakte, metanolni ekstrakti su pokazali veću efikasnost, ukazujući na prirodu bioaktivnih jedinjenja sa antibakterijskim delovanjem. Najefikasnijim su se pokazali 75% MeOH ekstrakti cijanobakterijskih sojeva<em>&nbsp;Oscillatoria</em>&nbsp;M2, <span id="cke_bm_336S" style="display: none;">&nbsp;</span><em>Calothrix</em><span id="cke_bm_336E" style="display: none;">&nbsp;</span> M2,<em>&nbsp;Lyngbya</em>&nbsp;T7 i&nbsp;<em>Cylindr<span id="cke_bm_337E" style="display: none;">&nbsp;</span>ospermum</em>&nbsp;K1 koji su ispoljili antibakterijsku aktivnost na 4 testirane bakterije. Hemijskom analizom masno kiselinskog sastava utvrđeno je da je masno kiselinski sadržaj cijanobakterijskih sojeva varirao u zavisnosti od soja. Najznačajniji konstituenti testiranih cijanobakterijskih sojeva bile su 18-to i 16-to ugljenične masne kiseline poput linolne kiseline i &alpha;-linoleinske. Najveći sadržaj linolne kiseline detektovan je kod sojeva <span id="cke_bm_344S" style="display: none;">&nbsp;</span><em>Phormidium</em><span id="cke_bm_344E" style="display: none;">&nbsp;</span> T11 i <em>Tolypothrix</em> K11 &scaron;to ukazuje na sojeve kao potencijalno značajne izvore esencijalnih masnih kiselina. Antiradikalska aktivnost detektovana je kod svih testiranih cijanobakterijskih sojeva. U DPPH eseju, etanolni ekstrakti soja<em> Calothrix</em> M2 ispoljili su najefikasniju sposobnost &bdquo;hvatanja&ldquo; DPPH&middot;radikala dok je u slučaju FRAP metode najveću redukujuću moć imao etanolni ekstrakt soj <em>Cylindrospermum</em> K1. Hemijskom analizom fenolnog sastava kod analiziranih cijanobakterijskih sojeva identifikovano je i kvantifikovano ukupno 21 fenolno jedinjenje. Fenolni sastav je varirao u zavisnosti od soja, a najče&scaron;će detektovana fenolna jedinjenja bila su luteolin-7-O-glukozid, bajkalin i kemferol. Soj sa najznačajnijom sposobno&scaron;ću produkcije fenolnih jedinjenja bio je&nbsp;<em>Phormidium&nbsp;</em>M1 kod koga je identifikovano prisustvo 11 fenolnih jedinjenja. Testirajem toksičnosti intracelularnih ekstrakata u biotestovima <span id="cke_bm_355S" style="display: none;">&nbsp;</span><span id="cke_bm_353S" style="display: none;">&nbsp;</span><em>A. salina</em><span id="cke_bm_355E" style="display: none;">&nbsp;</span><span id="cke_bm_353E" style="display: none;">&nbsp;</span>, <span id="cke_bm_356S" style="display: none;">&nbsp;</span><em>D. magna</em><span id="cke_bm_356E" style="display: none;">&nbsp;</span> i <em>D. rerio</em> ukupno 40<span id="cke_bm_357E" style="display: none;">&nbsp;</span>% testiranih sojeva ispoljilo je toksičan efekat. Najtoksičnijim sojem se pokazao soj&nbsp;<em>Nostoc</em> T7 koji je ispoljio tok<span id="cke_bm_354E" style="display: none;">&nbsp;</span>sičnost u sva tri testa. U slučaju biotesta&nbsp;<em>A. salina&nbsp;</em>najtoksičnijim sojevima pokazali su se&nbsp;<em>Nostoc</em><span id="cke_bm_368E" style="display: none;">&nbsp;</span><span id="cke_bm_366E" style="display: none;">&nbsp;</span>&nbsp;T7,<em>&nbsp;Oscillatoria&nbsp;</em>M2, <em>Oscillatoria&nbsp;</em>T18 i&nbsp;<em>Nostoc</em>&nbsp;K15. Cijanobakterijski sojevi koji su ispoljili najpotentniju aktivnost u biotestu&nbsp;<em>D. magna</em> bili su <span id="cke_bm_382S" style="display: none;">&nbsp;</span><span id="cke_bm_380S" style="display: none;">&nbsp;</span><em>Tolypothrix</em><span id="cke_bm_382E" style="display: none;">&nbsp;</span><span id="cke_bm_380E" style="display: none;">&nbsp;</span> K15, <span id="cke_bm_383S" style="display: none;">&nbsp;</span><em>Nostoc</em><span id="cke_bm_383E" style="display: none;">&nbsp;</span> T7 i <em>Calothrix</em> M2. <span id="cke_bm_384E" style="display: none;">&nbsp;</span><span id="cke_bm_381E" style="display: none;">&nbsp;</span>U biotestu sa embrionima zebrica, soj sa najznačajnijim teratogenim efektom bio je&nbsp; <em>Cylindrospermum</em>&nbsp;K1. U pogledu uticaja ekstrakta testiranog soja na ekspresiju gena&nbsp; kod model organizma&nbsp;<span id="cke_bm_392S" style="display: none;">&nbsp;</span><em>D. rerio</em><span id="cke_bm_392E" style="display: none;">&nbsp;</span>,&nbsp;<em>Cylindrospermum</em>&nbsp;K1 je ispoljio sposobnost modulacije biolo&scaron;kih procesa poput cirkadijalnog ritma kao i sposobnost p<span id="cke_bm_393E" style="display: none;">&nbsp;</span>rodukcije&nbsp;<br />jedinjenja sa estrogenim efektom. Rezultati analize toksigeničnosti testiranih cijanobakterijskih sojeva su pokazali da geni&nbsp;<em>mcyB</em>&nbsp;i&nbsp;<em>mcyE</em>&nbsp;koji su uključeni u&nbsp; produkciju cijanotoksina mikrocistina nisu detektovani ni u jednom od testiranih sojeva. Odsustvo dva gena <span id="cke_bm_399E" style="display: none;">&nbsp;</span>iz mcy genskog klastera ukazuje na to da su druga jedinjenja odgovorna za uočen toksični efekat u primenjenim biotestovima. Dobijeni<br />rezultati ukazuju na značaj ispitivanja zemlji&scaron;nih cijanobakterija, s obzirom na to da su rezultati ovog rada&nbsp; ukazali na velik metabolički diverzitet ispitivanih sojeva i izražen potencijal produkcije različitih bioaktivnih jedinjenja.</p> / <p>As one of the oldest groups of photoautotrophic microorganisms, cyanobacteria represent widespread prokaryotes&nbsp; with diverse metabolic strategies in order to survive and adapt to different environmental conditions. For this reason, cyanobacteria are significant as producers of various biologically active metabolites. However, most of the studies are mainly related to cyanobacteria of aquatic ecosystems. The subject of the research of this dissertation&nbsp; is to determine the diversity of autochthonous cyanobacteria of forest ecosystems and to characterize selected terrestrial cyanobacterial representatives of mountainous areas of Serbia isolated during three year monitoring by determining their basic ecophysiological, biochemical and genetic characteristics. The results obtained in this dissertation&nbsp; have show&nbsp; the diversity of soil cyanobacteria of forest habitats of the investigated mountain areas as well&nbsp; as their metabolic diversity and potential to produce&nbsp; various&nbsp; bioactive compounds. Based on taxonomically important features, 20 cyanobacterial strains have been identified&nbsp; to belong to the following genera:&nbsp; <em>Nostoc, Anabaena</em>, <em>Tolypothrix, Calothrix, Cylindrospermum, Lyngbya, Oscillatoria, Phormidium</em>. Identification of&nbsp; the&nbsp; isolated cyanobacteria using the molecular marker 16S rRNA in most cases (90%) confirmed the preliminary identification of genera based on&nbsp; morphological criteria. In terms of biomass production, the obtained results showed that the production of biomass in the selected tested cyanobacterial strains&nbsp; hepended on the applied cultivation conditions. It was found that nitrogen, glucose and sucrose acted towards the stimulation of biomass production in a large number of strains. The largest biomass production was detected in strain <em>Calothrix</em> M2 in the presence of nitrogen in the medium. In the presence of glucose and sucrose in the medium&nbsp; the highest increase in biomass production was recorded&nbsp; in&nbsp; cyanobacterial strain&nbsp;<em> Nostoc&nbsp;</em> T18. Also, the content of phycobiliproteins&nbsp; has been increased in most&nbsp; of the&nbsp; tested strains&nbsp; in the presence of glucose and sucrose in the medium.&nbsp; Examination of the carbohydrate content (glucose, fructose and xylose)&nbsp; showed that all three monosaccharides were present in all strains and that each strain had a specific carbohydrate profile&nbsp; whereby the content of monosaccharides in&nbsp; all tested strains declined in the following order: glucose ˃ fructose ˃ xylose. Among the tested strains, three strains namely <span id="cke_bm_329S" style="display: none;">&nbsp;</span><em>Nostoc </em><span id="cke_bm_329E" style="display: none;">&nbsp;</span>M1, <em>Phormidium </em>T11 and&nbsp; <em>Calothrix</em> M2 showed the exceptional ability to produce hexose and pentose. The antibacterial activity of intracellular extracts was recorded in 16 tested cyanobacterial strains and depended on the combination of cyanobacterial-bacterial strain and the type of applied extract. Compared to hexane extracts, methanol extracts showed greater efficiency, indicating on the nature of bioactive compounds with antibacterial activity. The most effective were 75% MeOH extracts of cyanobacterial strains&nbsp; <span id="cke_bm_338S" style="display: none;">&nbsp;</span><em>Oscillatoria</em><span id="cke_bm_338E" style="display: none;">&nbsp;</span>&nbsp; M2,&nbsp; <span id="cke_bm_339S" style="display: none;">&nbsp;</span><em>C<span id="cke_bm_330E" style="display: none;">&nbsp;</span>alothrix</em><span id="cke_bm_339E" style="display: none;">&nbsp;</span>&nbsp; M2,&nbsp; <em>Lyngby</em>a&nbsp; T7 an<span id="cke_bm_340E" style="display: none;">&nbsp;</span>d <em>Cylindrospermum</em>&nbsp; K1 which exhibited antibacterial activity on 4 tested bacteria.&nbsp; Results of the analysis of the fatty&nbsp; acid composition showed that the fatty acid content of tested cyanobacterial strains varied depending on the strain. The most significant constituents of the tested&nbsp; cyanobacterial strains were 18 and 16 carbonic fatty acids such as linoleic acid, &alpha;-linoleic. The highest content of linoleic acid was detected in two strains, <span id="cke_bm_350S" style="display: none;">&nbsp;</span><span id="cke_bm_348S" style="display: none;">&nbsp;</span><em>Phormidium</em><span id="cke_bm_350E" style="display: none;">&nbsp;</span><span id="cke_bm_348E" style="display: none;">&nbsp;</span> T11 and <span id="cke_bm_351S" style="display: none;">&nbsp;</span><em>Tolypot<span id="cke_bm_351E" style="display: none;">&nbsp;</span>hri</em>x K11, indicatin<span id="cke_bm_352E" style="display: none;">&nbsp;</span>g that these strains can be potentially significant sources of essential fatty acids.&nbsp; Results of antioxidant&nbsp; tests showed that all tested strains had antiradical activity. In the case of DPPH assay, ethanolic extracts&nbsp; of <em>Calothrix </em>M2 exhibited the most effective ability to scavenge DPPH &bull;radical while in the case of the FRAP method,&nbsp; ethanolic extract of&nbsp;<em> Cylindrospermum</em>&nbsp; K1 had&nbsp; the greatest reduction power. Accordi<span id="cke_bm_349E" style="display: none;">&nbsp;</span>ng to data obtained from&nbsp; chemical analysis of&nbsp; the phenolic composition of the analyzed cyanobacterial strains, a total of 21 phenol compounds were identified and quantified. The phenolic composition varied depending on the strain, and the most frequently detected phenolic compounds were luteolin-7-O-glucoside, baicalin and kaempferol. The strain with the&nbsp; most&nbsp; asignificant ability to produce phenolic compounds was<em> Phormidium</em> M1, in which the presence of 11 phenolic compounds was identified.&nbsp; The results of the toxicity of intracellular extracts obtained in <em>A. salina, D. magna and D. rerio&nbsp; </em>biotests, showed&nbsp; that a total of 40% of&nbsp; the&nbsp; tested strains exhibited a toxic effect.&nbsp; The most toxic strain was&nbsp;<em> Nostoc&nbsp;</em> T7 due to the fact that it showed toxicity in all three tests. In the case of&nbsp; <em>A. salina </em>biotest, the most potent strains were <span id="cke_bm_365S" style="display: none;">&nbsp;</span><em>Nostoc</em><span id="cke_bm_365E" style="display: none;">&nbsp;</span> T7, <em>Oscillatoria</em> M2,&nbsp; <em>Oscillatoria&nbsp;</em> T18 and <em>Nostoc</em> K15. Cyano<span id="cke_bm_366E" style="display: none;">&nbsp;</span>bacterial strains that exhibited the most prominent activity&nbsp; in&nbsp; <em>D. magna</em>&nbsp; biotest were <span id="cke_bm_378S" style="display: none;">&nbsp;</span><span id="cke_bm_376S" style="display: none;">&nbsp;</span><em>Tolypothrix </em><span id="cke_bm_378E" style="display: none;">&nbsp;</span><span id="cke_bm_376E" style="display: none;">&nbsp;</span>K15,<span id="cke_bm_379S" style="display: none;">&nbsp;</span><em> Nostoc</em><span id="cke_bm_379E" style="display: none;">&nbsp;</span> T7 and <em>Calothrix</em> M2. In bi<span id="cke_bm_380E" style="display: none;">&nbsp;</span><span id="cke_bm_377E" style="display: none;">&nbsp;</span>otest with zebrafish embryos, the strain with the most significant teratogenic effect was <em>Cylindrospermum</em> K1. Regarding the effect of cyanobacterialextract on gene expression in model organism<span id="cke_bm_388S" style="display: none;">&nbsp;</span><em> D. rerio,</em><span id="cke_bm_388E" style="display: none;">&nbsp;</span> cyanobacterial strain&nbsp; <em>Cylindrospermum</em>&nbsp; K1&nbsp; exhibited the ability to modulate biological processes such as circadian rhythm as well as <span id="cke_bm_389E" style="display: none;">&nbsp;</span>the ability to produce compounds with an estrogenic effect. The results of the&nbsp; toxicogenetic analysis of the tested cyanobacterial strains have shown that the genes&nbsp;<span id="cke_bm_394S" style="display: none;">&nbsp;</span><em> mcyB</em><span id="cke_bm_394E" style="display: none;">&nbsp;</span>&nbsp; and&nbsp; <em>mcyE&nbsp;</em> involved in the production&nbsp; of cyanotoxin microcystins have not been detected in any of the tested strains. The absence of two genes from the&nbsp; mcy&nbsp; gene cluster indicates<span id="cke_bm_395E" style="display: none;">&nbsp;</span> that other compounds are responsible for the observed toxic effect in applied biotests. The obtained results point&nbsp; out on the importance of the study of soil cyanobacteria, since the obtained results have indicated that the&nbsp; tested strains possess a large metabolic&nbsp; diversity and potential to produce various bioactive compounds.</p>

Aux Antilles Françaises, les animaux d’élevage sont susceptibles d’être exposés à la chlordécone (CLD), Polluant Organique Persistant (POP) présent dans leur environnement. Afin de sécuriser les Denrées Alimentaires d’Origine Animale (DAOA) destinées à la consommation humaine, nos travaux ont porté sur l’étude du devenir de la CLD chez la brebis. Les objectifs de ces travaux étaient de comprendre comment s’effectue l’élimination de la molécule depuis l’organisme animal c’est-à-dire de déterminer par quelle(s) voie(s), sous quelle(s) forme (s), en combien de temps, en quelle quantité et par quels mécanismes s’élimine la CLD. Pour cette étude, deux volets ont été considérés : un volet analytique et un volet toxicocinétique. En effet, les méthodes de dosage connues des métabolites de la CLD ne permettaient pas d’obtenir la sensibilité et la fiabilité attendues pour nos travaux. Ainsi, un développement analytique de méthodes de dosage de la CLD et de ses métabolites dans les matrices d’intérêt a été mené. Ces travaux analytiques se sont appuyés sur une méthodologie d’extraction de type Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS), une analyse par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) et l’utilisation d’étalons internes isotopiques. Ce développement permet de disposer de méthodes de dosage sensibles, rapides et fiables de la CLD et de ses métabolites dans différentes matrices animales. A l’issue de l’étape de développement analytique, les méthodes dans le foie, les fèces et les urines ont été validées à l’aide de profils d’exactitude établis selon la norme V03-110 et le guide SANTE de référence pour les pesticides. Pour exemple, dans la matrice foie, des limites de quantification de 1,36 µg kg-1 PF et de 2,50 µg kg-1 PF respectivement de chlordécone et de chlordécol (métabolite de la CLD) ont été retrouvées. Le deuxième volet de cette thèse s’est appuyé sur deux protocoles toxicocinétiques réalisés chez la brebis. A l’aide de ces expérimentations, il a été possible de combler une partie des lacunes sur la toxicocinétique de la CLD chez la brebis. Nos travaux ont démontré que la CLD est partiellement métabolisée en chlordécol (CLDOH) par la chlordécone réductase dans le foie des brebis. Par la suite, la CLD et le CLDOH peuvent être métabolisés à l’aide d’UDP-glucuronosyl-transferases et de sulfo-transferases en métabolites conjugués de la CLD et du CLDOH (CLD-C et CLDOH-C). Le CLDOH est un métabolite intermédiaire qui n’est quasiment jamais quantifié dans l’organisme animal hormis dans le tissu gras. L’élimination de la CLD se fait majoritairement via les fèces : 1/3 de la molécule parent CLD est éliminé sous forme de CLD et 1/6 est éliminé sous forme de CLDOH. La voie urinaire est une voie mineure d’élimination de la CLD. A l’aide de ces travaux, un modèle compartimental a été proposé. Sur la base de ce modèle, des travaux de modélisation seront possibles et permettront la proposition et la mise en place de stratégies de décontamination des ovins aux Antilles Françaises / In the French West Indies (FWI), farming animals can be exposed to CLD, a persistent organic pollutant (POP) bound to soil in contaminated areas. In order to produce safe animal products, this thesis was focused on the CLD’s fate in ewes. The aims were to determine by which way(s), in which form(s), in how much time, in which quantities and by which mechanisms, CLD would be eliminated from the animal body. In this thesis, two complementary approaches were followed. First it was necessary to improve the analytical methods especially for the metabolites in order to obtain more sensitive and reliable methods than the actual ones. In the analytical approach, methods for CLD and its metabolites were developed in various animal matrices suitable for the toxicokinetic studies. The extraction method was based on the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology and the analysis was performed with a liquid chromatography with tandem mass spectrometry (LC-MS/MS). Isotopic Standards were also used. Thanks to this work, sensitive, quick and reliable methods were obtained in animal matrices. The set-up methods in liver, feces and urine were validated with accuracy profiles according to the French Standard NF V03-110 and European Union guidelines. Limits of quantifications of 1.36 μg kg−1 and 2.50 μg kg−1 of fresh liver were respectively found for chlordecone and chlordecol (the CLD’s metabolite). Secondly, and thanks to the analytical development, in vivo toxicokinetic studies were performed to determine the fate of CLD in ewes. The second approach of this thesis was based on two toxicokinetic protocols realized in ewes. The results revealed that CLD is partially metabolized in chlordecol (CLDOH) by the chlordecone reductase in ewe’s liver. Then, CLD and CLDOH can be conjugated by UDP-glucuronosyl-transferases and sulfo-transferases in conjugated metabolites (CLD-C and CLDOH-C). It appeared that CLDOH is only an intermediate metabolite which is almost never quantified in the organism except in fat tissue. The major route of CLD elimination is in feces: 1/3 of the molecule is eliminated in its CLD’s form and 1/6 in its CLDOH’s form. The urinary way of CLD elimination is minor. Based on the obtained results, a compartmental model was proposed. It will allow to propose and to establish ovine decontamination strategies in the FWI

A dipirona é uma pró-droga com potente atividade antipirética e analgésica. Vários trabalhos mostram que assim como a indometacina, a dipirona reduz a resposta febril induzida pela endotoxina de E. coli, o LPS. Entretanto, a dipirona reduz respostas febris que são insensíveis a indometacina como a resposta febril induzida pela ET-1 e pelo vTs. Também foi demonstrado o efeito antipirético da dipirona sobre a resposta febril induzida por mediadores envolvidos na febre induzida pelo LPS. O objetivo deste estudo foi comparar o efeito antipirético da dipirona com o efeito de seus principais metabólitos ativos, 4-MAA e 4-AA, sobre a resposta febril induzida por citocinas, prostaglandinas, CRF e AEA. Nossos resultados mostraram que nas doses utilizadas, tanto a dipirona (120 mg/kg, i.p.) quanto os metabólitos 4-MAA e 4-AA (90 mg/kg, i.p.) não alteraram a temperatura corporal basal dos animais. Na febre induzida pelo LPS (50 µg/kg, i.p.), apenas o 4-MAA foi capaz de abolir a resposta enquanto que a dipirona e o 4-AA reduziram apenas a fase inicial. O tratamento com dipirona, 4-MAA e 4-AA foi efetivo para reduzir a resposta febril induzida após injeção i.c.v. de IL-6 (300 ng/rato), TNF-? (250 ng/rato) e PGF2? (750 ng/rato). Na febre induzida pela IL-1? (3,12 ng/rato, i.c.v.), apenas a dipirona e o 4-MAA inibiram esta resposta, enquanto que o 4-AA reduziu a febre apenas até a 2ªh. Também demonstramos que a redução da concentração de PGE2 no hipotálamo não está diretamente relacionada com o efeito antipirético desses fármacos, pois animais tratados com 4-AA apresentaram redução da concentração de PGE2 sem significante redução da resposta febril. Dipirona, 4-MAA e 4-AA não reduziram a resposta febril induzida pela PGE2 (250 ng/rato, i.c.v.) e CRF (5 µg/rato, i.c.v.). Entretanto animais estimulados com CRF apresentaram hipotermia quando tratados com 4-MAA e 4- AA. Nos animais estimulados com AEA (10µg/rato, i.c.v.), apenas o 4-MAA foi capaz de abolir a resposta febril, enquanto que a dipirona e 4-AA reduziram apenas o início desta resposta. Os resultados apresentados sugerem que, dependendo do estímulo, a dipirona, 4- MAA e 4-AA podem, além de inibir a síntese de PGE2, inibir a síntese/liberação de CRF e/ou opióides endógenos. / Dipyrone is a pro-drug with potent antipyretic and analgesic activity. Several studies show that as indomethacin, dipyrone reduces fever induced by endotoxin of E. coli, LPS. However, dipyrone reduces febrile responses that are insensitive to indomethacin as the fever induced by ET-1 and vTs. It has also been demonstrated the antipyretic effect of dipyrone on fever induced by pyrogenic mediators involved in LPS-induced febrile response. The aim of this study was to compare the antipyretic effect of dipyrone with the effect of its major active metabolites 4-MAA and 4-AA, on febrile response induced by cytokines, prostaglandins, CRF and AEA. Ours results showed that at the doses used, both dipyrone (120 mg/kg, i.p.) and 4-MAA e 4-AA (90 mg/kg, i.p.) did not alter basal body temperature of the animals. In LPS-induced fever (50 µg/kg, i.p.) only 4-MAA abolished the fever while dipyrone and 4-AA reduced only the initial phase. Pre-treatment with dipyrone, 4-MAA and 4-AA reduced the febrile response induced after i.c.v. injection of IL-6 (300 ng/rat), TNF-? (250 ng/rat) and PGF2? (750 ng/rat). The fever induced by IL-1? (3.12 ng/rat, i.c.v.) was reduced only by dipyrone and 4-MAA while 4-AA reduced only until the second hour. We also demonstrated that the reduction of PGE2 concentration in hypothalamus is not directly related to the antipyretic effects without significant reduction of fever response. Dipyrone, 4-MAA and 4- AA did not reduced the febrile response induced by PGE2 (250 ng/rat, i.c.v.) and CRF (5 µg/rat, i.c.v.). Though animals stimulated with CRF showed hypothermia when treated with 4-MAA and 4-AA.In animals injected with anandamide (10 µg/rat, i.c.v.), only 4-MAA abolished the fever response, and dipyrone and 4-AA only reduced the beginning of this response. These data suggest that, depending on stimulus, dipyrone, 4-MAA and 4-AA can, besides inhibiting PGE2 synthesis, inhibit synthesis/release of CRF and/or endogenous opioids.