Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis. / Ph. D.

Tesis por compendio / [EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CA] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / Compendio

Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-10-18T12:35:34Z No. of bitstreams: 1 2016 - Tatiana Pires Pereira.pdf: 2546753 bytes, checksum: c1673b0ae2c6812a3f60faa79b5fae75 (MD5) / Made available in DSpace on 2017-10-18T12:35:34Z (GMT). No. of bitstreams: 1 2016 - Tatiana Pires Pereira.pdf: 2546753 bytes, checksum: c1673b0ae2c6812a3f60faa79b5fae75 (MD5) Previous issue date: 2016-07-27 / Funda??o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do RJ - FAPERJ / This work was divided in four chapters, in which the first was performed with the goal of quantifying the condensed tannin (CT) content by the Stiasny?s reaction and to determine the classes of secondary metabolites present by the phytochemical prospection technique and magnetic resonance in the tropical forage legumes Cajanus cajan (guandu - GUA), Gliricidia sepium (gliricidia - GLI), Flemingia macrophylla (flemingia - FLE), Cratylia arg?ntea (cratilia - CRA), Mimosa caesalpineafolia (sabi?) (this legume divided into bark and leaf fraction ? SABc and SABf) among the treatments. The extracts obtained were divided into: total extract, number of Stiasny (NS), CT and non-tannins. The FLE, CRA, GUA,GLI, SABf and SABc had obtained the following values for total extract: 13.20; 13.06; 8.28; 14.73; 15.67 and 6.22%, respectively. The reactivity by NS, in the same order of legumes, was 11.25; 4.54; 7.37; 6.70; 23.06 and 71.62%, whereas the CT presented the following values: 1.52; 0.59; 0.61; 0.96; 3.6 and 4.43%, and non-tannin was 11.68; 12.46; 7.67; 13.75; 12.07 and 1.76%, respectively. The following classes of secondary metabolites were identified with greater evidence: saccharides, carbohydrates, non-protein amino acids and glicos?deos cardioativos. For the CT, the intensity was low for most of the legumes, with greater content in CRA, GUA and SABf. The wain compound in the extracts was methyl-inositol (sugar). The second chapter had the objective of assaying in the legumes mentioned above and one more specie, Stylosanthes spp. (estilosantes-EST), condensed tannin (CT) constituents, with the use of organic solvents, soluble CT (ECT), CT adhered to protein (PBCT), CT adhered to fiber (FBCT), and total CT (TCT), CT structural pro-pelargonidin (PP); prodelfinidin (PD) and procyanidin (PC), molecular weight (polymerization degree (DP), molecular distance distributed of the polymer (PDI); average weight of molecular mass (Mw), and average number of molecular mass (Mn), and the biological activity through precipitated proteins by phenols (PPP). The variables ECT, PBCT, and TCT presented were influenced by different species (P?0.05). The FBCT fraction was not found in the legumes. Molecular weights (DP, PDI, Mw e Mn) were affected by the different species (P?0.05), ranging from 737 to 1168 da. The structural characteristics (PP, PD, PC and PD:PC) varied among the species. In the third chapter I evaluated methanogenesis (total methane (CH4total)), incubated (CH4inc) and fermented (CH4ferm) and ruminal fermentation parameters total gas production (PGT), pH, ammonium (N-NH3), short-chain fatty acids (SCFA) and in vitro organic matter disappearance (IVOMD) as they related to CT present in the legumes and Urochloa brizantha cv. marandu hay as control (CTL). The effect of polyethylene glycol (PEG) on the leaf fraction of sabi? was tested as well, which had a CT content of 15.97%. No alteration in the pH (P?0.05) for the treatments evaluated. However, a decrease of total gas and methane production for all the treatments with presence of CT (P?0.05). When PEG was added, there was a 27.01 (8% PEG) and 35.01 (16% PEG) increase in total gas production and 3.59 (8% PEG) and 4.15 (16% PEG) of methane production. GUA, FLE, SABc and SABf were capable of modifying (P?0.05) the content of NH3-N (mg/dL), along with the CTL, which also presented lower values compared to legumes with no or only traces of CT (ETL, CRA and GLI). There was significant difference (P?0.05) for IVOMD between the legumes and control, it was observed lower disappearance (P?0.05) for FLE, GUA and SABf in relation to CTL, while SABc did disappear. The SABf IVOMD was affected by the addiction of PEG. There was lower digestibility for FLE, GUA and SABf in relation to the CTL, while the bark fraction of SAB did not disappear at all. On the SCFA profile, there was difference (P?0.05) among the treatments evaluated, with lower values for the legumes with presence of CT. In the fourth chapter I tested the effect of CT from the legumes in study (FLE, CRA, GUA, GLI, EST, SABf and SABc) on larval migration inhibition (LMI) in vitro, on the infective larvae L3 of the nematode Haemonchus contortus (HC), compared with Ivermectin and a negative control (rumen fluid and buffer). Among the legumes studied, SABf and GUA did not differ (P?0.05), with the greater (P?0.05) LMI percentage (34.75% and 34.33%) than the other entries. The legumes GUA, FLE and SABc did not differ (P?0.05), presenting moderate values of LMI (30.25%, 30.0% and 29.75%, respectively). Among the legumes studied, the lowest LMI percentage was CRA (18.46%), GLI (23.75%) and negative control (rumen fluid and buffer), with values near (P?0.05) from to Ivermectin (22.0%). / Este trabalho foi dividido em quatro cap?tulos. O primeiro realizou-se com o objetivo de quantificar o teor de tanino condensado (TC) atrav?s da Rea??o de Stiasny e conhecer as classes de metab?litos secund?rios presentes pela t?cnica de prospec??o fitoqu?mica e resson?ncia magn?tica nas leguminosas forrageiras tropicais Cajanus cajan (guandu-GUA), Gliricidia sepium (gliricidia-GLI), Flemingia macrophylla (flemingia-FLE), Cratylia arg?ntea (cratilia-CRA), Mimosa caesalpineafolia (sabi?) sendo que essa leguminosa tinha a fra??o casca e folha (SABc e SABf) entre os tratamentos. Os extratos obtidos foram divididos em: extrato total, n?mero de Stiasny (NS), TC e n?o taninos. A FLE, CRA, GUA, GLI, SABf e SABc apresentaram os valores para o extrato total 13,20; 13,06; 8,28; 14,73; 15,67 e 6,22%, respectivamente. A reatividade pelo NS, na mesma ordem das leguminosas, foi de 11,25; 4,54; 7,37; 6,70; 23,06 e 71,62%, j? o TC apresentou os seguintes valores 1,52; 0,59; 0,61; 0,96; 3,6 e 4,43% e o n?o tanino foi de 11,68; 12,46; 7,67; 13,75; 12,07 e 1,76%, respectivamente. Foram identificadas as seguintes classes de compostos secund?rios em maiores evid?ncias: os sacar?deos, carboidratos, amino?cidos n?o prot?icos e os glicos?deos cardioativos. J? para o TC, a intensidade foi baixa para grande parte das leguminosas, prevalecendo maior teor para CRA, GUA e SABf. Foi constatado como componente principal nos extratos o metil-inositol (a??car). O segundo cap?tulo teve como objetivo avaliar nas leguminosas citadas acima e mais uma esp?cie, o Stylosanthes spp (estilosantes-EST), analisar os constituintes do TC com uso de solvente org?nico, tanino sol?vel (TCE), tanino aderido ? prote?na (TCPB), tanino aderido ? fibra (TCFB) e taninos condensados totais (TCT), caracter?sticas estruturais tais como: propelargonidina (PP); prodelfinidina (PD) e procianidina (PC); peso molecular (grau de polimeriza??o?(DP); dist?ncia do peso molecular distribu?do do pol?mero (PDI); peso m?dio da massa molecular (Mw); n?mero m?dio da massa molecular (Mn); al?m de determinar a atividade biol?gica, atrav?s da t?cnica de prote?nas precipit?veis por fen?is (PPP). As vari?veis TCE, TCPB e TCT apresentadas foram influenciadas pelas diferentes esp?cies (P?0,05). A fra??o TCFB n?o foi constatada nas leguminosas. Os pesos moleculares (Mw) foram influenciados pelas diferentes esp?cies (P?0,05), variando de 737 a 1168 Da. As caracter?sticas estruturais (PP, PD, PC e PD:PC) tiveram varia??o entre as esp?cies estudadas. Objetivou-se com o terceiro cap?tulo avaliar a metanog?nese (metano total (CH4-total), incubado (CH4 inc.) e fermentado (CH4 ferm.) e os par?metros de fermenta??o ruminal (produ??o de g?s total (PGT), pH, am?nia (N-NH3), ?cidos graxos de cadeia curta (AGCC) e digestibilidade in vitro da mat?ria org?nica (DIVMO) frente aos TC presentes nas leguminosas e feno de Urochloa brizantha cv. marandu como controle (CTL). Foi testado tamb?m o efeito do polietileno glicol (PEG) sobre a fra??o folha do sabi?, que teve conte?do de TC de 15,97%. N?o foi observado altera??o no pH (P?0,05) para os tratamentos avaliados. No entanto, foram observadas diminui??o da produ??o total de g?s e produ??o de metano para todos os tratamentos com presen?a de TC (P?0,05). Para o tratamento com PEG houve aumento de 27,01 (8% PEG) e 35,01 (16% PEG) na produ??o total de g?s e 3,59 (8% PEG) e 4,15 (16% PEG) na produ??o de metano. GUA, FLE, SABc e SABf foram capazes de modificar (P?0,05) a concentra??o de N-NH3 (mg/dL) juntamente com o CTL, que tamb?m apresentou valores inferiores comparado as leguminosas com tra?os e aus?ncia do TC (ETL, CRA e GLI). Houve diferen?a (P?0,05) para DIVMO entre as leguminosas e o controle, observou-se menor digestibilidade (P?0,05) para FLE, GUA e SABf, em rela??o ao CTL, n?o sendo digest?vel o SABc. A DIVMO foi afetada pela adi??o de PEG na dieta do SABf. No perfil dos AGCC houve diferen?a (P?0,05) para os tratamentos avaliados, com menor valor para as leguminosas com presen?a de TC. O quarto cap?tulo teve como objetivo testar o efeito da t?cnica de inibi??o da migra??o larval (IML) in vitro do TC proveniente das leguminosas em estudo (FLE, CRA, GUA, GLI, EST, SABf e SABc) sobre as larvas infectantes L3 do nemat?de o Haemonchus contortus (HC) comparando com Ivermectina e controle negativo (l?quido ruminal e tamp?o). Entre as leguminosas estudadas o SABf e GUA n?o diferiram entre si (P?0,05), com as maiores porcentagens IML (34,75% e 34,33%). As leguminosas GUA, FLE e SABc n?o diferiram entre si (P?0,05), apresentando moderados valores de IML (30,25%, 30,0% e 29,75%, respectivamente). Entre as leguminosas estudadas a menor porcentagem de IML foi para CRA (18,46%), GLI (23,75%) e controle negativo (l?quido de r?men e tamp?o) valores pr?ximos do controle positivo com Ivermectina (22,0%).

<p>Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. </p><p>The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge <i>Geodia barretti</i> and its ability to inhibit larval settlement of the barnacle <i>Balanus improvisus</i> was determined. With an EC₅₀ value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from <i>Geodia barretti</i>, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT_1-7 receptors, whereas barettin possessed specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted with 5-HT₄. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC₅₀ value of 34 nM, displayed the highest settlement inhibition.</p><p>The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus <i>Agelas</i>. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified.</p><p>In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges <i>Geodia barretti</i> and <i>Agelas</i> spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.</p>

Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.

Abstract Host use and range of herbivorous insects are determined by several factors, of which nutritional quality and secondary chemistry have been shown to play very important roles. For herbivores feeding on lichens these traits are assumed to be more critical than for species feeding on higher plants, since lichens are nutritionally poor and often contain high concentrations of secondary metabolites. I examined the role of lichens' nutritional quality and secondary chemicals on the performance of lichen-feeding Lepidopteran larvae. I also tested whether females of lichenivorous species preferably oviposit on host species of the highest nutritional quality for the growth of larvae. Larvae of Eilema depressum performed best on Melanelia exasperata, which is of the highest nutritional quality, as indicated by the high N concentration and the absence of lichen secondary metabolites compared to the other lichens studied. Host nutritional quality did not promote the production of an additional generation. Larvae of E. depressum needed fewer instars and grew bigger on a high-quality diet than larvae reared on a diet of poorer quality. However, the main factor contributing to the wide variation in the number of larval instars was the question of whether or not larvae overwintered. Growth of Cleorodes lichenaria at the beginning of the larval period matched equally the nutritional quality of the hosts. However, the final larval period was shortest on Ramalina species, which was preferred by both females ovipositing their eggs and larvae searching for a host. In the field, larvae were found almost exclusively on Ramalina species. Larvae of E. depressum were not able to survive on intact thalli of Vulpicida pinastri and Hypogymnia physodes, but after removal of lichen's secondary metabolites, larval survival remained equally high as on other lichens. Larvae also showed a clear preference towards thalli with lowered concentrations of secondary metabolites in Parmelia sulcata, V. pinastri and H. physodes. Parietin in Xanthoria parietina was the only secondary metabolite that had no impact on the survival or host selection of E. depressum larvae. The present results show that the nutritional quality and some lichen secondary chemicals are important factors for the growth, survival and host selection of lichen-feeding Lepidopteran larvae. The preference-performance hypothesis is at least partly able to explain the host range of C. lichenaria, although it seems that there are also other factors, such as larval dispersal and host selection or top-down forces, that might contribute to host range of lichenivorous Lepidopteran larvae. Moreover, lichenivorous larvae seem to be partly responsible for their own host selection.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Vegetative growth, and essential oil yield and its components are strongly influenced by photoperiod, temperature, rainfall, and other factors that change according to the seasonal period. Thus, this study aimed to evaluate the vegetative and productive and qualitative performance of Aloysia triphylla When subjected to different periods of drought during the four seasons. The experiment was conducted in an agroclimatology laboratory greenhouse at UFSM, campus of Frederico Westphalen RS. The experiment was conducted with a complete randomized block design in a 5x4 factorial scheme, the treatments consisting of five periods of water stress (3, 6, 9, 12 days without irrigation or daily irrigation) and four seasons (winter , spring, summer and fall); the study had five replications. The evaluations were performed in the period that marked the half of each season samples were collected throughout the growing material. Periods of drought did not affect the growth and essential oil production. Growth characters showed the highest values in the summer and autumn seasons, with the worst results in the winter. The highest essential oil content was obtained in the summer in all treatments and the lowest in the winter season. Citral is the major component of this oil, and it decreases in winter and increases in autumn and summer. The winter favors the concentration of minor components, especially caryophyllene oxide and spathulenol. / O crescimento vegetativo, bem como a produção de óleo essencial e seus componentes é fortemente influenciado pelo fotoperíodo, temperatura, ocorrência de chuvas, dentre outros fatores que se modificam de acordo com o período sazonal. Com isso, o presente trabalho teve como objetivo avaliar o desempenho vegetativo e produtivo e qualitativo de Aloysia triphylla submetida a diferentes períodos de déficit hídrico, nas quatro estações do ano. O experimento foi conduzido em estufa plástica pertencente ao laboratório de agroclimatologia da UFSM campus de Frederico Westphalen - RS. O delineamento experimental utilizado foi o de blocos completos ao acaso em um esquema bifatorial 5x4, sendo os tratamentos compostos por cinco períodos de déficit hídrico (3, 6, 9, 12 dias sem irrigação ou com irrigação diária) e quatro estações do ano (inverno, primavera, verão e outono), com cinco repetições. As avaliações foram realizadas no período que marcou a metade de cada estação do ano sendo coletado todo o material vegetativo. Os períodos de déficit hídrico não influenciaram o crescimento e produção de óleo essencial. As variáveis de crescimento apresentaram os maiores valores na estação do verão e outono, decaindo na estação do inverno. O maior teor de óleo essencial foi obtido na estação do verão em todos os tratamentos avaliados e os menores teores na estação do inverno. O citral é o componente majoritário, sendo que seu teor diminui no inverno e aumenta no outono e no verão. A estação do inverno favoreceu a concentração de componentes minoritários, com destaque para o óxido de cariofileno e espatulenol.

La propagation des micro-organismes résistants aux antibiotiques menace le système mondial de santé publique. Pour lutter contre ce phénomène, le renouvellement des molécules utilisées en antibiothérapie est devenu une priorité mondiale. Les antibiotiques étant principalement d’origine microbienne, l’étude des micro-organismes et de leurs métabolites s’est donc renforcée et s’oriente vers des écosystèmes peu explorés comme les biotopes marins.Nous avons exploré les activités antimicrobiennes d’une collection de 183 champignons isoles de sédiments marins profonds et collectés entre 4 et 1884 mètres sous le plancher océanique. Le potentiel de production de métabolites de cette collection a été révélé par un criblage génétique ciblant les PolyKetide synthase (PKS), les Non-Ribosomal Peptide Synthetase (NPRS), les TerPene Synthase (TPS) et les hybrides PKS-NRPS. Après avoir regroupé les isolats en fonction de leur profil MSP PCR, 110 ont été sélectionnés pour un criblage fonctionnel, montrant une forte proportion de champignons filamenteux antimicrobiens (32%).Après extraction et fractionnement, les composés bioactifs de 3 souches ont été caractérisés aux niveaux structural et fonctionnel. Ainsi, O. griseum UBOCC-A-114129 produit la fuscine, la dihydrofuscine, la secofuscine et la dihydrosecofuscine, P. bialowiezense UBOCC-A-114097 produit l’acide mycophénolique et Penicillium sp. produit UBOCC-A-114109 la rugulosine.Parallèlement, des analyses en LC-HRMS, réalisées sur des extraits fongiques, ont révélé un grand nombre de métabolites non décrits dans les bases de données. Les champignons des sédiments marins constituent donc un réservoir de structures originales à explorer. / The spreading of antimicrobial resistant microorganisms jeopardizes global health caresystem. To counteract this threat the renewal of antibiotic molecules is a global priority. Antibioticcompounds are mainly originated from microorganisms, so microorganisms and their secondarymetabolites received an increasing interest. The search for new natural antimicrobial compoundsfrom microorganisms gained untapped ecosystems as marine biosphere.We investigated the antimicrobial properties of a fungal collection. The 183 fungal isolateswere collected from deep subseafloor sediment and isolated between 4 and 1,884 meters belowthe seafloor. Secondary metabolites production potential was studied for all isolates in thecollection by screening genes coding PolyKetide Synthase (PKS), Non-Ribosomal Peptide Synthetase(NRPS), TerPene Synthase (TPS) and hybrid PKS-NRPS. After isolates dereplication according to theirMSP-PCR fingerprinting, an antimicrobial screening was performed for 110 isolates, highlighting ahigh proportion of filamentous fungi with antimicrobial properties (32%).After extraction and bio-guided fractionation bioactive metabolites isolated from 3 strains,were characterized in a structural and functional manner: O. griseum UBOCC-A-114129 producedfuscin, dihydrofuscin, secofuscin and dihydrosecofuscine, P. bialowiezense UBOCC-A-114097synthetized mycophenolic acid and Penicillium sp. UBOCC-A-114109 produced rugulosin.In the meantime, LC-HRMS analysis, performed on fungal extracts, showed a great proportionof metabolites not detected in interrogated databases. So, deep subseafloor fungi, represent anuntapped reservoir of original structures to explore.

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