A esquistossomose mansônica é a segunda maior endemia parasitária do mundo em termos de extensão das áreas endêmicas e do número de pessoas infectadas com 200 milhões de pessoas acometidas. Esta doença é causada pelo parasito trematódeo Schistosoma mansoni, o qual apresenta adequados mecanismos de resposta ao estresse envolvendo a regulação da expressão gênica e proteica, reparo ou substituição de moléculas danificadas, recuperação do balanço redox, controle do ciclo celular e apoptose. O sistema ubiquitina- proteassoma é importante para manter a homeostase proteica durante o estresse celular. Inibidores do proteassoma podem interferir em processos como crescimento, progressão do ciclo celular e replicação, e os seus efeitos vem sendo caracterizados em muitos parasitos. Nosso laboratório demonstrou que MG132 reduz o número de esquistossômulos, a carga parasitária e a ovoposição em camundongos infectados com S. mansoni. Neste trabalho, são descritos os efeitos in vitro do estresse oxidativo, choque térmico e estresse químico em vermes adultos de S. mansoni. Observou-se alteração no perfil de expressão proteica durante estresse oxidativo e térmico, sendo identificadas dezoito proteínas upreguladas nestas condições. Estas proteínas estão envolvidas em muitas vias intracelulares como dobramento de proteínas, proteólise, ligação a íons cálcio, regulação de proteínas e resposta a estresse. Além disso, o estresse oxidativo gerou mudanças em vermes adultos de S. mansoni em processos como produção de ovos, motilidade, morfologia do tegumento, viabilidade e pareamento dos vermes. O estresse químico induzido com Curcumina, IBMX e MG132 aumentou a produção de ROS intracelular e alterou o perfil de expressão de enzimas antioxidantes em S. mansoni. As enzimas SmGPx1 e SmPGx2 tiveram a expressão aumentada no estresse com Curcumina e IBMX, enquanto que SmSOD e SmTGR foram induzidas no estresse com Curcumina. As enzimas do proteassoma SmHul5 e SmUbp6 tiveram a expressão modulada durante o estresse oxidativo, choque térmico e estresse químico. Em adição, a análise de expressão no ciclo de vida de S. mansoni revelou que estes genes apresentam um nível alto de expressão em esporocistos, esquistossômulos e miracídios. Estes resultados sugerem que estas proteínas acessórias do proteassoma participam da resposta ao estresse e desenvolvimento do parasito. O nível de expressão de SmHul5 e SmUbp6 foi cerca 9 e 16 vezes menor em relação ao controle no estresse químico induzido com IBMX, respectivamente, sugerindo a desmontagem do proteassoma. Por outro lado, Curcumina, MG132, estresse oxidativo e choque térmico aumentaram o nível de expressão de SmHul5 e SmUbp6. Além disso, o nível de expressão da proteína de maturação do proteassoma (SmPOMP) aumentou no estresse com Curcumina, MG132 e estresse oxidativo, sugerindo a síntese de novas populações de proteassoma. Em relação ao estresse oxidativo, nós demonstramos o aumento no nível proteico de proteassoma 20S e da subunidade alfa-3 do proteassoma sugerindo que em S. mansoni as proteínas oxidadas são degradadas pelo proteassoma 20S. Além do mais, nós observamos que vermes adultos de S. mansoni parecem utilizar mecanismos de resposta similares para diferentes estresses. Nossos resultados demonstraram que o estresse oxidativo, choque térmico e estresse químico modificam o perfil de expressão de genes relacionados ao sistema ubiquitina-proteassoma e sugerem que o proteassoma é importante para as respostas celulares ao estresse neste parasito. / Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma) and affecting 200 million people worldwide. This disease continues to rank, following malaria, at the second position of the world\'s parasitic diseases in terms of the extent of endemic areas and the number of infected people. There are different types of stress and the organisms have many mechanisms to respond to these stressor agents. The responses involve the regulation of gene and protein expression and consist in events such as repair or substitution of damaged molecules, recovery of redox balance, cell cycle control and apoptosis. The proteasomal system is important to support the protein homeostasis during the cellular stress. Effect of proteasome inhibitors has been described in many protozoans, either inhibiting growth or cell cycle progression, or blocking replication. Our laboratory\'s results have shown that MG132 reduces the number of lung stage schistosomula, the worm burden and consequently decreases oviposition in S. mansoni-infected mice. Here, we describe the in vitro effects of oxidative stress, heat shock and chemical stress in S. mansoni adult worms. We report that the oxidative stress and heat shock cause drastic changes in the protein profile of S. mansoni adult worms, and we identified a total of eighteen upregulated proteins in these conditions. These proteins are involved with many intracellular pathways as protein folding, proteolysis, calcium ion binding, regulator proteins and stress response. In addition, oxidative stress induced with H2O2 generated significative changes in the adult worms concerning process such as egg production, motor activity, tegument morphology, viability and pairing of worms. Chemical stress induced with Curcumin, IBMX and MG132 increases ROS production and changes the gene expression profile of antioxidant enzymes of S. mansoni adult worms. The enzymes SmGPx1 and SmGPx2 were upregulated in Curcumin and IBMXinduced chemical stress, and both SmSOD and SmTGR were upregulated- Curcumin. The proteasomal enzymes SmHul5 and SmUbp6 had their gene expression modified during oxidative stress, heat shock and chemical stress. Besides of, expression analyses in the S. mansoni life cycle indicate that genes are different express in sporocyst, schistosomula and miracidia. These results suggest these accessory proteins proteasome participates of stress response and parasite development. The expression level of SmHul5 and SmUbp6 were 16 and 9 times less than the control in chemical stress induced by IBMX, and we suggest that these results are due to the proteasome disassembling. On the other hand, Curcumin, MG132, oxidative stress e heat shock increases the expression of SmHul5 and SmUbp6. Furthermore, the expression level of maturation proteasome protein (SmPOMP) increases in stress induced by Curcumin, MG132 and oxidative stress suggesting new proteasome synthesis. In addition, we demonstrate increase the both 20S level and alpha-3 subunit proteasome in the oxidative stress, suggesting that in S. mansoni oxidized protein formed due to oxidative damage are degrade by proteasome 20S. We observed that S. mansoni adult worms utilize similar mechanisms to respond different stresses. Ours results demonstrate that oxidative stress, heat shock and chemical stress modified the expression profile of genes related with the ubiquitinproteasome system and suggest that the proteasome is important to responses the cellular stresses in the parasite.

Os produtos do metabolismo secundário, também conhecidos por produtos naturais, representam uma fonte inexplorada de compostos com atividade biológica. Os micro-organismos, entre eles os endófitos, são fontes promissoras de obtenção dessas substâncias. Assim sendo, essa pesquisa visou obter compostos de importância biotecnológica produzidos por fungos endofíticos isolados de folhas das plantas Anthurium alcatrazense, Begonia venosa e B. fischeri. Para isso, 5 linhagens de fungos endofíticos isolados dessas plantas (códigos P7BDA1F2, P8BDA1F1, AM29, D28 e D29) foram estudadas. A identificação desses micro-organismos foi realizada por meio de análises morfológicas e moleculares, revelando serem estas linhagens Hymenochaete-like, Trichoderma sp., Neopestalotiopsis sp., Aspergillus sp. e Diaporthe sp., respectivamente. A partir do extrato bruto de Hymenochaete-like (código P7BDA1F2) foram isolados os compostos 5,7-dimetoxiftalida e metil orselinato, os quais foram testados contra Leishmania (L.) infantum e alvo do proteassoma e não apresentaram atividade. A partir do extrato bruto de Trichoderma sp. (código P8BDA1F1) foram isolados os compostos trilongins BI-BIV. Estes apresentaram atividade inibitória ao fitopatógeno C. gloeosporioides, com MIC de 40, 320, 160 e 310 μM, respectivamente. As trilongins BI-BIV foram testadas contra a subunidade ChTL do proteassoma e apresentaram os valores de IC50 de 6,5 ± 2,7; 4,7 ± 1,8; 6,3 ± 2,2; e 2,7 ± 0,5 μM. Os compostos também foram testados ex vivo contra os amastigotas intracelulares de Leishmania (L.) infantum, mas não apresentaram seletividade. A partir do extrato bruto de Neopestalotiopsis sp. (código AM29), foi isolado um composto de massa molecular 366,0570 Da (que pode ser inédito na literatura), o qual apresentou atividade inibitória ao fitopatógeno P. sojae, com MIC de 312 μg mL-1. A partir dos extratos brutos de Aspergillus sp. e Diaporthe sp. foram isolados 9 compostos, cujas frações precursoras apresentaram atividade contra as bactérias formadoras de biofilme S. aureus e P. aeruginosa. Para a identificação desses compostos, análises adicionais precisam ser realizadas. Este é o primeiro relato do isolamento dos compostos 5,7-dimetoxiftalida e metil orselinato do basidiomiceto Hymenochaete-like. Também está sendo relatada pela primeira vez a atividade antifúngica das trilongins a C. gloeosporioides e contra o alvo do proteassoma, assim como o isolamento de um possível novo composto de Neopestalotiopsis sp. e sua atividade contra P. sojae. / Secondary metabolism products, also known as natural products, represent an unexplored source of compounds with biological activity. Microorganisms, including endophytes, are promising sources of these substances. Thus, this research aimed to obtain compounds with biotechnological importance produced by endophytic fungi isolated from leaves of the plants Anthurium alcatrazense, Begonia venosa and B. fischeri. To this end, 5 endophytic fungal strains isolated from these plants (codes P7BDA1F2, P8BDA1F1, AM29, D28 and D29) were studied. The identification of these microorganisms was carried out by morphological and molecular analyzes, revealing that these strains are Hymenochaete-like, Trichoderma sp., Neopestalotiopsis sp., Aspergillus sp. and Diaporthe sp., respectively. From the crude extract of Hymenochaete-like (code P7BDA1F2) the compounds 5,7-dimethoxyphthalide and methyl orselinate were isolated, which were tested against Leishmania (L.) infantum and proteasome target and showed no activity. From the crude extract of Trichoderma sp. (code P8BDA1F1) the trilongins BI-BIV were isolated. These compounds presented inhibitory activity to the plant pathogen C. gloeosporioides, with MIC of 40, 320, 160 and 310 μM, respectively. The trilongins BI-BIV were tested against the ChTL subunit of the proteasome and showed IC50 values of 6.5 ± 2.7, 4.7 ± 1.8, 6.3 ± 2.2, 2.7 ± 0, 5 μM. The compounds were also tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum, but did not show selectivity. From the crude extract of Neopestalotiopsis sp. (code AM29), a compound with molecular mass 366.0570 Da (which can be unpublished in the literature) was isolated, which presented inhibitory activity to the plant pathogen P. sojae, with MIC of 312 μg mL-1. From the crude extracts of Aspergillus sp. and Diaporthe sp. 9 compounds were isolated, whose precursor fractions showed activity against the biofilm forming bacteria S. aureus and P. aeruginosa. For the identification of these compounds, additional analyzes need to be performed. This is the first report of the isolation of the compounds 5,7-dimethoxyphthalide and methyl orselinate from the basidiomycete Hymenochaete-like. The antifungal activity of trilongins to C. gloeosporioides and against the proteasome target is also being reported for the first time, as well as the isolation of a possible new compound from Neopestalotiopsis sp. and its activity against P. sojae.

Neste trabalho está descrito a síntese e avaliação biológica de uma série de compostos de selênio(IV) e telúrio(IV). Esta série foi planejada para que diferentes fatores estruturais pudessem ser avaliados e as possíveis relações entre a estrutura e atividade biológica dos compostos pudessem ser determinadas. Para tanto, selênio, telúrio, cloro e bromo foram diferentemente combinados em um esqueleto carbônico simples, contendo ou não um centro assimétrico. Os compostos de interesse foram sintetizados empregando metodologias quimio-enzimáticas, quando necessário, e reações clássicas da química do selênio e telúrio, levando aos compostos de interesse em poucas etapas e com bons rendimentos. No caso dos ensaios biológicos, parâmetros como potência relativa, constante de inibição de segunda-ordem, mecanismo de inibição, CI50 e viabilidade celular foram determinados dentro das possibilidades experimentais envolvendo cada enzima. As possíveis combinações deram origem a 12 compostos que foram avaliados como inibidores de cisteíno catepsinas B, K, V e S onde a potência relativa dos mesmos pode ser determinada. Para as cisteíno catepsinas V e S, as constantes de inibição de segunda-ordem foram determinadas e ficou evidenciado que a combinação entre telúrio e bromo leva aos compostos mais potentes para estas proteases, enquanto que a combinação entre selênio e cloro origina os inibidores menos potentes. A combinação, selênio e bromo, ou telúrio e cloro forneceu inibidores com potências intermediárias. Este foi o primeiro estudo descrevendo compostos de selênio(IV) como inibidores de proteases. Os mesmos compostos também foram avaliados como inibidores do proteassomo 20S, uma treonino protease, onde se pode observar pela primeira vez que compostos de selênio e telúrio atuam como inibidores desta protease. Os valores de CI50 dos compostos foram determinados e novamente os compostos de telúrio mostraram-se mais potentes do que seus congêneres de selênio. Por outro lado, ensaios em células demonstraram que os compostos de telúrio são direcionados a outro alvo biológico, diferentemente dos compostos de selênio que continuaram a inibir o proteassomo em um lisado celular. Em ensaios de viabilidade celular ficou evidenciado que os compostos de selênio foram mais citotóxicos do que os de telúrio, o que se mostrou muito interessante para desenvolvimento de um agente anticancer onde a resposta biológica desejada é a morte celular. / The synthesis and biological evaluation of a series of selenium(IV) and tellurium(IV) compounds have been described in this research. This series was designed to allow different structural factors to be evaluated, and the possible strutucture-activity relationships determinated. Selenium, tellurium, chlorine and bromine were differently combined in a carbon backbone with or without an asymmetric center. The compounds were synthetized by using both chemo-enzymatic methodology and classical selenium and tellurium chemistry. From biological assays, relative potency, second-order inactivation constant, inhibition mechanism, IC50 and cell viability were determinated according to the experimental possibilities involving each enzyme protocol. The 12 compounds synthesized from the possible combinations among selenium, tellurium, chlorine and bromine were evaluated as cysteine cathepsins B, K, V and S inhibitors, and their relative potencies were determined. By determining the second-order inactivation constant for cysteine cathepsins V and S, it was shown that a tellurium and bromine combination led to most powerfull inhibitors. Selenium and chlorine combination led to less potent inhibitiors, while selenium and bromine, and tellurium and chlorine led to inhibitors with intermediate potency. Those compounds were also evaluated as 20S proteasome inhibitors, a threonine protease. We first observed selenium and tellurium-containing compounds acting as inhibitors of 20S proteasome. The IC50 values were determinate and tellurium compounds were more potent again. On the other hand, tellurium compound did not inhibit proteasome in cells, while selenium-containing compound does it. By cell viability assays it was verified that selenium-containing compounds were more cytotoxic than their tellurium analogs. This data is interesting for someone that wishes to develop an anti-cancer agent where the biological response desired is death of cancerous cells.

Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische Änderung der katalytischen Aktivität bewirkte die Assoziation des Proteasomen-Aktivators. Innerhalb der ersten zwölf Stunden einer IFN gamma Stimulation wurde das Epitop vermehrt mit der Unterstützung des Proteasomen-Aktivators generiert, nach 24 Stunden zunehmend durch freies 20S Proteasom. Die Ergebnisse der vorgestellten Arbeit zeigen, dass Strukturvarianten des Proteasoms zusammen mit PA28 redundant funktionieren und eine hohe proteolytische Plastizität des UPS gewährleisten. / The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces a heterogeneity of 20S proteasomes in the early stage of stimulation, acting in combination with the proteasome activator in a redundant manner; and provides a high proteolytic placticity of the proteasome system.

Dans cette thèse, j’ai cherché à étudier l’impact du contenu et de la structure du nucléole sur les fonctions nucléolaires chez A. thaliana. Pour cela je me suis appuyée sur deux cas concrets : 1- J’ai réalisé le protéome du nucléole et caractérisé une de ces activités non-ribosomales / 2- J’ai étudié l’impact du fer nucléolaire dans la biogenèse des ribosomes.D’une part, le protéome nucléolaire d’A. thaliana m’a permis d’identifier des protéines nucléolaires dont les fonctions connues sont extra-ribosomales. Ainsi j’ai démontré que l’activité du protéasome 26S peut être régulée par le nucléole. Plus précisément l’activité du protéasome diminue lors d’une déstructuration du nucléole. De plus, j’ai constaté que le protéasome 26S, conjointement avec la protéine Nucléoline, pourrait avoir un rôle dans la transcription et/ou la maturation des ARNr.D’autre part, j’ai démontré que l’absence de fer nucléolaire (chez des plantes mutantes nas1,2,4) provoque une augmentation des structures nucléolaires propices à la transcription (les centres fibrillaires). Cette observation est corrélée à la transcription de l’ADNr du NOR2, normalement réprimé. Et, de manière inattendue, est liée avec l’hyperméthylation des promoteurs des ADNr en contexte CHH. Il se peut alors que le fer régule des facteurs impliqués dans les mécanismes épigénétiques responsables de la répression ou de l’activation des ADNr. / The aim of this thesis work is to highlight the impact of both nucleolus content and structure on nucleolar functions in A. thaliana. For this I followed two approaches: 1- I performed nucleolus proteome and characterized one of its non-ribosomal activity / 2- I studied nucleolar iron impact on ribosomes biogenesis.Firstly, the A. thaliana nucleolar proteome allowed me to identify nucleolar proteins with non-ribosomal functions. Among these, I showed that 26S proteasome activity can be regulated by nucleolus. More precisely, proteasome activity decreases with nucleolus disorganization. Moreover, I also showed that 26S proteasome, together with Nucleolin, might play a role in ribosomal RNA transcription and/or maturation.Secondly, I proved that loss of nucleolar iron (in nas1,2,4 mutant plants) induces an increase of nucleolar transcriptional structures (fibrillar centers). This observation is correlated with the transcription of normally silenced rDNA from NOR2 and, interestingly, with hypermethylation of rDNA promoters in CHH context. And so, iron might regulate factors implicated in epigenetic pathways responsible of either rDNA transcription or repression.

Os produtos do metabolismo secundário, também conhecidos por produtos naturais, representam uma fonte inexplorada de compostos com atividade biológica. Os micro-organismos, entre eles os endófitos, são fontes promissoras de obtenção dessas substâncias. Assim sendo, essa pesquisa visou obter compostos de importância biotecnológica produzidos por fungos endofíticos isolados de folhas das plantas Anthurium alcatrazense, Begonia venosa e B. fischeri. Para isso, 5 linhagens de fungos endofíticos isolados dessas plantas (códigos P7BDA1F2, P8BDA1F1, AM29, D28 e D29) foram estudadas. A identificação desses micro-organismos foi realizada por meio de análises morfológicas e moleculares, revelando serem estas linhagens Hymenochaete-like, Trichoderma sp., Neopestalotiopsis sp., Aspergillus sp. e Diaporthe sp., respectivamente. A partir do extrato bruto de Hymenochaete-like (código P7BDA1F2) foram isolados os compostos 5,7-dimetoxiftalida e metil orselinato, os quais foram testados contra Leishmania (L.) infantum e alvo do proteassoma e não apresentaram atividade. A partir do extrato bruto de Trichoderma sp. (código P8BDA1F1) foram isolados os compostos trilongins BI-BIV. Estes apresentaram atividade inibitória ao fitopatógeno C. gloeosporioides, com MIC de 40, 320, 160 e 310 μM, respectivamente. As trilongins BI-BIV foram testadas contra a subunidade ChTL do proteassoma e apresentaram os valores de IC50 de 6,5 ± 2,7; 4,7 ± 1,8; 6,3 ± 2,2; e 2,7 ± 0,5 μM. Os compostos também foram testados ex vivo contra os amastigotas intracelulares de Leishmania (L.) infantum, mas não apresentaram seletividade. A partir do extrato bruto de Neopestalotiopsis sp. (código AM29), foi isolado um composto de massa molecular 366,0570 Da (que pode ser inédito na literatura), o qual apresentou atividade inibitória ao fitopatógeno P. sojae, com MIC de 312 μg mL-1. A partir dos extratos brutos de Aspergillus sp. e Diaporthe sp. foram isolados 9 compostos, cujas frações precursoras apresentaram atividade contra as bactérias formadoras de biofilme S. aureus e P. aeruginosa. Para a identificação desses compostos, análises adicionais precisam ser realizadas. Este é o primeiro relato do isolamento dos compostos 5,7-dimetoxiftalida e metil orselinato do basidiomiceto Hymenochaete-like. Também está sendo relatada pela primeira vez a atividade antifúngica das trilongins a C. gloeosporioides e contra o alvo do proteassoma, assim como o isolamento de um possível novo composto de Neopestalotiopsis sp. e sua atividade contra P. sojae. / Secondary metabolism products, also known as natural products, represent an unexplored source of compounds with biological activity. Microorganisms, including endophytes, are promising sources of these substances. Thus, this research aimed to obtain compounds with biotechnological importance produced by endophytic fungi isolated from leaves of the plants Anthurium alcatrazense, Begonia venosa and B. fischeri. To this end, 5 endophytic fungal strains isolated from these plants (codes P7BDA1F2, P8BDA1F1, AM29, D28 and D29) were studied. The identification of these microorganisms was carried out by morphological and molecular analyzes, revealing that these strains are Hymenochaete-like, Trichoderma sp., Neopestalotiopsis sp., Aspergillus sp. and Diaporthe sp., respectively. From the crude extract of Hymenochaete-like (code P7BDA1F2) the compounds 5,7-dimethoxyphthalide and methyl orselinate were isolated, which were tested against Leishmania (L.) infantum and proteasome target and showed no activity. From the crude extract of Trichoderma sp. (code P8BDA1F1) the trilongins BI-BIV were isolated. These compounds presented inhibitory activity to the plant pathogen C. gloeosporioides, with MIC of 40, 320, 160 and 310 μM, respectively. The trilongins BI-BIV were tested against the ChTL subunit of the proteasome and showed IC50 values of 6.5 ± 2.7, 4.7 ± 1.8, 6.3 ± 2.2, 2.7 ± 0, 5 μM. The compounds were also tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum, but did not show selectivity. From the crude extract of Neopestalotiopsis sp. (code AM29), a compound with molecular mass 366.0570 Da (which can be unpublished in the literature) was isolated, which presented inhibitory activity to the plant pathogen P. sojae, with MIC of 312 μg mL-1. From the crude extracts of Aspergillus sp. and Diaporthe sp. 9 compounds were isolated, whose precursor fractions showed activity against the biofilm forming bacteria S. aureus and P. aeruginosa. For the identification of these compounds, additional analyzes need to be performed. This is the first report of the isolation of the compounds 5,7-dimethoxyphthalide and methyl orselinate from the basidiomycete Hymenochaete-like. The antifungal activity of trilongins to C. gloeosporioides and against the proteasome target is also being reported for the first time, as well as the isolation of a possible new compound from Neopestalotiopsis sp. and its activity against P. sojae.

O proteassomo é um complexo proteico responsável pela degradação de proteínas poli-ubiquitinadas. É constituído por uma unidade catalítica denominada de proteassomo 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o proteassomo 26S. O 20SPT é capaz de degradar proteínas por um processo independente de ATP e ubiquitina. Este mecanismo é considerado preventivo de agregação proteica, uma vez que o acúmulo de proteínas oxidadas está diretamente associado ao envelhecimento e doenças neurodegenerativas. Foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação nos resíduos de Cys 76 e Cys 221 da subunidade 5. Dados obtidos por microscopia eletrônica de transmissão mostraram que a S-glutatiolação promove a abertura da câmara catalítica e consequentemente o aumento da degradação de proteínas. Recentemente, foram obtidas linhagens com mutações sítio-específicas pela substituição dos dois resíduos de Cys glutatioláveis pela Ser. Ensaios realizados com a linhagem C221 mostraram um aumento da longevidade e resistência ao estresse oxidativo quando comparada com a linhagem selvagem, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Foi verificado também que a população de proteassomo isolada da linhagem C221 apresenta maior proporção da forma aberta da câmara catalítica. Resultados opostos foram observados na linhagem C76S. No entanto, constatamos um aumento de proteínas oxidadas e de agregados proteicos na linhagem C221 em comparação a selvagem. Esses dados não condizem com o aumento do tempo de vida cronológico desta linhagem, porém acreditamos que esses agregados estejam relacionados a um tipo de sequestro de proteínas potencialmente prejudiciais, como será discutido neste trabalho. / The proteasome is a protein complex responsible for the degradation of poly-ubiquitinated proteins. It comprises a catalytic unit called 20S proteasome (20SPT) and regulatory units (19S) coupled at one or both ends to form the 26S proteasome. The 20SPT is able to degrade proteins by an ATP and ubiquitin independent process. This mechanism is considered preventive of protein aggregation, since the accumulation of oxidized proteins is directly related to aging and neurodegenerative diseases. It was observed by our group that the yeast 20SPT (S. cerevisiae) is modified by S-glutathiolation in the residues Cys 76 and Cys 221 of the 5 subunit. Data obtained by transmission electron microscopy showed that the S-glutathiolation promotes the opening of the catalytic chamber and consequently increased degradation of oxidized proteins. Recently, strains were obtained by site-specific mutations by replacing the two glutathiolable Cys residues by Ser. Tests performed with the C221 strain showed an increased longevity and resistance to oxidative stress when compared to the wild type strain, whereas the C76 strain showed a slower growth. It was also found that the isolated population of the 5-C221S proteasome presents the highest frequency of open catalytic chamber conformation. Opposite results were observed in the C76S lineage. However, we noted an increased pool of oxidized proteins and protein aggregates in the C221 strain compared to the wild type. These data do not match with the increase of chronological life span observed in this lineage, but we believe that these aggregates are related to a kind of \"sequestration\" of potentially damaging proteins, as will be discussed in this work.

The work in this thesis is divided into two parts; the first is the synthesis of a ‘switch-on’ fluorophore to measure cell viability, and the second is the development of a fluorescent detection method for protein−peptide affinity assays applied in the identification of protein-protein inhibitors. Tetrazolium salts are often used in cytotoxicity assays as indicators of cell viability as they are reduced to deeply coloured formazans exclusively in healthy cells. However, measuring the absorbance of the formazan is prone to bias from other coloured species in the cell media, requires solubilisation and can be difficult to quantify. A preferable method of detection is direct fluorescence as it is easily quantified, more sensitive and would ideally remove the need to solubilise the insoluble dye. The aim of this project was to synthesise a tetrazolium salt that could be reduced to a soluble fluorescent formazan in healthy cells as an indicator of cell viability. A number of fluorescent formazans were synthesised by incorporation of a fluorophore. The corresponding tetrazolium salts were non-fluorescent and could be reduced to the formazan in vitro. Several formazans were synthesised to attempt to increase the emission wavelength and intensity to overcome cellular autofluorescence. Protein-protein interactions have been implicated in the pathogenesis of many human diseases but until recently were considered undruggable. However, peptides have emerged as ideal compounds for targeting the large and relatively featureless protein interfaces. Work focussed on the discovery of peptide inhibitors for the E3 ubiquitin ligase stationary-phase kinase associated protein (Skp2). Potential peptide inhibitors were identified using CelluSpot synthesis and array technology to screen peptide libraries. Qualitative analysis of the protein affinity assay results by enhanced chemiluminescent detection was found to be misleading, and so a quantifiable and more sensitive fluorescent detection method was developed.

Neste trabalho está descrito a síntese e avaliação biológica de uma série de compostos de selênio(IV) e telúrio(IV). Esta série foi planejada para que diferentes fatores estruturais pudessem ser avaliados e as possíveis relações entre a estrutura e atividade biológica dos compostos pudessem ser determinadas. Para tanto, selênio, telúrio, cloro e bromo foram diferentemente combinados em um esqueleto carbônico simples, contendo ou não um centro assimétrico. Os compostos de interesse foram sintetizados empregando metodologias quimio-enzimáticas, quando necessário, e reações clássicas da química do selênio e telúrio, levando aos compostos de interesse em poucas etapas e com bons rendimentos. No caso dos ensaios biológicos, parâmetros como potência relativa, constante de inibição de segunda-ordem, mecanismo de inibição, CI50 e viabilidade celular foram determinados dentro das possibilidades experimentais envolvendo cada enzima. As possíveis combinações deram origem a 12 compostos que foram avaliados como inibidores de cisteíno catepsinas B, K, V e S onde a potência relativa dos mesmos pode ser determinada. Para as cisteíno catepsinas V e S, as constantes de inibição de segunda-ordem foram determinadas e ficou evidenciado que a combinação entre telúrio e bromo leva aos compostos mais potentes para estas proteases, enquanto que a combinação entre selênio e cloro origina os inibidores menos potentes. A combinação, selênio e bromo, ou telúrio e cloro forneceu inibidores com potências intermediárias. Este foi o primeiro estudo descrevendo compostos de selênio(IV) como inibidores de proteases. Os mesmos compostos também foram avaliados como inibidores do proteassomo 20S, uma treonino protease, onde se pode observar pela primeira vez que compostos de selênio e telúrio atuam como inibidores desta protease. Os valores de CI50 dos compostos foram determinados e novamente os compostos de telúrio mostraram-se mais potentes do que seus congêneres de selênio. Por outro lado, ensaios em células demonstraram que os compostos de telúrio são direcionados a outro alvo biológico, diferentemente dos compostos de selênio que continuaram a inibir o proteassomo em um lisado celular. Em ensaios de viabilidade celular ficou evidenciado que os compostos de selênio foram mais citotóxicos do que os de telúrio, o que se mostrou muito interessante para desenvolvimento de um agente anticancer onde a resposta biológica desejada é a morte celular. / The synthesis and biological evaluation of a series of selenium(IV) and tellurium(IV) compounds have been described in this research. This series was designed to allow different structural factors to be evaluated, and the possible strutucture-activity relationships determinated. Selenium, tellurium, chlorine and bromine were differently combined in a carbon backbone with or without an asymmetric center. The compounds were synthetized by using both chemo-enzymatic methodology and classical selenium and tellurium chemistry. From biological assays, relative potency, second-order inactivation constant, inhibition mechanism, IC50 and cell viability were determinated according to the experimental possibilities involving each enzyme protocol. The 12 compounds synthesized from the possible combinations among selenium, tellurium, chlorine and bromine were evaluated as cysteine cathepsins B, K, V and S inhibitors, and their relative potencies were determined. By determining the second-order inactivation constant for cysteine cathepsins V and S, it was shown that a tellurium and bromine combination led to most powerfull inhibitors. Selenium and chlorine combination led to less potent inhibitiors, while selenium and bromine, and tellurium and chlorine led to inhibitors with intermediate potency. Those compounds were also evaluated as 20S proteasome inhibitors, a threonine protease. We first observed selenium and tellurium-containing compounds acting as inhibitors of 20S proteasome. The IC50 values were determinate and tellurium compounds were more potent again. On the other hand, tellurium compound did not inhibit proteasome in cells, while selenium-containing compound does it. By cell viability assays it was verified that selenium-containing compounds were more cytotoxic than their tellurium analogs. This data is interesting for someone that wishes to develop an anti-cancer agent where the biological response desired is death of cancerous cells.

Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.