Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Four research projects were carried out and they are described in this dissertation. Glycogen synthase kinase-3 beta (GSK3β) plays a pivotal and central role in the pathogenesis of Alzheimer's disease (AD) and protein kinase C (PKC) controls the function of other proteins via phosphorylation and involves in tumor promotion. In pursuit of identifying novel GSK3β and/or PKC inhibitors, substituted quinoline molecules were designed and synthesized based on the structure-activity-relationship studies. Synthesized molecules were evaluated for their neural protective activities and selected molecules were further tested for inhibitory activities on GSK3β and PKC enzymes. Among these compounds, compound 2 was found to have better GSK3β enzyme inhibitory and MC65 cell protection activities at low nanomolar concentrations and poor PKC inhibitory activity whereas compound 3 shows better PKC inhibitory activity. This demonstrates the potential for uses of quinoline scaffold in designing novel compounds for AD and cancer. Pharmacokinetics and distribution profiles of two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were assessed using HPLC/MS. Plasma samples of mice and rats fed with TP70 via different routes over various times were analyzed to quantify the amounts of TP70 in plasma of both species. Distribution profiles of TP70 in various tissues of mice were studied and results show that TP70 penetrated the blood brain barrier and accumulated in the brain tissue in significant amounts. Similarly, the amount of CP2 in plasma of mice was analyzed. The HPLC analysis revealed that both compounds have good PK profiles and bioavailability, which would make them suitable candidates for further in vivo efficacy studies. Nanodelivery of specific dsRNA for suppressing the western corn rootworm (WCR, Diabrotica virgifera virgifera) genes was studied using modified chitosan or modified polyvinylpyrrolidinone (PVP) as nanocarriers. Computational simulation studies of dsRNA with these polymers revealed that nanoparticles can be formed between dsRNA and modified chitosan and PVP polymers. Nanocarriers of hydroxylated PVP (HO-PVP) and chitosan conjugated with polyethylene glycol (PEG) were synthesized, and analyzed using IR spectroscopy. Particle sizes and morphology were evaluated using AFM and encapsulation was studied using UV spectroscopy. However, the formation of stable nanoparticles with dsRNA could not be achieved with either of the polymers, and further efforts are ongoing to discover a better nanocarrier for nanodelivery of siRNA by using chitosan-galactose nanocarrier. In our efforts to discover a novel class of tripeptidyl anti-norovirus compounds that can strongly inhibit NV3CLpro, a set of tripeptidyl molecules were synthesized by modifying the P1 - P3 of the substrate peptide including a warhead. It was found that the replacement of P1 glutamine surrogate with triazole functionality does not improve the inhibitory activities of the compounds. In addition, the synthesis of a known dipeptidyl compound (GC376) was carried out for evaluating its efficacy on feline infectious peritonitis (FIP) in cats.

Sementes das plantas pertencentes à família Leguminosae são excelentes fontes de inibidores de proteases. O gênero Acacia é um dos membros mais importantes deste grupo. Neste trabalho, foram descritos novos inibidores de proteases das sementes de Acacia plumosa Lowe. A partir do extrato salino das sementes maduras, os inibidores foram purificados por cromatografia de exclusão molecular em coluna Superdex-75 (equilibrada e eluída com PBS) e cromatografia de troca iônica em coluna Mono-S, equilibrada e eluída com o tampão Acetato de Sódio 50 mM (pH 5.0) num gradiente linear de \'NA\'\'CL\' 0-0.5 M. Quatro frações (eluídas por volta de 0.1 8, 0.22, 0.33 e 0.37 M de \'NA\'\'CL\') apresentaram atividade anticoagulante e ação inibitória sobre serinoproteases, estas frações foram denominadas ApTIA, ApTIB, ApTIC e ApTID, respectivamente. Em condições nativas, a espectrometria de massas mostrou as massas moleculares de três deles (A, B e C): 19.709; 19.869 e 20.378 Dáltons, enquanto que em SDS-PAGE na presença de \'beta\'-mercaptoethanol, foram observadas duas cadeias para cada um dos inibidores. A análise dos primeiros 10 resíduos de aminoácidos da região N-Terminal das duas cadeias das formas A, B, C revelou identidade com inibidores do tipo Kunitz, e também mostrou dois resíduos diferentes na ApTIC, em relação as formas A e B. Estes dados levam a interpretação de que estes inibidores são diferentes isoformas encontradas nesta semente. O espectro de dicroísmo circular foram compatíveis com proteínas que majoritariamente apresentam elementos- β e não-ordenados em sua estrutura, apresentando máximos positivos por volta de 230 nm e mínimos em 202 nm. Os três isoinibidores foram muito estáveis em pHs ácidos e alcalinos, e suas estruturas foram afetadas somente acima de 75oC. As constantes de associação (KA) e de dissociação(KD) determinadas por SPR (num sistema BIACORE) com enzimas proteolíticas indicaram que a afinidade destes inibidores por tripsina foi até 20 vezes maior que para quimotripsina (tripsina: KA2.57x109 M-1 e quimotripsina: KA 1.34x108M-1), e o complexo tripsina-inibidor mostrou maior estabilidade (tripsina: KD por volta de 0,5 nM e quimotripsina: 6 nM). Estes inibidores também apresentaram ação inibitória sobre o crescimento dos fungos Aspergillus niger, Thielaviopsis paradoxa, Colletotrichum sp P10 e Fusarium moniliforme, mostrando que provavelmente a inibição de suas serinoproteases possa ser um mecanismo de controle das suas proliferações. / Seeds of plants belonging to Leguminosae family are rich sources of protease inhibitors. The Acacia genus is one of the most important members of this group. In this work, novel protease inhibitors from Acacia plumose Lowe seeds have been described. From the saline extract of mature seeds, the inhibitors were purified by size exclusion chromatography on Superdex-75 column (equilibrated and eluted with PBS) and ionic exchange chromatography on Mono-S column, equilibrated and eluted with Sodium Acetate 50 mM (pH 5.0) in a linear gradient of NaCl 0-0.5M. Four fractions (eluted around 0.18, 0.22, 0.33 and 0.37 M of NaCl) presented anticoagulant activity and inhibitory action on serineprotease, these fractions were denoted ApTIA, ApTIB, ApTIC and ApTID, respectively. In native conditions, mass spectrometry showed the molecular weights of three of them (A, B and C): 19,709; 19,869 and 20,378 Daltons, while in SDS-PAGE in ?-mercaptoethanol presence, two chains for each inhibitor were observed. The N-terminal analysis of the first 10 amino acid residues of both chains of the isoforms A, B, and C revealed identity with Kunitz protease inhibitors and also showed two different residues in ApTIC, comparing with A and B isoforms. These data indicate that the inhibitors are different isoforms present in this seeds. The circular dichroism spectra were compatible with proteins that majority present unordered and beta-elements in these structures, presenting positive maxima around 230 nm and minima about 202 nm. The three isonhibitors were very stable at acids and alkalines pH, and their structures are only affected over 75ºC. The association (KA) and dissociation constants (KD) determined by SPR (BIACORE system) with proteolytic enzymes indicated that the affinity of these inhibitors for trypsin was up to 20 times bigger than for chymotrypsin (trypsin: KA 2.57x109 M-1 and chymotrypsin: KA 1.37x108 M-1), and the complex inhibitor-trypsin showed higher stability (trypsin: KD around 0,5 nM and chymotrypsin: 6 nM). These inhibitors also presented inhibitory action on the fungi growth of Aspergillus niger, Thielaviopsis paradoxa, Colletotrichum sp P10 e Fusarium moniliforme showing that probably the inhibition of their serineproteases can be a mechanism of control of their proliferation.

Orientador: Marcelo Menossi Teixeira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T11:53:59Z (GMT). No. of bitstreams: 1 Mondego_JorgeMauricioCosta_D.pdf: 1365745 bytes, checksum: d2f496fe8deacf2a20e24327a8f483dc (MD5) Previous issue date: 2005 / Resumo: O café é um dos principais produtos agrícolas mundiais. O Brasil é um dos maiores países produtores e consumidores de café. Assim sendo, a cafeicultura possui extrema importância econômica em nosso país. Um dos principais fatores que causam prejuízo à lavoura do café é o ataque do bicho mineiro (Leucoptera coffeella), pois Coffea arabica, principal espécie cultivada do café, é suscetível a essa praga. O Instituto Agronômico de Campinas (IAC) empreende um projeto de melhoramento de Coffea arabica, visando a resistência ao bicho mineiro, através de cruzamentos com C. racemosa, espécie naturalmente resistente a L. coffeella.Neste trabalho isolamos genes diferencialmente expressos durante o ataque do bicho mineiro a plantas de uma progênie híbrida derivada de cruzamentos entre Coffea arabica e C. racemosa. Foram construídos macroarranjos de DNA contendo 1536 ESTs de bibliotecas de subtração enriquecidas em genes preferencialmente expressos em plantas resistentes infestadas. Membranas foram hibridadas com sondas de cDNA obtidas a partir de RNA total de folhas suscetíveis e resistentes ao bicho mineiro, em diferentes momentos da infestação (controle não infestado, pósoviposição e pós-eclosão). Após análises estatísticas e clusterização hierárquica, 21 cDNAs induzidos em pelo menos um tratamento foram selecionados como diferencialmente expressos durante a infestação do bicho mineiro. A expressão diferencial de cinco genes (PR-8, CAX9, SPC25, psaH, BEL) foi confirmada através de RNA blot contendo RNA de um segundo experimento de infestação, demonstrando a eficiência dos macroarranjos de DNA na seleção de genes diferencialmente expressos. O padrão de expressão dos cinco genes citados foi verificado em diferentes órgãos do cafeeiro e durante o desenvolvimento do fruto do café. Nossos resultados sugerem que o mecanismo de resistência ao bicho mineiro é derivado de uma maior expressão basal de genes relacionados a defesa em plantas resistentes do que em plantas suscetíveis, e que plantas resistentes possuem um mecanismo de sinalização de defesa disparado pela oviposição de L. coffeella. Dentre os cDNAs selecionados, destacamos SSH101B04, cuja proteína deduzida é similar a inibidores de protease do tipo Kunitz STI (Soybean Trypsin Inhibitor). Devido a sua alta similaridade com proteínas do tipo miraculina, esse gene foi denominado CoMir (Coffea Miraculin). CoMir foi induzido após a oviposição em plantas resistentes, mas não foi induzido após a eclosão da lagarta do minador em plantas resistentes nem em plantas suscetíveis. Através de ensaios de RNA blot foi verificado que CoMir é expresso em folhas, botões florais verdes e brancos e em frutos verdes imaturos. Ensaios de hibridação in situ demonstraram que CoMir é expresso no metaxilema de folhas, de pétalas e do estigma, e no estômio, endotécio, tapete e feixe vascular da antera. Ensaios de localização subcelular demonstraram que a proteína CoMir localizou-se preferencialmente no apoplasma e no citoplasma de células de epiderme de cebola (Allium cepa). Nossos resultados sugerem que CoMir é uma proteína reguladora de proteólise durante o desenvolvimento do café, que é mobilizada para defesa após a oviposição de L. coffeella / Abstract: Coffee is one of the most important crops in the world. Brazil is one of the biggest coffee producer and consumer countries. Therefore, coffee plantations have great relevance in our country. One of the main factors that affect coffee plantations is the attack of the coffee leaf miner (Leucoptera coffeella). This is due to the susceptibility of Coffea arabica, the main cultivated species. The Agronomic Institute of Campinas (IAC) develops a Coffea arabica breeding program aiming the resistance to the infestation of coffee leaf miner, using crosses with C. racemosa, a resistant species. In this work, we have isolated differentially expressed genes during L. coffeella attack to plants of a hybrid progenie between C. arabica and C. racemosa. We have produced cDNA arrays containing ESTs from subtracted cDNA libraries enriched in genes preferentially expressed in infested resistant plants. Arrays were probed with samples from susceptible and resistant leaves, in different treatments (control noninfested, after oviposition and after caterpillar eclosion). After statistical analysis and hierarchical clustering, 21 cDNA clones induced in at least one treatment were selected as differentially expressed during coffee leaf miner infestation. The differential expression of five genes (PR-8, CAX9, SPC25, psaH, BEL) was confirmed by RNA blot containing samples from a second infestation experiment, demonstrating the efficiency of DNA arrays in the identification of differentially expressed genes. The expression profile of these five genes was verified in different organs of coffee plants and during coffee fruit development. Our results suggest that the resistance mechanism against coffee leaf miner is derived from a higher basal expression of defense/stress genes in resistant plants, and that resistant plants have a defense signaling mechanism triggered by L. coffeella oviposition. Among the selected cDNAs, we identified SSH101B04, which deduced protein is similar to Kunitz STI (Soybean Trypsin Inhibitor) protease inhibitors. The gene was named CoMir due to its high similarity to miraculin-like proteins. CoMir was induced after oviposition in resistant plants, but it was not induced after larval eclosion in susceptible and resistant plants. RNA-blot experiments showed that CoMir was expressed in leaves, green flower buds, white flower buds and early green fruits. In situ hybridization showed that CoMir is expressed in the metaxylem vessels of leaves, petals and stigma and in the stomium, endothecium and vascular bundles of anthers. Subcellular localization assays demonstrated that CoMir was localized in the apoplasm and citoplasm of onion (Allium cepa) epidermal cells. Our results suggest that CoMir is a protein that regulates proteolysis during coffee development that is mobilized to defense after L. coffeella oviposition / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

A resistência que apresentam as serpentes peçonhentas às suas próprias peçonhas, assim como a resistência observada em alguns animais está bem documentada, e é atribuída a fatores solúveis presentes no seu plasma, soro ou músculo. No caso das serpentes peçonhentas estes fatores as amparam de sofrer danos decorrentes da própria peçonha. No presente trabalho foi isolado do soro da serpente Bothrops alternatus um inibidor de metaloproteases, denominado BaltMPI, mediante cromatografia em DEAE Sepharose(TM), Superdex(TM) 200, MonoQ(TM) 5/50 GL e cromatografia de fase reversa em C18, com uma recuperação proteica de 0,3%. A massa molecular determinada para o BaltMPI por SDS - PAGE foi de 60,5 kDa, e por espectrometria de massas MALDI/TOF de 42,4 kDa. Seu ponto isoelétrico, determinado por focalização isoelétrica, é 5,27. Portanto, o BaltMPI, é um SVMPIs com caráter ácido, pertencente aos SVMPIs de baixa massa molecular. Os primeiros 60 aminoácidos da região N-terminal do BaltMPI foram determinados mediante degradação de Edman, e apresentou alto grau de homologia com a sequência para a mesma região de outros SVMPIs isolados a partir do soro de serpentes peçonhentas. Outros segmentos da sequência também foram determinados após clivagem com tripsina e posterior análise por espectrometria de massas. Posteriormente realizou-se o alinhamento da sequência parcial determinada para o BaltMPI, com a sequência do BJ46a, um SVMPI proveniente da Bothrops jararaca, encontrando alta homologia entre elas. Tanto o soro da serpente, quanto o inibidor, BaltMPI, inibiram a atividade hemorrágica da Batroxase, uma SVMP da classe P-I, e a da BjussuMP-I, uma SVMP da classe P-III. Já, em relação à DHM da Batroxase, foi determinado que o BaltMPI tem uma DIHM de 5ug, e uma CE50% de 0,857ug. O BaltMPI também apresentou um efeito inibitório sobre a ação proteolítica da Batroxase, sobre os substratos: fibrinogênio, fibrina e azocaseína. Sobre a atividade fibrinogenolítica da serinoprotease BjSP, o BaltMPI não apresentou um efeito inibitório, corroborando assim com a especificidade descrita para inibir as SVMPs que possuem os SVMPIs. O BaltMPI inibiu a ação hemorrágica e proteolítica da Batroxase, ao formar um complexo mediante ligações não covalentes com esta SVMP. O inibidor BaltMPI, ao igualmente aos demais SVMPIs descritos, é estável em uma ampla faixa de pH (1 - 9), e a temperaturas elevadas, sendo que em temperaturas acima de 60°C foi observada uma diminuição na sua capacidade de inibir a atividade hemorrágica da Batroxase. A inibição da atividade hemorrágica da Batroxase, quando avaliado o potencial do BaltMPI como complemento à soroterapia, foi menor à inibição observada ao realizar os testes com incubação prévia entre o inibidor e a metaloprotease. No entanto, os resultados obtidos são promissores, reforçando o grande potencial que os SVMPIs possuem, tanto como ferramentas moleculares, como opção para o tratamento dos acidentes ofídicos, em especial o acidente botrópico, no qual as SVMPs tem papel fundamental na fisiopatologia observada, e que conduz à alta morbidade associada com este tipo de acidente. / Resistance exhibited by snakes to their own venom, as well as resistance observed in some animals has been well recorded, and has also attributed to soluble factors present in the plasma, serum or muscle. In the case of poisonous snakes, these factors protect them from damages caused by their own venom. An inhibitor, named as BaltMPI, was isolated from the snake´s serum of Bothrops alternates. This inhibitor was isolated through several chromatographic steps including DEAE Sepharose(TM), Superdex(TM) 200, MonoQ(TM) 5/50 GL and C-18 reverse phase, with a protein yield of 0.3%. Molecular mass of 60.5 kDa for BaltMPI was determined by SDS - PAGE and 42.4 kDa by MALDI/TOF mass spectrometry. Its isoelectric point, determined by isoelectric focalization, was 5.27. According to the obtained data, it was established that BaltMPI is an SVMPIs of acid character, belonging to low molecular mass SVMPIs. The first 60 aminoacids from the N-terminal region were determined by Edman degradation. This parcial sequence showed high homology to the corresponding sequence of other SVMPIs isolated from poisonous snake´s serum. Other segments from the sequence were also determined after cleavage with tripsine and MS analyses. Consequently, alignment of the partial sequence of BaltMPI with the sequence of BJ46a, a SVMPI from Bothrops jararaca, was made finding high homology. Both, snake´s serum and BaltMPI, inhibited the hemorragic activity of Batroxase, a class P-I SVMP, and of BjussuMP-I, a class P-III SVMP. When compared to the Batroxase DHM, it was determined a DIHM of 5?g and a CE50% of 0.857?g. BaltMPI also exhibited an inhibitory activity against the proteolitic effect of Batroxase on fibrinogen, fibrin and azocasein as substrates. On the fibrinogenolitic activity of serineprotease BjSP, BaltMPI did not showed inhibitory effect, demonstrating its specificity for inhibiting SVMPs that SVMPIs possess. BaltMPI inhibits the hemorragic and proteolitic action of Batroxase by forming a complex through non covalent linkages with that SVMP. BaltMPI, as well as other reported SVMPIs, is stable in a very high range of pH (1-9), and at high temperatures, although above 60°C a decrease in its inhibition capacity to the hemorrhagic activity of Batroxase. Inhibition of the hemorrhagic activity of Batroxase, when BaltMPI potencial as serum-therapy complement was evaluated, was less than the inhibition observed when tests by previously incubating inhibitor and metalloprotease were made. However, the results obtained are encouraging, highlighting the potential that these kind of proteins, SVMPIs, have as molecular tools, optional treatments for ophidic accidents, specially bothropic accident, in which SVMPs have a fundamental role in the observed physiopathology, leading to a high associated morbidity to this kind of accidents.

As cianobactérias são micro-organismos reconhecidos por seu potencial em produzir cianotoxinas que afetam não só o ecossistema e a outros organismos dos ambientes aquáticos, mas também aos seres humanos, agindo em diversos órgãos e tecidos. Cerca de 600 metabólitos secundários produzidos por cianobactérias já foram descritos na literatura, sendo que muitos deles possuem potencial biológico. Os peptídeos de baixo peso molecular, produzidos por cianobactérias chamados cianopeptídeos, dos quais se podem citar as anabaenopeptinas, aeruginosinas, microviridinas, cianopeptolinas e microgininas, são compostos provindos do metabolismo secundário de cianobactérias e são descritos como inibidores de proteases e fosfatases em alguns sistemas biológicos. Sendo assim, o objetivo do estudo foi identificar a ocorrência de cianopeptídeos em cianobactérias brasileiras e testar seu efeito de inibição da atividade sobre enzimas proteases. Os objetos de estudo foram: uma linhagem da espécie Sphaerospermopsis torques-reginae, duas de Cylindrospermopsis raciborskii, duas de Microcystis sp, uma de Oscillatoria e uma de Pseudanabena sp. A partir dos resultados obtidos pode-se afirmar que do total de linhagens analisadas, ao menos 4 destas parecem produzir os cianopeptídeos de interesse, quando avaliados por cromatografia líquida de alta eficiência com detector de arranjo de diodos (HPLC-DAD). Em seguida, culturas de duas linhagens de C. raciborskii (uma produtora e outra não produtora de saxitoxina) foram amostradas a cada 3 dias, para a avaliação do crescimento celular, produção de cianopeptídeos e de saxitoxina e suas variantes. Não foi possível confirmar a produção de cianopeptídeos nas duas linhagens desta espécie. Por outro lado, foi evidenciado um aumento da produção de saxitoxinas quando cultivada num meio sem nitrogênio em comparação com a condição controle. Quando analisada a linhagem de Microcystis sp. (LTPNA 08), produtora de microcistinas, foi possível confirmar por cromatografia líquida acoplada a espectrometria de massas (LC-MS) a produção de dois cianopeptídeos, sendo estes, duas microgininas. Desta forma, desenvolveu-se um método cromatográfico para a separação desses compostos e purificação por cromatografia líquida semi-preparativa, onde foi possível a obtenção de frações enriquecidas de microgininas e microcistinas-RR e LR, com cerca de 70%, 86% e 97% de pureza. Por fim, realizaram-se ensaios avaliando a atividade da enzima conversora de angiotensina (ECA) e aminopeptidase M (AMP M) com as frações isoladas de microgininas e microcistina-LR. A atividade da ECA foi ± 50% inibida pelas frações testadas de cianopeptídeos, microcistina-LR isolada e microcistins-lR comercial. A atividade da AMP M foi 100% e 24,5% inibida quando incubada com 20 µM da fração de microgininas e microcistina-LR, respectivamente. Desta forma, as microgininas isoladas de cianobactérias brasileiras, mostraram-se como importantes inibidores da ECA e AMP M, podendo, no futuro, serem utilizadas como compostos para o tratamento de patologias cardiovasculares e renais. / Cyanobacteria are micro-organisms recognized for their potential to produce cyanotoxins that affect not only the ecosystem and other organisms of aquatic environments, but also humans, acting in various organs and tissues. Around 600 secondary metabolites produced by cyanobacteria have been described in the literature; many of them have biological potential. Low molecular weight peptides produced by cyanobacteria are called cyanopeptides, among them we can cite the anabaenopeptins, aeruginosins, microviridins, cyanopeptolins and microginins, these compounds are derived from secondary metabolisms of cyanobacteria and apparently cause inhibition of proteases and phosphatases in some biological systems. Therefore, this study targeted the identification of the occurrence of cyanopeptides in Brazilian cyanobacterias and testing its effect on the inhibition of proteases activity. The targets of study were: a strain of species Sphaerospermopsis torques-reginae, two strains of Cylindrospermopsis raciborskii, two strains of Microcystis sp. and, one strain of Pseudanabena and Oscillatoria sp. From the results obtained in this study it can be stated that at least four of the total of strains analyzed appear to produce cyanopeptides of interest, when analyzed by high-performance liquid chromatography whit phodo diodo array detector (HPLC-PDA). The cultures of two strains of C. raciborskii (a producer of saxitoxin and a non-producer) were sampled every 3 days for assessment of cell growth, production of cyanopeptides and saxitoxins. It was not possible to confirm the production of cyanopeptides in strains of this species. Nevertheless, an increase in production of saxitoxins was shown when cultivated in an environment without nitrogen, as compared to the control condition. When the strain of Microcystis sp. (LTPNA 08), a producer of microcystins, was analyzed, the production of two cyanopeptides was confirmed by using liquid chromatography-mass spectrometry (LC-MS). After confirmation, a method using HPLC-PDA was used to do the separation and purification of these compounds by semi-preparative chromatography, in which it was possible to obtain an enriched fraction of microginins, microcystin-RR and microcystin-LR with approximately 70%, 86% and 97% purity, respectively. Lastly, inhibition experiments were run with angiotensin-converting enzyme (ACE) and aminopeptidase M (AMP M) with the isolated fractions. The microginins fractions, MC-LR commercial and MC-LR isolated, showed an inhibition of ± 50% of the angiotensin-converting enzyme. The activity of AMP M was 100% and 24.5% inhibited when incubated with microginins and microcystin-LR fractions in a concentration of 20 µM, respectively. Thus, isolated microginins from Brazilian cyanobacteria have exhibited properties as potential therapeutic agents in development of inhibitors of ACE and AMP M, which can be a benefit of using these molecules in the treatment of cardiovascular and renal pathologies.

Cada subtipo de HIV-1 tem um padrão mutacional próprio. Dados sobre mutações de resistência aos antiretrovirais foram obtidos com o subtipo B, primeiro em prevalência no Brasil. O segundo em algumas regiões é o subtipo F. Foram analisados padrões mutacionais em seqüências brasileiras de protease do subtipo F e levantou as seqüências deste subtipo disponíveis na base de dados de Stanford. A análise de dois grupos de seqüências (pacientes não tratados e tratados com inibidores de protease) mostrou 19 mutações associadas ao tratamento comuns ao subtipo B e 17 duplas de mutações associadas ao tratamento que diferem das descritas para o subtipo B, indicando a necessidade de estudos sobre rotas mutacionais no subtipo F. / Each HIV-1 subtype has a specific mutation pattern. Data on HIV-1 antiretroviral resistance mutations were obtained with subtype B, the first in prevalence in Brazil. The second in some regions is subtype F. Mutation patterns of Brazilian subtype F protease sequences were analyzed and performed a research of the sequences of Stanford Database. The analysis of two groups of sequences (untreated and treated patients with protease inhibitors) showed 19 treatment associated mutations also common in subtype B and 17 combinations of statistically treatment associated mutations that were quite different to those described for subtype B, indicating the need of studies to evaluate specific mutation pathways of subtype F.

M.Sc. / Proteinases are involved in many cellular reactions involving protein degradation, such as degradation of storage proteins and protein degradation during senescence processes. Their action can be inhibited by proteinase inhibitors. Information is still limited about the regulation of these inhibitors in plants and their possible interaction with proteinases under stress conditions. To obtain a better understanding of the physiological role of a proteinase inhibitor in plants under stress, the expression of a papaya cysteine proteinase inhibitor (cystatin) and its relation to proteinase expression was investigated in more detail. For this purpose, expression of the inhibitor was studied in papaya plants exposed to different physiological stress conditions, such as high/low temperature, and treatment with selected chemicals, such as glutathione, OTC (L-2- Oxothiazolidine-4-carboxylate), bestatin ([(2S, 3R)-3-amino-2-hydroxy-4-phenyl butanoylj-L-leu) and 2.4-D (2,4-dichiorophenoxyacetic acid). Using detection tools like activity gel electrophoresis, immunoblotting and enzymatic assays, the production of the cystatin under stress was monitored in different papaya explants, such as roots, leaves and embryos. Inhibitor production increased under different stress conditions when compared to untreated controls. However, this increase was not dramatic in any of the stresses applied. Exact quantification of the increase by using immunoblotting as the only specific tool to determine cystatin expression, was difficult. Neither activity gel electrophoresis nor enzymatic assays were successful to further quantify the exact cystatin levels. Higher cystatin expression was accompanied with a decrease in proteinase activity. Transgenic tobacco plants carrying the gene for a rice cystatin had a significantly lower cysteine proteinase activity when compared to non-transgenic tobacco plants after prolonged cold stress. Furthermore, protein degradation and leaf yellowing as a consequence of cold treatment were prevented in transgenic plants. An attempt to obtain a transformed papaya plant to study silencing of cystatin expression under stress was unsuccessful. In this study, the protective role of a cystatin in cold stress was described for the first time.

Doctor of Philosophy / Department of Entomology / C. M. Smith / Brenda Oppert / Coleopteran insects compensate for dietary protease inhibitors by a number of mechanisms. To study this compensation response at the molecular level, the digestive proteases of Tenebrio molitor were studied. Biochemical studies of the pH optima and inhibitor sensitivity of proteases indicated the cysteine proteases were mostly in the anterior and serine proteases were in the posterior midgut of T. molitor larvae. Expressed Sequence Tags (ESTs) from T. molitor larval midgut cDNA libraries contained sequences encoding putative digestive proteases. Of a total of 1,528 cDNA sequences, 92 cDNAs encoded proteases, and 50 full-length cDNAs were grouped into serine, cysteine and metallo protease classes. Sequences tmt1a, tmt1b and tmt1c were identified as genes encoding isoforms of T. molitor trypsin, and tmc1a encoded T. molitor chymotrypsin. The general distribution cysteine protease transcripts in the anterior and serine protease transcripts in the posterior midgut, of T. molitor larvae, was in agreement with the biochemically-characterized compartmentalization of proteases. Expression analyses of selected transcripts demonstrated varied expression patterns across five developmental stages of T. molitor, with maximal expression of most protease transcripts in first instar larvae. Dietary serine and cysteine protease inhibitors fed in combination to early-instar T. molitor larvae caused a significant delay in larval growth in 21-day-old larvae. Real-time quantitative PCR analysis of RNA isolated from larvae fed different protease inhibitor treatments indicated that dietary inhibitors affected the expression of serine and cysteine proteases. Larvae fed soybean trypsin inhibitor, a serine protease inhibitor, compensated by the hyperproduction of proteases from the same class, as well as the upregulation of cysteine proteases. A cysteine protease inhibitor, E-64, caused a reduction in the hyperproduction of all proteases, and, in combination with the soybean trypsin inhibitor, lowered the compensation response of T. molitor larvae to negligible levels. These data suggest that T. molitor larvae are more sensitive to the effects of cysteine protease inhibitors, perhaps because these proteases are the first line of defense for larvae against plant protease inhibitor. The bioassay and molecular studies suggested that combinations of inhibitors that target both serine and cysteine proteases are needed to effectively control larval infestations of T. molitor.

A series of the Baylis-Hillman adducts have been obtained by reacting protected O-benzylated and unprotected substituted salicylaldehydes with methyl acrylate or tertbutyl acrylate, respectively, using DABCO as catalyst. Treatment of the Baylis-Hillman adducts with HCl in a mixture of acetic acid and acetic anhydride afforded the corresponding 3-(chloromethyl)coumarin derivatives with yields of up to 94%. Similar use of HI afforded the corresponding 3-(iodomethyl)coumarins but, depending on the reaction time, the reduced 3-methyl analogues could also be obtained. Arbuzov reactions of the 3-(halomethyl)coumarin derivatives have been undertaken to afford 4-phosphorylated and 1’-phosphorylated derivatives, regioselectivity being dependent on the halide-leaving group. The 3-(chloromethyl)coumarin derivatives have been subjected to nucleophilic (SN) attack by benzylamine to give the corresponding 3- [(benzylamino)methyl]coumarin derivatives in yields of up to 74%. Further treatment of the 3-[(benzylamino)methyl]coumarin derivatives with chloroacetyl chloride afforded the chloroacetamide derivatives, which exhibit hindered rotation about the amine C(O)-N bond. The acetamide derivatives have also been subjected to Arbuzov reaction conditions to afford the phosphorylated derivatives in yields of up to 86%. In a preliminary modelling study, hydrolysed analogues of the synthesized phosphorylated derivatives have been docked into the active site of the HIV-1 protease enzyme using the Cerius-2 Ligandfit software module to provide an insight into potential receptor-ligand hydrogen bonding interactions.

Coronaviruses have been responsible for several global disease outbreaks over the last 20 years, including the “Severe Acute Respiratory Syndrome” in 2002/2003, the “Middle East Respiratory Syndrome” in 2012, and the “Coronavirus Disease of 2019 (COVID19)”. These viruses are highly contagious and can cause multiple medical disorders upon contraction, such as common cold or lower respiratory infections. SARS-CoV-2, the newly emerged coronavirus variant of 2019, has been confirmed as the cause of the ongoing COVID19 pandemic, which infected over 167 million people worldwide and, by the end of May 2021, has a death toll of over 3 million people. Even though several SARS-CoV-2 vaccines have made it to the market, no proven options have yet been discovered for treating COVID19 infections. The aim of this project is, therefore, to improve the potency of two active SARS-CoV-2 main protease (Mpro) inhibitors (ML188 and X77) by performing a structure-activity-relationship study where two specific sites of the inhibitors are altered. The inhibition activity of these compounds is then tested on isolated SARS-CoV-2 Mpro. The four-component Ugi reaction was utilized to synthesize the ML188 and X77 analogs, which were purified by column chromatography before testing. During this project, six pure analogs were successfully synthesized and will be sent shortly for testing. Inhibitors with good activity against SARS-CoV-2 Mpro will be further tested for their antiviral activity in cell-based infection assays. The results obtained from this study will later be used to perform a second structure-activity-relationship study to further improve the potency of the two inhibitors by developing a 2nd generation library.