HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57. To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.

Indiana University-Purdue University Indianapolis (IUPUI) / The proteasome is responsible for breaking down the majority of the proteins in the cell. However, a complete understanding of how this large multi-subunit protease is assembled is currently lacking. Proper and timely assembly of the proteasome is critical for the functioning of the ubiquitin-proteasome pathway, defects in which have been associated with several different cancers. A recently discovered heterodimeric proteasome assembly chaperone, Pba1p-Pba2p, has been suggested to prevent the assembly process from straying off path. Pba1p-Pba2p associates with proteasomal assembly intermediates via C-terminal HbYX motifs. The HbYX motif is a tri-peptide sequence containing a hydrophobic residue (Hb) followed by a tyrosine (Y), then any amino acid (X). This motif was originally identified in proteasomal activators, and shown to mediate the association of activators with the proteasome by inserting into intersubunit pockets on either end of the proteasome. There are seven unique intersubunit binding pockets, located between neighboring α subunits on the proteasome, to which a HbYX-containing protein can bind; which of these pockets Pba1p-Pba2p binds to remains elusive. I attempted to identify where Pba1p and Pba2p bind via a crosslinking approach. Specific residues were mutagenized to cysteines on Pba1p, Pba2p, and the individual α subunits in order to generate crosslinkable species. By exposing yeast cells expressing these crosslinkable proteins to mild oxidizing conditions, I attempted to trap the Pba1p and Pba2p α intersubunit pocket interactions. In order to optimize crosslinking conditions, the assay was modified several ways. Additionally, measures were taken to increase detection of the crosslinked species via immunoblotting. Despite the efforts to improve the crosslinking and detection, I was unable to successfully detect a crosslinked species. However, crosslinking is a reasonable method to identify the Pba1p and Pba2p proteasomal binding sites, having been successfully used to identify binding sites for other HbYX-motif-containing proteins; further assay optimization should yield Pba1p and Pba2p proteasomal crosslinks.

Les inhibiteurs de la protéase virale (IPs) sont utilisés avec succès dans le cadre d'une thérapie anti-VIH-1. L'efficacité de ce traitement est incontestable notamment en terme de réduction de la charge virale et de maintient du taux de lymphocytes CD4 circulants. Depuis l'incorporation des IPs dans la prise en charge thérapeutique, on note une réduction significative de la morbidité et de la mortalité ainsi qu'un allongement de la durée de vie des patients. Cependant, la prise de ces molécules anti-rétrovirales s'accompagne de nombreux effets secondaires. Les plus préoccupants d'entre eux sont : une lipodystrophie partielle, une hyperlipidémie, une insulino-résistance, une athérosclérose prématurée, ainsi que des infarctus du myocarde. Les patients sont également confrontés à l'apparition de maladies généralement associées à l'âge telles que la neurodégenérescence, l'ostéopénie et le développement de tumeurs malignes. Les mécanismes cellulaires et moléculaires impliqués dans ces altérations métaboliques n'ont pas encore été élucidés. L'objectif de cette étude était d'étudier les effets cellulaires de quatre IPs (Atazanavir, Lopinavir, Ritonavir et Saquinavir) sur la cellule musculaire striée squelettique humaine. Ces travaux ont permis d'identifier une augmentation de la production d'espèces oxygénées réactives (ERO), une altération morphologique du réticulum sarco/endoplasmique (RS/RE) ainsi qu'une augmentation de l'expression de CHOP, un marqueur du stress de ce compartiment et une diminution de l'expression et de la localisation dans les microdomaines membranaires (lipid rafts) de la cavéoline 3 et de la flotilline 1. Enfin, l'utilisation d'un antioxydant, le Resvératrol protége le myotube primaire humain de ces différentes altérations engendrées par les IPs. Ces données suggèrent un rôle central de la surproduction d'ERO dans le développement du stress du RE/RS et de la perte de localisation des protéines résidantes des microdomaines membranaires. De plus , en l'absence de persceptive vaccinale concrète, le Resvératrol, au travers de ces effets protecteurs, pourrait se révéler un atout de choix dans l'atténuation des effets secondaires des IPs en contribuant ainsi à l'amélioration de la prise en charge du patient séropositif. / HIV protease inhibitors (PI) have been successfully used in highly active antiretroviral therapy (HAART) of HIV-1 infection, the most effective treatment currently available. Incorporation of protease inhibitors in HAART has significantly reduced the morbidity and mortality and prolonged the lifespan of patients with HIV infection. Protease inhibitor benefits are unfortunately compromised by a number of clinically important adverse side-effects. Most patients on HAART develop a metabolic syndrome associated with partial lipodystrophy, hyperlipidemia, insulin resistance, premature atherosclerosis and myocardial infarction. Moreover, these patients face a growing number of other age-related comorbidities, such as neurodegeneration, osteopenia and malignancies. The cellular and molecular mechanisms underlying protease inhibitor-associated metabolic abnormalities remain elusive, but they seem to be related to overproduction of reactive oxygen speci es (ROS), induction of endoplasmic reticulum stress and activation of the unfolded protein response (UPR). The objective of this thesis was to determine the cell effects of four PIs (Atazanavir, Lopinavir, Ritonavir and Saquinavir) in cultures of primary human skeletal myotubes. This study showed that PIs increased ROS production, altered sarco/endoplasmic reticulum (SR/ER) morphology, increased expression of C/EBP homologous protein, a SR/ER stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. In addition, we showed that the antioxidant Resveratrol protected the human primary myotube of these iatrogenic effects. These data suggest a central role of the overproduction of ERO in the development of SR/ER stress and mislocalization of lipid raft proteins induced by PIs. Besides, in absence of vaccine, Resveratrol may be used by a potentiel therapeutic agent to attenuate PI-induced side effects

The main purpose of the master thesis project is to investigate the influence of selected serine protease inhibitors (SPI) on the catalytic action of the serine proteases chymotrypsin and trypsin, in a conjugated and non-conjugated state. The inhibitors included for this study were extracted from Solanum tuberosum, i.e.common potato. The purification method included in this study consist of crude extraction by mixer, followed by a salt-out procedure with ammonium sulphate. Further purification steps were cation exchange chromatography and, finally, gel filtration to obtain SPI of high purity. The purified sample was then characterized by SDS-page and kinetic activity measurement of trypsin and chymotrypsin action on synthetic substrate derivate, N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPA) and N-Succinyl-L-phenylalanine-p-nitroaniline (SFpNA) respectively. The characterization showed inhibitory inactivation of both pancreatic proteases. This would indicate successful extraction of SPI. To investigate inhibitory action in a conjugated state, either enzyme or inhibitor was immobilized onto aluminium oxide membranes. Then two different experimental setups were tested, called experiment 1 and 2. In experiment 1, the inhibitor was immobilized and the interaction was monitored from a retention shift of enzyme flow-through compared to a blank column, using detection at 280 nm of the enzyme. In experiment 2 the enzyme was instead immobilized and a mixture of inhibitor and substrate was circulated with monitoring of the catalytic activity. The main goal was thus to measure the effects on the kinetics in the conjugated state compared to enzyme and inhibitor in the free state. The result from both experiment 1 and 2 did not yield consistent and reliable result so the discussed method should be regarded as preliminary results. The study also includes investigation of inhibitor-enzyme interaction as revealed by molecular mass data to determine complex formation. This part was conducted with static light scattering analysis to determine the stoichiometry for the interaction between pancreas proteases and the inhibitor. Results from light scattering showed promising indication of many-to-one interaction between enzyme and inhibitor, which have been seen by previous studies. It should be considered a preliminary result as complex formation does not exclude aggregation of enzymes or inhibitor in the solution.

La voie de signalisation Notch est conservée au cours de l'évolution. Elle joue un rôle clé dans le développement, et elle est impliquée dans de nombreuses décisions de destin cellulaire, dans le maintien des cellules souches, et dans le contrôle de la prolifération et de la différenciation cellulaires. Une dérégulation de la signalisation Notch est impliquée dans diverses maladies et cancers, y compris les tumeurs solides, comme les cancers du sein et du col de l'utérus, et les leucémies, comme la Leucémie Aiguë Lymphoblastique des cellules T (LAL-T). Notch est un récepteur transmembranaire activé par des ligands transmembranaires de la famille DSL (Delta/Serrate/Lag-2). Bien que plusieurs mutations oncogéniques ont été identifiées au niveau du récepteur Notch, de nombreux cancers modulés par Notch demeurent ligand-dépendants. Étonnamment, les mécanismes moléculaires régulant l'activation du ligand sont encore relativement peu caractérisés par rapport à ceux qui régissent le récepteur Notch lui-même. Utilisant un essai de co-culture avec un rapporteur luciférase de Notch, nous avons effectué le premier crible d'ARNi pan-génomique visant spécifiquement à identifier des régulateurs des ligands de Notch dans la cellule émettrice du signal. Nous avons ainsi pu découvrir de nouvelles classes de régulateurs communs pour les ligands Delta-like1 et 4. Ces régulateurs comprennent des inhibiteurs de protéases, des facteurs de transcription, et des gènes divers à fonction inconnue, tels que Tmem128 « Transmembrane protein 128 », ou à fonction préalablement caractérisée tels que la co-chaperonne moléculaire Cdc37 « Cell division cycle 37 homolog ». Par la suite, nous avons développé des cribles secondaires fonctionnels où nous avons démontré l'importance de ces régulateurs pour des événements Notch-dépendants, comme la différenciation des cellules T normales, et la survie des cellules souches pré-leucémiques isolées à partir d'un modèle murin de LAL-T. En outre, nous avons prouvé que les régulateurs les plus forts du crible de survie sont également nécessaires pour l'activité d'auto-renouvellement des cellules souches pré-leucémiques. Finalement, nous avons entamé une caractérisation moléculaire préliminaire de deux régulateurs nouvellement identifiés; Tmem128 et Cdc37 afin d'étudier leur mécanisme d'action sur les ligands. En conclusion, cette étude nous a permis d'identifier de nouveaux régulateurs de la voie Notch qui pourraient servir de cibles thérapeutiques potentielles dans les cancers; tel qu'illustré par le modèle LAL-T. La compréhension des détails moléculaires sous-jacents aux fonctions de ces régulateurs sera essentielle afin de développer des inhibiteurs pharmacologiques pour bloquer leur action et entraver la signalisation Notch dans le cancer. / The Notch signalling pathway is evolutionarily conserved. It plays a key role in development and it is involved in multiple cell fate decisions, in the maintenance of stem cells, and in the regulation of cell proliferation and differentiation. Misregulation of Notch signalling is implicated in various diseases and cancers including solid tumours, such as breast and cervical cancers, and leukemias, such as T-cell Acute Lymphoblastic Leukemia (T-ALL). Notch is a transmembrane receptor activated by transmembrane ligands of the DSL family (Delta/Serrate/Lag-2). Whereas oncogenic mutations have been identified in the Notch receptor, many Notch-mediated cancers remain ligand-dependent. Strikingly, the molecular mechanisms that regulate ligand activation are still poorly characterized as compared to those regulating the Notch receptor itself. Using a co-culture assay with a luciferase Notch reporter, we performed the first genome-wide RNAi screen aiming specifically at identifying regulators of Notch ligands in the signal-sending cell. We thereby unraveled new classes of common regulators for both Delta-like1 and 4 ligands. These regulators include protease inhibitors, transcription factors and various genes of unknown function such as Tmem128 (Transmembrane protein 128), or of previously characterized function such as the molecular co-chaperone Cdc37 (Cell division cycle 37 homolog). We next developed functional secondary screens where we demonstrated that our hits are important for Notch-mediated events, such as normal T-cell differentiation, and survival of pre-leukemic stem cells (pre-LSCs) isolated from a mouse model of T-ALL. Moreover, we showed that top hits from the pre-LSC survival screen are also required for the self-renewal activity of pre-LSCs. Finally, we performed a preliminary molecular characterization of two newly identified regulators; Tmem128 and Cdc37 in order to investigate their mechanism of action on Delta-like ligands. Altogether, this study led to the identification of novel Notch pathway regulators that could serve as potential therapeutic targets in Notch cancers, as exemplified by the T-ALL model. Elucidating the finer details that underlie the molecular functions of these regulators will be critical to develop pharmacological inhibitors to counteract their action and impede Notch signalling in cancer.

A Dengue e a Febre Aftosa são infecções virais que ocorrem tanto no Brasil como no mundo, apresentando enorme impacto socioeconômico. As proteases virais são reconhecidas como alvos de grande interesse para o planejamento de antivirais, devido ao seu papel fundamental no ciclo de vida de muitos vírus, incluindo-se os flavivirus (vírus da Dengue DENV) e os picornavirus (vírus da Febre Aftosa FA). Com o objetivo de buscar e identificar novos inibidores de proteases virais (da NS2B/NS3pro do DENV ou da Lbpro do vírus da FA) propusemos modelos de busca virtual, incluindo diferentes filtros de seleção (ex. farmacofórico, drug-like, similaridade e ancoramento) aplicados, sequencialmente, a bancos de dados de compostos comerciais (280x103 a 23x106 compostos). A seleção final dos compostos foi sempre feita por inspeção visual. Para NS2B/NS3pro do DENV, construíram-se quatro modelos de busca virtual (Modelo I-DENV a Modelo IV-DENV). O primeiro foi construído, baseando-se na estrutura cristalográfica ligada a um inibidor peptidomimético, e aplicado ao banco ZINC. Ao final, dez compostos foram comprados e submetidos a testes de inibição enzimática, frente à NS2B/NS3pro, para validação experimental deste modelo. Dois compostos mostraram alguma atividade inibitória (IC50 150 - 300 µM). Visando-se melhorar estes resultados, a flexibilidade da NS2B/NS3pro foi incluída, usando simulações de dinâmica molecular (DM), e um novo banco de dados construído (ZINC-Curated). Através de uma análise extensiva do banco ZINC-Curated, usando ferramentas estatísticas/quimiométricas, confirmou-se que este foi enriquecido com compostos com características de fármacos. Outros três modelos de busca virtual foram construídos incluindo-se diferentes informações obtidas nas simulações de DM. O modelo II-DENV foi feito usando ancoramento e aplicado ao banco ZINC-Curated, selecionando dezesseis compostos. Nenhum deles apresentou atividade inibitória significativa frente à NS2B/NS3pro do DENV. Os modelos III-DENV e IV-DENV utilizaram modelos farmacofóricos, que tiveram seus desempenhos previamente avaliados usando dados de literatura, e foram aplicados aos bancos NCI e ZINC-Curated, respectivamente. O modelo III-DENV selecionou quinze compostos, tendo quatro deles apresentado atividade inibitória (IC50 30 - 100 µM). O modelo IV-DENV selecionou dezoito compostos, sendo quatro ativos frente a esta protease (IC50 4 - 90 µM), representando uma taxa de acerto de ~22 %. Ainda, uma série de treze análogos estruturais do composto mais ativo foi construída, sendo três deles também ativos. Portanto, as modificações incluídas na busca virtual permitiram melhorar, significativamente, os resultados obtidos. Para Lbpro do vírus da FA, construíram-se dois modelos de busca virtual (Modelos I-FA e II-FA). O primeiro foi construído usando sua estrutura cristalográfica, sem ligantes, e uma série in house de potenciais inibidores covalentes. Seis compostos foram selecionados e testados frente à Lbpro, tendo dois deles baixa atividade inibitória (IC50 300 - 600 µM). A partir da disponibilidade da estrutura da Lbpro com ligante, o modelo IIFA foi construído e aplicado ao banco ZINC-Curated, selecionando quinze compostos. Estes foram adquiridos e testados frente à Lbpro, não apresentando atividade inibitória significativa. Assim, as modificações incluídas ainda não foram suficientes para selecionar inibidores mais potentes. No entanto, estes modelos/resultados contribuíram para o entendimento da(s) interação(ões) no sítio ativo da Lbpro. / Dengue and Food-and-mouth disease are viral infections that occur in Brazil and in the world, causing a huge socioeconomic impact. Viral proteases are recognized as targets for antiviral design, because they are crucial for the life cycle of many viruses, such as flavivirus (Dengue virus DENV) and picornavirus (Food-and-mouth disease virus FMDV). In order to discovery novel inhibitors of viral proteases (of NS2B/NS3pro of DENV or of Lbpro of FMDV) virtual screening models were proposed comprising a sequence of different filters (e.g. pharmacophore, drug-like, similarity and docking) applied to databases of commercial compounds (280x103 to 23x106 compounds). In all models, the final selection of compounds was always done by visual inspection. For DENV NS2B/NS3pro, four virtual screening models were proposed (Model I-DENV to Model IV-DENV). Model I-DENV was built, based on the crystal structure bound to a peptidemimetic inhibitor, and applied to ZINC database. Finally, ten compounds were purchased and submitted to enzymatic assays against this protease to the experimental validation of this model. Two compounds showed some inhibitory activity (IC50 150 - 300 µM). In order to improve these results, NS2B/NS3pro flexibility was included, using molecular dynamics (MD) simulations, and a novel database was built (ZINC-Curated). Throughout an exhaustively analysis of ZINC-Curated, using statistical/chemometrics tools, we confirmed that this new database was enriched with drug like compounds. Other three virtual screening models were built including different information from MD simulations. Model II-DENV was built using docking and applied to the ZINC-Curated database, selecting sixteen compounds. None of them showed a significant inhibitory activity against DENV NS2B/NS3pro. Models III-DENV and IV-DENV were built using pharmacophore models, which have their performance previously evaluated using literature data, and applied to NCI and ZINC-Curated databases, respectively. Model III-DENV selected fifteen compounds, showing four of them inhibitory activity (IC50 30 - 100 µM). Model IV-DENV selected eighteen compounds. Four of them were active against this protease (IC50 4 - 90 µM), representing a hit rate of ~22 %. Moreover, a set of thirteen structural analogues of the most active compound were built, being three of them also active. Thus, the modifications done in the virtual screening procedure really improved our results. For FMDV Lbpro, two virtual screening models were built (Models I-FMDV and II-FMDV). The first one was based on the crystal structure, without ligands, and used a set of in house potential covalent inhibitors. Six of the in house compounds were selected and tested against this protease. Two of them showed a weak inhibitory activity (IC50 300 - 600 µM). Later on, the Lbpro bound with ligands was available being therefore used to build another model. Model II FMDV was applied to the ZINC-Curated database, selecting fifteen compounds that were purchased and also tested against the target protease. But none of them showed a significant inhibition. Thus, the incorporated changes were not enough to retrieve active compounds. However, these models/results contributed to better understand Lbpro binding site interactions

Introdução: Demonstramos previamente que em modelo experimental de enfisema pulmonar induzido por instilação de elastase, o inibidor de serinoprotease rBmTI-A promoveu a melhora da destruição tecidual em camundongos. Considerando que o tabagismo é o principal fator de risco para o desenvolvimento da Doença Pulmonar Obstrutiva Crônica (DPOC) e que o modelo de exposição à fumaça de cigarro é considerado o que melhor mimetiza esta doença em humanos, este estudo teve por objetivo verificar a ação do inibidor para serinoproteases rBmTI-A sobre os processos fisiopatológicos envolvidos no desenvolvimento do enfisema pulmonar, em modelo de exposição ao tabaco. Métodos: Para a indução do enfisema pulmonar, os animais foram expostos à fumaça de cigarro (duas vezes ao dia/ 30 minutos/ 5 dias por semana/ durante 12 semanas), e os animais controle permaneceram expostos ao ar ambiente. Dois protocolos de tratamento com o inibidor rBmTI-A foram realizados. No primeiro, os animais receberam duas administrações do inibidor rBmTI-A ou de seu veículo (Solução Salina 0,9%) por via intranasal, sendo a primeira após 24h do término das exposições ao cigarro e outra, 7 dias após à primeira instilação do inibidor. No segundo protocolo, os animais receberam 3 administrações do inibidor rBmTI-A, durante o tempo de exposição (1ª dose: 24h antes do início da exposição à fumaça de cigarro; 2ª dose: um mês após o início da exposição; 3ª dose: dois meses após o início). Após o término dos protocolos de exposição e tratamento, os animais foram submetidos aos procedimentos para coleta dos dados de mecânica respiratória e avaliação do Intercepto Linear Médio (Lm). Para o segundo protocolo, realizamos também as medidas para quantificação de fibras de colágeno e elástica, da densidade de células positivas para MAC-2, MMP-12 e 9, TIMP-1, Gp91phox e TNFalfa; no parênquima através de imunohistoquímica, contagem de células polimorfonucleares além da expressão gênica de MMP-12 e 9 no pulmão através de RT-qPCR. Resultados e Discussão: O tratamento com o inibidor para serinoprotease rBmTI-A atenuou o desenvolvimento do enfisema pulmonar apenas no segundo protocolo, quando foi administrado durante a exposição à fumaça de cigarro. Embora os grupos Fumo-rBmTIA e Fumo-VE apresentem aumento de Lm comparados aos grupos controles, houve uma redução deste índice no grupo Fumo-rBmTIA comparado ao grupo Fumo-VE. O mesmo comportamento foi observado para as análises de proporção em volume de fibras de elástica e colágeno no parênquima. Além disto, observamos aumento de macrófagos, MMP-12, MMP-9 e TNFalfa; nos grupos expostos à fumaça de cigarro, mas o tratamento com o inibidor rBmTI-A diminuiu apenas a quantidade de células positivas para MMP-12. Na avaliação da expressão gênica para MMP-12 e 9, não observamos diferença entre os grupos experimentais e o mesmo comportamento foi observado para a quantidade de células polimorfonucleares no parênquima. Além disso, observamos aumento de GP91phox e TIMP-1 nos grupos tratados com rBmTIA. Conclusões: Tais resultados sugerem que o inibidor rBmTI-A não foi efetivo como tratamento da lesão após a doença instalada. Entretanto, atenuou o desenvolvimento da doença quando administrado durante a indução do enfisema, possivelmente através do aumento de GP91phox e TIMP-1, acompanhados pela diminuição de MMP-12. / Introduction: We have previously showed that in an elastase-induced model of emphysema, the treatment with a serine protease inhibitor rBmTI-A, resulted in an improvement of tissue destruction in mice. Considering that smoking is the main risk factor for the development of COPD, and the cigarette smoke (CS) exposure is considered the best model to reproduce physiopathologic similarities with such disease in humans, this study aimed to verify the rBmTI-A treatment on the physiopathological processes involved in the development of cigarette smoke-induced emphysema. Methods: To induce pulmonary emphysema, animals were exposed to cigarette smoke (twice a day/ 30 minutes/ 5 days per week/ for 12 weeks) and the control animals were exposed to room air. Two treatment protocols with rBmTI-A inhibitor were performed. In the first one, animals received two administrations of rBmTI-A inhibitor or its vehicle (Saline Solution 0.9%) by nasal instillation, one dose at 24 hours after the end of exposure to tobacco smoke and another one, 7 days after the first instillation of the inhibitor. In the second protocol, animals received 3 rBmTI-A inhibitor administrations during the exposition time (1st dose: 24 hours before the start of exposure to cigarette smoke; 2nd dose: one month after the start of exposure, 3rd dose: two months after the start). After the end of exposure and treatment protocols, animals were submitted to procedures for collection of respiratory mechanics and evaluation of the Mean Linear Intercept (Lm). For the second protocol, we also measured the volume proportion of collagen and elastic fibers, the density of positive cells for MAC-2, MMP-12 and -9, TIMP-1, GP91phox and TNF-alfa in lung parenchyma by immunohistochemistry. Also, we evaluated the measurement of polymorphonuclear cells and the lung gene expression for MMP-12 and 9 by RT-qPCR. Results and Discussion: Treatment with the serine protease inhibitor rBmTI-A attenuated the development of emphysema only in the second protocol, when it was administered during exposure to cigarette smoke. Although Smoke-rBmTIA and Smoke-VE groups showed an increase of Lm measure compared to Control groups, there were a decrease in the Smoke-rBmTIA group compared to Smoke-VE group. The same response was observed for the analysis of volume proportion of elastic and collagen fibers in parenchyma. In addition, we observed an increase of macrophages, MMP-12, MMP-9 and TNF-alfa; in groups exposed to cigarette smoke, but treatment with rBmTI-A inhibitor only decreased the number of positive cells for MMP-12. We did not observed difference between the experimental groups in lungs gene expression for MMP- 12 and 9, and the same behavior was observed for the amount of polymorphonuclear cells in parenchyma. Moreover, we observed an increase of GP91phox and TIMP-1 in groups treated with rBmTI-A. Conclusions: These results suggest that rBmTI-A inhibitor was not effective for treatment of parenchymal lesions after established disease. However, this inhibitor attenuated the development of disease when administered during the induction of emphysema, possibly by an increase of GP91phox and TIMP-1, accompanied by a decrease of MMP-12

INTRODUÇÃO: Os corticosteróides são considerados padrão-ouro no tratamento da asma, porém, existem asmáticos graves que não obtém controle dos sintomas, o que suscita a busca por novas terapias. Os inibidores de proteinases têm sido estudados no controle de diversos processos inflamatórios, dentre estes, encontra-se Crataeva tapia Bark Lectin (CrataBL). OBJETIVO: Avaliar se a proteina bifuncional de planta, CrataBL, que tem função lectínica e se liga a carboidratos, modula a hiperresponsividade brônquica à metacolina, inflamação, remodelamento e estresse oxidativo nas vias aéreas e nos septos alveolares de camundongos com inflamação pulmonar alérgica crônica. MÉTODOS: Trinta e dois camundongos machos Balb-c SPF (6-7 semanas, 25-30 g) foram divididos em 4 grupos: C (controle), OVA (sensibilizados - 50 ug de ovalbumina intraperitoneal (i.p) nos dias 0 e 14 e desafiados - 1% de ovalbumina nos dias 22, 24, 26, 28); C+CR (controle tratados com CrataBL - 2 mg/kg/i.p dos dias 22 a 28); OVA+CR (sensibilizados e desafiados com ovalbumina e tratados com CrataBL - 2 mg /kg/i.p dos dias 22 a 28). No dia 29, realizamos: (i) hiperresponsividade à metacolina - resposta máxima de resistência (Rrs) e elastância (Ers) do sistema respiratório; (ii) quantificação do número total de células, macrófagos, linfócitos e células polimorfonucleares no fluido do lavado broncoalveolar (FLBA); (iii) análise histopatológica do pulmão por morfometria para quantificação de eosinófilos, fração de volume de fibras colágenas e elásticas; (iiii) imunohistoquímica para quantificação de células positivas para IL-4, IL-5, IL-13, IFN-y, MMP-9, TIMP-1, TGF-beta, iNOS, NF-kB e fração de volume de 8-iso-PGF2alfa nas vias aéreas e nos septos alveolares e ELISA para quantificação da concentração de IL-4, IL-5 e IFN-y. A significância foi considerada quando p < 0,05. RESULTADOS: Houve atenuação da resposta máxima de Rrs e Ers no grupo OVA+CR comparado ao grupo OVA (p < 0,05). O tratamento com CrataBL nos animais sensibilizados atenuou o número de células totais, macrófagos, linfócitos e células polimorfonucleares no FLBA, o número de eosinófilos, células positivas para IL-4, IL-5, IL-13, IFN-y, iNOS, MMP-9, TIMP-1, TGF-beta, NF-kB e fração de volume de 8-iso-PGF2alfa, fibras colágenas e elásticas tanto nas vias aéreas quanto nos septos alveolares quando comparados ao grupo OVA. O tratamento com CrataBL atenuou os níveis de concentração de IL-4, IL-5 e IFN-y em comparação ao grupo OVA (p < 0,05), a partir do método ELISA. CONCLUSÕES: CrataBL atenuou a hiperresponsividade brônquica, a inflamação, o remodelamento e o estresse oxidativo nesse modelo experimental de inflamação pulmonar alérgica crônica. Contudo, mais estudos são necessários para verificar se este inibidor pode ser uma potencial ferramenta terapêutica para a asma / RATIONALE: Corticosteroids are considered the gold standard in the treatment of asthma, however, there are severe asthmatics who do not get control of symptoms, which raises the search for new therapies. The proteinase inhibitors have been studied in the control of various inflammatory processes, including such inhibitors is Crataeva tapia Bark Lectin (CrataBL). OBJECTIVE: To evaluate the proteinase inhibitor CrataBL modulates the bronchial responsiveness to methacholine, inflammation, remodeling and activation of oxidative stress in the airways and alveolar septa of mice with chronic allergic pulmonary inflammation. METHODS: Thirty two SPF Balb-c male mice (6-7 weeks, 25-30 g) were divided into 4 groups: control (C), OVA (sensitized - 50 ug ovalbumin intraperitoneal (i.p) on days 0 and 14 and challenged - 1% ovalbumin on days 22, 24, 26, 28); C+CR (control treated with CrataBL - 2 mg / kg / i.p of 22 to 28); OVA+CR (sensitized and challenged with ovalbumin and treated with CrataBL - 2 mg / kg / i.p from day 22 to 28). On the 29th, we held: (i) hyperresponsiveness to methacholine and maximal responses were obtained resistance (Rrs) and elastance (Ers) of the respiratory system; (ii) quantification of total cells, macrophages, polymorphonuclear cells and lymphocytes in bronchoalveolar lavage fluid (BALF); (iii) histopathological analysis of the lungs by morphometry to quantify the eosinophils, volume fraction of collagen and elastic fibers; (iiii) immunohistochemistry for quantification of positive IL-4, IL-5, IL-13, IFN-y, MMP-9, TIMP-1, TGF-beta, iNOS, NFk-beta cells and volume fraction of 8-iso-PGF2? in airway and alveolar septa and ELISA for quantification of concentration of IL-4, IL-5 e IFN-y (p < 0.05). RESULTS: There was attenuation of the maximal response Rrs and Ers in the OVA+CR group compared to OVA (p < 0.05). Treatment with CrataBL in sensitized animals attenuated the number of total cells, macrophages, lymphocytes, and polymorphonuclear cells in BALF, the number of eosinophil positive IL-4, IL-5, IL-13, IFN-y, iNOS, MMP -9, TIMP-1, TGF-beta, NF-kB cells and volume fraction of 8-iso-PGF2alfa, collagen and elastic fibers in both the airways and alveolar septa compared to OVA group. Treatment with CrataBL attenuated concentration levels of IL-4, IL-5 and IFN-y compared to OVA group (p < 0.05) from the ELISA method.CONCLUSIONS: CrataBL attenuated bronchial hyperresponsiveness, inflammation, remodeling and oxidative stress in this experimental model of chronic allergic pulmonary inflammation. However, more studies are needed to determine if this inhibitor can be a potential therapeutic tool for asthma

L’étude du retentissement d’un xénobiotique en général ou d’un médicament en particulier sur la gestation humaine est difficile à appréhender. Elle doit prendre en compte des considérations éthiques, indispensables lors de toute recherche chez la femme enceinte, mais aussi pour le cas de l’induction de la parturition, de la complexité des mécanismes physiologiques impliqués. S’il existe un relatif consensus expérimental à partir de modèles animaux pour évaluer leur retentissement sur la fertilité et l’embryogenèse, il n’y a pas à ce jour d’attitude pour évaluer un risque induit d’un accouchement prématuré. Hors la naissance prématurée est la première cause de mortalité du nouveau-né et concerne quinze millions de naissances par an au niveau mondial. Les étiologies sont multiples : infectieuses dont VIH,grossesses multiples, addictions. La littérature récente fait part aussi de cette problématique pour certains médicaments indispensables comme les inhibiteurs de protéase chez les femmes enceintes infectées par le VIH. Nous avons réalisé trois études spécifiquement chez la femme enceinte : modulation du passage placentaire des inhibiteurs de protéase du VIH, modulation des hormones stéroïdiennes placentaires et materno-foetales par la mifépristone, variation des protéines sériques maternelles dans les jours précédents la parturition. C’est à l’issue de ces travaux que nous émettons plusieurs hypothèses sur l’incidence d’un médicament lors de la parturition humaine et les moyens envisagés pour l’étudier de manière la moins invasive possible. / Effects of a xenobiotic or drug on human gestation are difficult to approach. This is due tonecessary ethical considerations with regards to research on pregnant women as well as thecomplexity of physiological processes involved in the case of induced parturition. While thereis a relative experimental consensus with animal models to assess their impact on fertility andembryogenesis, there is currently no method to assess the risk of induced preterm labor.Preterm labor is the leading cause of newborn deaths at the rate fifteen million births per yearglobally. The etiologies are multiple: infectious including HIV, multiple pregnancies,addictions. Recent publications also discuss this issue in the context of necessary drugs suchas protease inhibitors for HIV infected pregnant women. We realised three studies specificallyin pregnant women: modulation of the placental transfer of protease inhibitors of the HIV,modulation of the feto-maternal and placental steroid hormones by mifepristone, and a studyabout variation of the maternal serum proteins in the previous days of the parturition. Also,regarding our studies and the literature, we make several hypotheses on possible interferencesbetween drugs and human parturition, disturb its signal, and methods proposed for its study ina minimally invasive manner.