Peptídeos com ação inibitória sobre a enzima conversora de angiotensina I (ECA) e com o potencial de reduzir a pressão arterial têm sido obtidos a partir de diferentes tipos de alimentos ou matérias-primas, sendo grande o interesse em aproveitar resíduos da indústria alimentícia como fontes desses peptídeos. Neste aspecto, as amêndoas de cupuaçu (Theobroma grandiflorum S.), que são em sua maioria descartadas pela indústria, apresentam um teor considerável de proteínas e poderiam ser aproveitadas como fontes de peptídeos inibidores da ECA. Assim, o objetivo deste trabalho foi verificar se o concentrado proteico obtido a partir de amêndoas fermentadas de cupuaçu após ser submetido à hidrólise com a enzima pepsina poderia gerar peptídeos com ação inibitória sobre a ECA in vitro. Foi observado que após a hidrólise do concentrado proteico com pepsina por 1h foi obtido um efeito de 50 % de inibição da ECA, em ensaio realizado com o substrato Abz-FRK(Dnp)-P-OH. Posteriormente, esse hidrolisado foi submetido ao fracionamento por cromatografia em fase reversa (RP-HPLC) e resultou em cinco frações (F1-F5), das quais a terceira teve uma subfração (F3.1) com quatro novos peptídeos identificados por LC-MS/ MS com potencial em inibir a ECA. Esses quatro peptídeos (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) foram sintetizados e submetidos aos ensaios para a determinação do IC50 e Ki. Os peptídeos YRLAF e LGYFK, que apresentaram mecanismo de inibição do tipo competitivo e acompetitivo, respectivamente, tiveram valores de IC50 de 4.73 e 11.11µM, e de Ki de 9.14 e 8.15 µM. Dentre os peptídeos identificados merece destaque VTTVVTGLTF que demonstrou ser um inibidor do tipo acompetitivo e apresentou as menores IC50 (0.70 µM) e Ki (2.79 µM). Em contraste, FWVAM atuou como substrato da ECA e não peptídeo inibidor. A partir dos resultados obtidos neste estudo fica demonstrado que as amêndoas fermentadas de cupuaçu podem ser fonte de peptídeos com ação inibitória da ECA, com potencial efeito anti-hipertensivo a ser, futuramente, investigado a partir de estudos in vivo. / Angiotensin converting enzyme (ACE) inhibitory peptides with the potential to reduce blood pressure have been obtained from different types of food or raw materials, and there is a great interest in utilize residues from the food industry as sources of peptides. In this regard, cupuassu almonds (Theobroma grandiflorum S.), which are mostly discarded by the industry, has a considerable protein content and could be used as source of ACE-inhibiting peptides. Thus, the objective of this work was to verify if the protein concentrate obtained from fermented almonds of cupuassu after being submitted to the hydrolysis with the enzyme pepsin could generate peptides with inhibitory activity on ACE in vitro. In this study it was observed that after hydrolysis of the protein concentrate with pepsin for 1 h, a 50% effect of ACE inhibition was obtained in an assay performed with the Abz-FRK (Dnp) -P-OH substrate. Posteriorly, the hydrolyzate was subjected to fractionation by reverse phase chromatography (RP-HPLC) and resulted in five fractions (F1-F5), of which the third had a subfraction (F3.1) with four new peptides identified by LC-MS / MS with the potential to inhibit ACE. These four peptides (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) were synthesized and assayed for IC50 and Ki. The YRLAF and LGYFK peptides, which showed a competitive and uncompetitive type inhibition mechanism respectively, presented IC50 values of 4.73 and 11.11µM, and the values for Ki were 9.14 and 8.15 µM. Among the peptides identified, it is possible to highlight VTTVVTGLTF, which was shown to be an inhibitor of the uncompetitive type and presented the lowest value for IC50 (0.70 µM) and Ki (2.79 µM). While FWVAM acted as a substrate of the ACE and not as an inhibitory peptide. From the results obtained in this study it is demonstrated that cupuassu fermented almonds can be a source of peptides with ACE inhibitory activity with potential antihypertensive effect to be further investigated from in vivo studies.

L’intégrité des synapses neuronales est primordiale pour le développement et le maintien des capacités cognitives. Des mutations dans des gènes codant pour des protéines synaptiques ont été trouvées chez des patients atteints de déficience intellectuelle (DI), qui est une maladie neurodéveloppementale ayant des conséquences sur les fonctions intellectuelles et adaptatives. Ce travail de thèse porte sur l’étude de l’un de ces gènes, IL1RAPL1, dont les mutations sont responsables d’une forme non-syndromique de DI liée au chromosome X, et sur le rôle de la protéine IL1RAPL1 dans la formation et le fonctionnement des synapses. IL1RAPL1 est une protéine trans-membranaire qui est localisée dans les synapses excitatrices où elle interagit avec les protéines post-synaptiques PSD-95, RhoGAP2 et Mcf2l. De plus, IL1RAPL1 interagit en trans- avec une protéine phosphatase présynaptique, PTPd, via son domaine extracellulaire. Nous avons étudié les conséquences fonctionnelles de deux nouvelles mutations qui affectent le domaine extracellulaire d’IL1RAPL1 chez des patients présentant une DI. Ces mutations conduisent soit à une diminution de l’expression de la protéine, soit à une réduction de l’interaction avec PTPd affectant ainsi la capacité d’IL1RAPL1 à induire la formation de synapses excitatrices. En absence d’IL1RAPL1, le nombre ou la fonction des synapses excitatrices est diminué, ce qui mène à un déséquilibre entre les transmissions synaptiques excitatrice et inhibitrice dans des régions spécifiques du cerveau. Dans le cas particulier de l’amygdale latérale, nous avons montré que ce déséquilibre conduit à des défauts de mémoire associative chez la souris déficiente en Il1rapl1. L’ensemble des résultats qui font partie de ce travail montre que l’interaction IL1RAPL1/PTPd est essentielle pour la formation des synapses et suggère que les déficits cognitifs des patients avec une mutation dans il1rapl1 proviennent du déséquilibre de la balance excitation/ inhibition. Ces observations ouvrent des perspectives thérapeutiques visant à rétablir cette balance dans les réseaux neuronaux affectés. / Preserving the integrity of neuronal synapses is important for the development and maintenance of cognitive capacities. Mutations on a growing number of genes coding for synaptic proteins are associated with intellectual disability (ID), a neurodevelopmental disease characterized by deficits in adaptive and intellectual functions. The present work is dedicated to the study of one of those genes, IL1RAPL1, and the role of its encoding protein in synapse formation and function. IL1RAPL1 is a trans-membrane protein that is localized at excitatory synapses, where it interacts with the postsynaptic proteins PSD-95, RhoGAP2 and Mcf2l. Moreover, the extracellular domain of IL1RAPL1 interacts trans-synaptically with the presynaptic phosphatase PTPd. We studied the functional consequences of two novel mutations identified in ID patients affecting this IL1RAPL1 domain. Those mutations lead either to a decrease of the protein expression or of its interaction with PTPd, affecting in both cases the IL1RAPL1-mediated excitatory synapse formation. In the absence of IL1RAPL1, the number or function of excitatory synapses is perturbed, leading to an imbalance of excitatory and inhibitory synaptic transmissions in specific brain circuits. In particular, we showed that this imbalance in the lateral amygdala results in associative memory deficits in mice lacking Il1rapl1. Altogether, the results included in this work show that IL1RAPL1/PTPd interaction is essential for synapse formation and suggest that the cognitive deficits in ID patients with mutations on IL1RAPL1 result from the imbalance of the excitatory and inhibitory transmission. These observations open therapeutic perspectives aiming to reestablish this balance in the affected neuronal circuits.

O presente estudo teve como principal objetivo avaliar a capacidade de produção contínua de hidrogênio, sob condições termofílicas (55ºC), a partir de diferentes concentrações de vinhaça de cana-de-açúcar em reator anaeróbio de leito fluidificado (RALF) utilizando argila expandida como material suporte para adesão microbiana. Foram utilizados cinco reatores de idêntica configuração, denominados R5, R10, R15, R20 e R30, com variação na concentração afluente de 5000, 10.000, 15.000, 20.000 e de 30.000 mg DQO L-1, respectivamente (com cargas orgânicas volumétricas- TCO entre 15 e 720 kg DQO m-3 d-1). O tempo de detenção hidráulica (TDH) reduziu de 8, 6, 4, 2 e 1 h. Na estratégia de operação de um deles, (R5) investigou-se os efeitos da co-fermentação de porcentagens de glicose e de vinhaça no substrato de alimentação. Os outros quatro reatores foram operados mediante a adição de glicose como co-substrato ao afluente, apenas durante o período de partida operacional. Em todos os reatores, foi observado comportamento de elevação na produção volumétrica de H2 (PVH) a partir da diminuição do TDH. A máxima PVH obtida foi de de 1,96 L h-1 L-1 (R10; TDH de 1h; TCO de 240 kg DQO m-3 d-1). No entanto, verificou-se diminuição do rendimento de H2 (HY) e no conteúdo de H2 no biogás, com a aplicação dos TDH reduzidos, em todos os reatores, alcançando valor máximo de 4,62 mmol g DQOadicionada-1 no TDH de 8 h (R5) e 57.51% de H2 no TDH de 6 h (R20). O reator operado com a maior concentração de vinhaça (R30) apresentou menores valores de produção de H2, atribuídos aos compostos inibitórios (elevada concentração de ácidos voláteis no afluente - butírico e acético). As análises de clonagem e sequenciamento dos consórcios bacterianos termofílicos revelaram semelhanças (99%) com cepas produtoras de H2, como Thermoanaerobacterium thermosaccharolyticum, Clostridium cellulosi, Lactobacillus fermentum e Megasphaera elsdenii. A capacidade de produzir H2, a distribuição dos ácidos e a estrutura da comunidade bacteriana foram fatores influenciados pelo aumento da concentração de vinhaça e das TCO aplicadas. / The present study aimed to evaluate the ability of continuous hydrogen production under thermophilic conditions (55 °C), from different sugar cane vinasse concentrations in anaerobic fluidized bed reactor (AFBR) using expanded clay as support material for microbial adhesion. Five reactors of identical configuration were used, denominated R5, R10, R15, R20 and R30, followed by influent variation concentration of 5000; 10,000; 15,000; 20,000 and 30,000 mg COD L-1, respectively (with organic loading rate- OLR varying from 15 to 720 kg COD m-3 d-1). The hydraulic retention time (HRT) decreases from 8, 6, 4, 2 and 1 h. The strategy applied to one of those (R5), investigated the co-fermentations effects across the application of different percentages of glucose and stillage in the fed substrate. The other four reactors were operated by adding glucose as co-substrate to the influent just during the start-up period. In all reactors, it was observed a volumetric H2 production (HPR) increase behavior from the HRT decrease. The maximum HPR obtained was 1.96 L h-1 L-1 (R10; HRT of 1h; OLR of 240 kg COD m-3 d-1). However, there was a decrease in H2 yield (HY) and H2 content in biogas, with the application of reduced HRT for all reactors, reaching maximum value of 4.62 mmol g CODadded-1 at HRT of 8 h (R5) and 57.51% of H2 at HRT of 6 h (R20). The reactor operated with the highest concentration of stillage (R30) showed lower values for H2 production, attributed to inhibitory compounds (high concentration of volatile acids in the affluent - butyric and acetic). The cloning and sequencing analyzes of the thermophilic bacterial consortium showed similarity (99%) with H2 producing strains, such as Thermoanaerobacterium thermosaccharolyticum, Clostridium cellulosi, Lactobacillus fermentum and Megasphaera elsdenii. The ability to produce H2, the distribution of organic acids and the bacterial community structure analyzed in all reactors were factors influenced by the increasing stillage concentration and OLR applied.

Decision trees are one of the most commonly used tools in decision analysis, knowledge representation, machine learning, etc., for its simplicity and interpretability. We consider an extension of dynamic programming approach to process the whole set of decision trees for the given decision table which was previously only attainable by brute-force algorithms. We study decision tables with many-valued decisions (each row may contain multiple decisions) because they are more reasonable models of data in many cases. To address this problem in a broad sense, we consider not only decision trees but also inhibitory trees where terminal nodes are labeled with “̸= decision”. Inhibitory trees can sometimes describe more knowledge from datasets than decision trees. As for cost functions, we consider depth or average depth to minimize time complexity of trees, and the number of nodes or the number of the terminal, or nonterminal nodes to minimize the space complexity of trees. We investigate the multi-stage optimization of trees relative to some cost functions, and also the possibility to describe the whole set of strictly optimal trees. Furthermore, we study the bi-criteria optimization cost vs. cost and cost vs. uncertainty for decision trees, and cost vs. cost and cost vs. completeness for inhibitory trees. The most interesting application of the developed technique is the creation of multi-pruning and restricted multi-pruning approaches which are useful for knowledge representation and prediction. The experimental results show that decision trees constructed by these approaches can often outperform the decision trees constructed by the CART algorithm. Another application includes the comparison of 12 greedy heuristics for single- and bi-criteria optimization (cost vs. cost) of trees. We also study the three approaches (decision tables with many-valued decisions, decision tables with most common decisions, and decision tables with generalized decisions) to handle inconsistency of decision tables. We also analyze the time complexity of decision and inhibitory trees over arbitrary sets of attributes represented by information systems in the frameworks of local (when we can use in trees only attributes from problem description) and global (when we can use in trees arbitrary attributes from the information system) approaches.

Camundongos C3H/HePAS infectados com Trypanosoma cruzi Sylvio X10/4 desenvolvem cardiomiopatia chagásica crônica semelhante aquela observada em pacientes portadores da doença de Chagas. A análise das células que infiltram o coração na fase crônica mostra uma maior frequência de leucócitos e maior expressão de PD-1 e PD-L1, moléculas inibitórias do sistema imune. Visando restaurar a resposta, tratamos camundongos primeiramente com anticorpos anti-PD-L1 e posteriormente com anticorpos anti-PD-L1 e anti-PD-1 em diferentes experimentos, que falharam em exercer uma diferença significativa na patologia cardíaca, parasitemia do sangue ou do coração associado ao aumento da resposta imune. Para fornecer co-estímulo além de bloquear interações inibitórias, realizamos o tratamento com anti-PD-1 e anti-PD-L1 associado a T. cruzi irradiado, que aumentou a patologia cardíaca e a bradicardia, assim como diminuiu a parasitemia sanguínea, sem mudanças no perfil das células T. O bloqueio das interações inibitórias associado ao desafio e ao co-estímulo das células T, leva a uma piora no funcionamento cardíaco associado à diminuição da parasitemia nos camundongos crônicos. / C3H/HePAS mice infected with T. cruzi Sylvio X10/4 parasites develop a chronic cardiomyopathy like the observed in human chagasic patients. Flow cytometry analysis of heart-infiltrating cells in chronically infected mice revealed higher leukocyte frequency with increased PD-1 and PD-L1 expression. In order to restore the immune response, mice were treated with anti-PD-L1 or with anti-PD-L1 and anti-PD-1, but neither exerted effects at heart pathology and parasitemia nor restored T cell response. Hence, we evaluated the possibility of using irradiated T. cruzi challenge to provide parasite antigen and co-stimulatory signaling to exhausted cells. Thus, association of anti-PD-1 and anti-PD-L1 treatment with irradiated T. cruzi challenge increased heart pathology and bradycardia as well as reduced blood parasitemia. Additionally, the treatment does not change T cell phenotype. Blocking both inhibitory interactions and provide antigen with co-stimulation seems to cause a loss in heart function despite decreasing parasitemia.

Introduction: Impaired inhibitory control, one of the core executive functions, is common among individuals with mental illness. However, inhibitory control is essential for successful treatment and recovery. Inhibitory control is extremely vulnerable to developmental disruption during adolescence, a time when mental illness is first diagnosed. An acute bout of exercise has been shown to improve inhibitory control in healthy adolescents, however, to our knowledge there are no studies evaluating this effect in adolescents with mental illness. Purpose: The primary goal of this project was to examine the effect of an acute bout of high-intensity interval training on inhibitory control immediately, and 30 minutes following exercise in adolescents hospitalized for mental illness. The secondary goal was to assess the feasibility of using this type of exercise as an adjunct to current treatment. Methods: Participants were recruited through the inpatient mental health unit at the Children’s Hospital of Eastern Ontario. They performed exercise and control conditions in a randomized, counterbalanced manner. The Colour-Word Stroop Task was assessed pre, post, and 30-minutes-post on both days. The exercise condition included a 12 minute HIIT circuit, consisting of body weight exercises performed in a 1:1 work to rest ratio. The control condition involved reading magazines. Repeated-measures ANOVA evaluated changes in Interference Cost, the reaction time cost of responding to trials where the ink and colour do not match, and overall accuracy. Feasibility was assessed through recruitment and completion rates, as well as changes in affect and acceptability of the high-intensity interval training. Results: There was a significant interaction between condition and time for the Interference Cost measure, F(1.6,43.3)=13.6, p<.0001, η2=.34. Interference Cost was similar for both conditions at baseline (Mdiff = 12.4±11.11, p=.28). Interference Cost was significantly reduced immediately (Mdiff = 78.8±14.91, p<.001) and 30-minutes post-exercise (Mdiff = 59.6±15.14, p=.001) compared to control. Response accuracy did not differ by time, F(2,54)=.14, p<=.87, η2=.01 nor condition, F(1,27)=2.25, p=.15, η2=.08. After exercise, participants increased positive affect (mean difference = 4.3±8.09, p=.009) and were willing to perform the exercise before therapy sessions (rating = 6.4±2.75 out of 10). Conclusion: These findings suggest that high-intensity interval training could be used to improve inhibitory control in adolescents with mental illness, which has the potential to enhance the efficacy of their treatment. Future research should determine the impact of individual factors, such as diagnosis, medication, age of illness onset, length of hospitalization, and treatment history, on inhibitory control improvement after exercise.

Type-A Î3-amino-butyric acid receptors (GABA_ARs) are pentameric ligand-gated ion channels (pLGICs), which mediate the majority of fast inhibitory neurotransmission in the animal central nervous system. Their dysfunction is related to numerous conditions including epilepsy, insomnia, anxiety, panic disorders, depression and schizophrenia. GABA_ARs are therefore major targets of clinically important drugs, including benzodiazepines and the intravenous general anaesthetics etomidate and propofol, as well as endogenous modulators, for example neurosteroids. Despite recent progress in structural biology of pLGICs, GABA_AR structures remain notoriously elusive. Structural information available at the beginning of this project was limited to the benzamidine-bound homopentameric GABA_AR-Î23, in a desensitised conformation. A large number of fundamental questions, including the molecular architecture of physiological, heteromeric GABA_ARs, their signalling mechanisms, the binding and action modes of their numerous ligands, remained to be answered. During this DPhil project, I employed structural biology techniques (X-ray crystallography and single particle cryo-electron microscopy) to further the molecular understanding of human GABAARs. I used subunit-specific llama nanobodies to aid crystallization of homomeric GABA_A-β3 receptors, which led to a 3.16 Å structure in complex with the general anaesthetic etomidate. This structure elucidates the binding mode of the etomidate, the basis for its subunit selectivity and illustrates conformational changes it triggers. I then used cryo-electron microscopy to determine the first structure of a heteromeric GABA_AR, the human α1b3g2, bound to an activating llama nanobody at a medium (5.2 Å) resolution. The numerous other insights obtained range from unambiguously establishing the subunit arrangement and stoichiometry, to proposing a mechanism for receptor assembly and discovering an unexpected role played by N-linked glycans in this process. The work described here opens multiple avenues for future research. Immediate opportunities include high resolution structural characterization of heteromeric GABA_ARs, via cryo-electron microscopy, further development of nanobodies as novel, high affinity and subunit specific tools to modulate GABA-ergic signalling, and structural characterization of numerous small-molecule modulators, of clinical and physiological relevance, bound to human GABA_ARs.

Many parasites have evolved to release products that inhibit host defence mechanisms such as enzymes in the mammalian host, in order to promote and sustain their survival within the host. The human filarial nematode Brugia malayi produces larval microfilariae, which circulate in the blood stream. Their most abundant secreted product is a serine protease inhibitor Bm-SPN-2. Serine protease inhibitors (Serpins) are reported to be involved in how the nematodes avoid host immune defences, and in the case of Bm-SPN-2, the protein was found to specifically inhibit the enzymatic activity of human neutrophil elastase and cathepsin G in a dose-dependent manner. More recently, these two enzymes have been linked to the activation of a major innate cytokine IL-33, which is stored as a full-length 270-aa protein in the cell nucleus, and released as an active C-terminal domain upon stimulation. As full-length (FL) human and murine IL-33 are not commercially available, soluble murine and human FL-IL-33 were produced in transfected HEK 293T cells, following mutation of the nuclear binding motif. In this form, IL-33 is no longer retained in the nucleus and can be purified as a soluble protein. It was confirmed that once cleaved, recombinant human IL-33 was able to induce significant IL-6 secretion by mast cells. Bm-SPN-2 was then shown to block human full-length IL-33 cleavage by inhibiting human neutrophil cathepsin G in a dose dependent manner, supporting the hypothesis that Bm-SPN-2 may act in vivo to prevent IL-33 activation and the promotion of the TH2 immune response. However, in the in vivo setting, it was unexpectedly found that IL-33R (ST2) gene deficiency did not enhance the survival of B. pahangi microfilariae. Furthermore, in the absence of IL-33R, murine immune responses to microfilariae were not significantly altered compared to wild-type BALB/c mice, other than in a significant increase in IL-33 expression. Hence while Bm-SPN-2 can act in vitro to forestall one of the key events in TH2 induction, this has not yet been shown to be crucial to the immune response to the parasite in vivo.

Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.

Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.