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The heterogeneity of response to SARS-CoV-2 infection is directly linked to the individual genetic background. Genetic variants of inflammasome-related genes have been pointed as risk factors for several inflammatory sterile and infectious disease. In the group of inflammasome receptors, NLRP1 stands out as a good novel candidate as severity factor for COVID-19 disease.To address this question, we performed an association study of NLRP1, DPP9, CARD8, IL1B, and IL18 single nucleotide variants (SNVs) in a cohort of 945 COVID-19 patients.The NLRP1 p.Leu155His in the linker region, target of viral protease, was significantly associated to COVID-19 severity, which could contribute to the excessive cytokine release reported in severe cases.Inflammasome genetic background contributes to individual response to SARS-CoV-2.

Communications biology 5(1), 805 (2022). doi:10.1038/s42003-022-03737-7, SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections., Published by Springer Nature, London

Bioactive peptides can provide health benefits due to different mechanisms. The aims of the present study are to produce bioactive peptides from bovine and goat milk subjected to the proteolytic activity of Aspergillus oryzae and Aspergillus flavipes enzymes, as well as to assess their putative antimicrobial and antioxidant activity. Bioactive peptides were successfully generated from proteases of fungi cultivated in solid-state fermentation. The generated peptides were effective against all tested bacteria and fungi. There was antioxidant activity, up to 92.5% DPPH reduction and ORAC stabilization at 52.5 μmol μL−1 of Trolox Equivalent. The generation of milk-specific sequences peptides in the samples was obtained through 2D-PAGE fractioning followed by mass spectrometry (ESI-MS/MS). Based on results in the present study, milk bioactive peptides presenting broad antimicrobial action and antioxidant activity spectra can be cost-effectively produced through solid-state fermentation. The herein addressed approach can be valuable for the pharmaceutical and food industries.

Tesis doctoral.-- Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Farmàcia), The present thesis has established a new and state of the art mammalian protein expression system at the laboratory which has then contributed with high-yield expression for the purification and crystallization of three glycoproteins, namely TGFβ2, ADAMTS13 and Neprosin, which adopt important biomedical roles in human physiology, cardiovascular disorders, and celiac disease treatment, respectively. Proteases are major players in the physiology and pathology of all living organisms. Their often highly sophisticated regulation mechanisms are therefore essential for proper function, and to prevent misdirected spatial and/or temporal proteolytic activity, which in turn may trigger disease. This regulation is achieved through a wide variety of mechanisms including protein-protein interaction, substrate allosteric activation, or their biosynthesis as inactive precursor, so called zymogens. In the present thesis a variety of regulatory mechanisms of proteases and inhibitors were studied by using a combination of biochemical, biophysical and structural techniques. In the first project we comprehensively illuminate the biochemistry of the VWF:ADAMTS-13 axis. ADAMTS13 regulates the multimeric form of Von Willebrand Factor (VWF) in blood circulation by specific, shear-dependent proteolysis. Despite circulating in a seemingly latent form with a very long active plasma half-life, ADAMTS13 is resistant to plasma inhibitors and VWF is the only known substrate, and which involves an allosteric activation of ADAMTS13 by substrate binding. This unprecedented specificity has been attributed to extensive exosite interactions between the AD13-MDTCS domains, where the Disintegrin-like domain (D) approaches to Metalloprotease (M), Cysteine-rich (C), and Spacer (S) domain once the VWF is bound achieving a “tight” conformation. Structural analysis in solution revealed that the enzyme adopts a highly flexible unbound, latent structures and VWF peptide-bound, active structures that significantly differes from the AD13-MDTCS crystal structure. We integrate the experimental results with computational approaches. Thus, we hypothesize that the interaction culminates in a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and ultimately in cleavage. In the second project a novel family of glutamate peptidases was studied and the crystal structure determined of both the active and zymogen form, establishing the active site, catalytic mechanism and protease class annotation, and describing a unique catalytic dyad composed by two glutamates while showing a close structural homology with eqolisins.

Orientador: Prof Dr. Alex Maiorka Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa : Curitiba, 04/02/2021 Inclui referências Resumo: O farelo de soja (FS) é uma importante fonte de proteína em alimentos, entretanto, não possui uma composição nutricional homogênea. Este importante ingrediente é uma importante fonte de antinutrientes proteicos dietéticos e apresenta variações nas suas características químicas e físicas. A adição de enzimas exógenas, como a fitase e a protease, reduz a influência negativa de antinutrientes proteicos e do fitato. O objetivo deste estudo foi avaliar a suplementação de dietas de frango de corte com altos níveis de fitase e a utilização de FS com diferentes conteúdos proteicos no desempenho, digestibilidade ileal dos nutrientes, mio-inositol plasmático, liberação de fosfatos in vitro, e composição mineral óssea, e avaliar o efeito de diferentes níveis de proteína bruta e aminoácidos em dietas de frangos de corte com ou sem inclusão de uma protease exógena. Foram alojados um total de 1.200 pintinhos de um dia de idade, provenientes de um incubatório comercial, alojados em unidades experimentais de 2,06 m2 (12,1 frangos de corte/m2), equipados com bebedouros nipple e comedouros tubulares. As aves receberam ração e água ad libitum. As dietas foram baseadas em milho e farelo de soja, fareladas. O estudo foi dividido em dois, sendo que no primeiro experimento, os tratamentos dietéticos foram um arranjo fatorial completo 2 x 2, que consistiu em 2 farelos de soja com diferentes concentrações de proteína (45% e 47% de proteína bruta), e 2 doses de fitase (1.000 e 2.500 unidades de fitase/kg - FYT), totalizando quatro tratamentos dietéticos com 12 repetições de 25 aves cada e divididas em duas fases de: fase inicial (1 a 21 dias) e fase de crescimento/terminação (22 a 42 dias). No segundo experimento, os boxes com os pintinhos foram distribuídos em um arranjo fatorial 2x2 inteiramente casualizado. Os fatores avaliados consistiram em 2 dietas: uma dieta basal e uma dieta reduzida (com redução dos níveis de proteína bruta e aminoácidos); e 2 doses de suplementação de protease: com ou sem a suplementação exógena de 15.000 unidades de protease (PROT)/kg; totalizando 4 tratamentos com 12 repetições de 25 aves cada. Em conclusão, maiores doses de fitase para frangos de corte melhoram o desempenho, o mioinositol, e a composição mineral óssea. A digestibilidade ileal dos nutrientes pode ser aumentada com doses de fitase mais elevadas quando em combinação com farelo de soja de maior qualidade nutricional. As dietas à base de milho e farelo de soja com menor teor de proteína bruta e aminoácidos tiveram efeitos negativos sobre o ganho de peso e conversão alimentar de frangos de corte de 1 a 42 dias de idade. Aos 42 dias, o rendimento de carcaça e de peito foi reduzido, e a porcentagem de gordura abdominal aumentada com dietas de baixos níveis de proteína bruta e aminoácidos. A inclusão de 15000 PROT/kg de uma protease exógena não teve efeito sobre o desempenho de crescimento e variáveis de carcaça, mas melhorou a solubilidade da proteína dietética in vitro. Palavras-chave: Farelo de Soja, Fitase, Fósforo, Protease, Proteína. Abstract: The soybean meal is an important source of protein in feed, however, it does not have a homogeneous nutritional composition. This important ingredient is an important source of dietary protein antinutrients and has variations in its chemical and physical characteristics. The addition of exogenous enzymes such as phytase and protease reduces the negative influence of protein antinutrients such as trypsin inhibitors and phytate. The objective of this study was to evaluate the supplementation of broiler diets with high levels of phytase and the use of soybean meal with different protein contents in the performance, ileal digestibility of nutrients, plasma myo-inositol, phosphate release in vitro, and bone mineral composition, and to evaluate the effect of different levels of crude protein and amino acids in broiler diets with or without the inclusion of an exogenous protease. A total of 1,200 day old chicks from a commercial hatchery were housed in experimental units of 2.06 m2 (12.1 broilers/m2, equipped with nipple drinkers and tubular feeders. The chickens received feed and water ad libitum. The diets were based on corn and soybean meal. The study was divided in two, and in the first experiment, the dietary treatments were a complete 2 x 2 factor arrangement, consisting of 2 soybeans meal with different protein concentrations (45% and 47% crude protein), and 2 doses of phytase (1,000 and 2,500 units of phytase/kg - FYT), totaling four dietary treatments with 12 repetitions of 25 birds each and divided into two phases: initial phase (1 to 21 days) and growth/finisher phase (22 to 42 days). In the second experiment, the boxes with the chicks were distributed in a 2x2 factorial arrangement entirely randomized. The factors evaluated consisted of 2 diets: a basal diet and a reduced diet (with reduction of crude protein and amino acids levels); and 2 doses of protease supplementation: with or without exogenous supplementation of 15,000 units of protease (PROT)/kg; totaling 4 treatments with 12 repetitions of 25 birds each. In conclusion, higher doses of phytase for broilers improve performance, the supply of myo-inositol, and the bone mineral composition. The ileal digestibility of nutrients can be increased with higher doses of phytase when combined with higher nutritional quality soybean meal. Corn-based diets and soybean meal with lower crude protein and amino acid content had negative effects on weight gain and feed conversion of broilers from 1 to 42 days of age. At 42 days, carcass and breast yields were reduced, and the percentage of abdominal fat increased with low levels of crude protein and amino acids diets. The inclusion of 15000 PROT/kg of an exogenous protease had no effect on growth performance and carcass variables, but improved the solubility of dietary protein in vitro. Key-words: Phosphorus, Phytase, Protease, Protein, Soybean Meal.

[eng] The present thesis has established a new and state of the art mammalian protein expression system at the laboratory which has then contributed with high-yield expression for the purification and crystallization of three glycoproteins, namely TGFβ2, ADAMTS13 and Neprosin, which adopt important biomedical roles in human physiology, cardiovascular disorders, and celiac disease treatment, respectively. Proteases are major players in the physiology and pathology of all living organisms. Their often highly sophisticated regulation mechanisms are therefore essential for proper function, and to prevent misdirected spatial and/or temporal proteolytic activity, which in turn may trigger disease. This regulation is achieved through a wide variety of mechanisms including protein-protein interaction, substrate allosteric activation, or their biosynthesis as inactive precursor, so called zymogens. In the present thesis a variety of regulatory mechanisms of proteases and inhibitors were studied by using a combination of biochemical, biophysical and structural techniques. In the first project we comprehensively illuminate the biochemistry of the VWF:ADAMTS-13 axis. ADAMTS13 regulates the multimeric form of Von Willebrand Factor (VWF) in blood circulation by specific, shear-dependent proteolysis. Despite circulating in a seemingly latent form with a very long active plasma half-life, ADAMTS13 is resistant to plasma inhibitors and VWF is the only known substrate, and which involves an allosteric activation of ADAMTS13 by substrate binding. This unprecedented specificity has been attributed to extensive exosite interactions between the AD13-MDTCS domains, where the Disintegrin-like domain (D) approaches to Metalloprotease (M), Cysteine-rich (C), and Spacer (S) domain once the VWF is bound achieving a “tight” conformation. Structural analysis in solution revealed that the enzyme adopts a highly flexible unbound, latent structures and VWF peptide-bound, active structures that significantly differes from the AD13-MDTCS crystal structure. We integrate the experimental results with computational approaches. Thus, we hypothesize that the interaction culminates in a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and ultimately in cleavage. In the second project a novel family of glutamate peptidases was studied and the crystal structure determined of both the active and zymogen form, establishing the active site, catalytic mechanism and protease class annotation, and describing a unique catalytic dyad composed by two glutamates while showing a close structural homology with eqolisins. Neprosin was recently discovered in the fluid of the carnivorous pitcher plant Nepenthes ventrata. Given its high activity at low pH, thermic stability and great specificity, neprosin is especially useful in detoxifying gluten proteins including wheat gliadin and the 33-mer immunogenic fragment into non-toxic peptides in a human stomach environment. Additionally, we determined its inhibitory profile against a cohort of peptidase inhibitors. Future approaches will study the effect of neprosin processing of the 33-mer on inflammatory responses in cells and tissue explants to elucidate if neprosin indeed presents an effective and harmless supplementation approach for gluten intolerant patients. In the third project the main protease inhibitor of the blood plasma has been studied; human α2 Macroglobulin (hα2Ms) has a great role on the human physiology by modulating the activity of cytokines such as TGFβ2. We characterised the interaction by a variety of biochemical, biophysical, and binding techniques. Intriguingly, during our crystallographic studies of the complex, we obtained a crystal structure of a TGFβ2 dimer in a novel dimeric assembly of biological unknown function. Overall, the present thesis contributes substantially to the field of structural biochemistry, expanding previous knowledge at the molecular level and adding new regulatory mechanisms, which will hopefully pave the way for the design of specific drugs for therapeutic interventions as well as lead to new approaches for treating coeliac disease through an enzyme supplementation strategy.

[eng] The plant pathogen Ralstonia solanacearum is the causal agent of the devastating disease known as bacterial wilt. This disease affects more than 200 plant species in over 50 families, including economically important crops such as tomato, potato, banana or pepper. R. solanacearum is a soil-borne and vascular pathogen. It enters its hosts through wounds in the roots and lateral root emerging sites. Then, the pathogen traverses the cortical apoplast until it reaches the vascular cylinder and colonizes to very high numbers the xylem vessels. The combination of bacterial growth and the secretion of a mucus-like exopolysaccharide clogs the infected vessels, blocking its water flow and causing the wilt disease symptoms that lead to the dead of the plant. To date, the most reliable management strategy to control R. solanacearum has been the use of genetic resistance. In this thesis we attempted to shed light on the old question of bacterial wilt resistance, which has haunted plant epidemiologists for more than one hundred and twenty years, using novel proteomic techniques. We thought that the best way to address this challenge was to follow a “divide and conquer” approach: by identifying the key bottlenecks of resistance (Chapter 1), we would be able to precisely dissect which proteins are involved in different tissues and stages of the infection (Chapters 2 and 3). Finally, in Chapter 4 we delved into the tomato P69 family of subtilisin-like proteases, as many of its members appeared upregulated during infection. In Chapter 1 we identified the plant tissues and organs that represented major constraints during R. solanacearum colonization. Using simple and very visual tools –bacterial reporter strains and plant grafting–, we highlighted the root system as the first and most conspicuous barrier. The roots consist of an epidermis and a cortical parenchyma that R. solanacearum must traverse before reaching the vasculature, a movement that occurs through the intercellular space or apoplast. In this chapter, we showed how the roots of resistant plants (H7996) effectively prevented R. solanacearum entry and invasion, single-handedly reducing the colonization and consequent development of wilt disease symptoms in grafted plants. A reduction in colonization was also evident within the xylem vessels of H7996. Grafting susceptible roots onto resistant hypocotyls allowed us to confront the resistant stems with high bacterial inoculum using the conventional –and more natural– soil-drench inoculation method. Once the bacteria faced the resistant tissue, a decrease in colonization was observed, indicating that H7996 can also restrict R. solanacearum movement/growth along the xylem in a root-independent manner. In addition, we observed how this resistant tomato variety impeded the escape of R. solanacearum from colonized xylem vessels towards the apoplastic space of the neighboring parenchymatic cells, a situation clearly promoted in susceptible plants (Marmande) and that facilitated tissue decay in the latest stages of infection. Altogether, the data presented in this chapter highlighted these two plant environments, the apoplast and the xylem, as the two main battlefields of infection. In Chapters 2 and 3 we used quantitative mass spectrometry approaches to investigate the apoplastic and xylem sap proteomes in response to R. solanacearum. In Chapter 2 we focused on the active proteome of the apoplast using activity-based molecular probes that covalently bound to the active site of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs). Upon infection, the activity of PLCPs and SHs was higher in the apoplast of H7996 than in Marmande. We found that two well-known PLCPs, Rcr3 and Pip1, and many SHs from distinct protease families experienced significant changes after R. solanacearum inoculation and in the comparison of the two tomato varieties. Additionally, a protein network analysis suggested that the apoplastic proteome of H7996 might be more naturally prepared to face intruders, as many protein interactions did not substantially change upon infection. In Chapter 3 we focused on the xylem sap proteome and, similar to the apoplast, we observed many differentially accumulated proteins (DAPs) in response to infection. A clear “downregulation” was detected in Marmande, which could either be due to a deliberated inhibition caused by R. solanacearum or to tissue damage resulting from the infection process. On the other hand, cell wall modifying enzymes, including peroxidases and glycoside hydrolases (GHs), were overaccumulated in H7996. Moreover, the overaccumulation of many tomato and R. solanacearum GHs both in the apoplast and the xylem highlighted the importance of the cell wall as an effective barrier in plant-pathogen interactions, particularly against R. solanacearum. Finally, in the survey of both proteomes –apoplast and xylem–, we identified members of the P69 family of subtilases to overaccumulate upon R. solanacearum infection. This 10 gene family of proteases was comprehensibly addressed in Chapter 4. We analyzed the conservation of this family and found that they are only present in Solanaceae, being P69A and P69D the two most conserved P69 genes. P69s were not only induced by R. solanacearum but responded to unrelated plant pathogens, as illustrated by several transcriptomic and proteomic studies. Additionally, their overall high percentage of identity (>71%) appeared to partially translate in their cleavage specificity, although some particularities could be drawn for P69A, P69B and P69D. Conversely, substrate specificity might also be determined by their differential post-translational N-glycosylation status, and/or by the higher sequence divergence within their protease- associated domain, which could influence protein-protein interactions. Finally, a CRISPR/Cas9 mutagenic approach was conducted to obtain a P69D single mutant and two separate P69-cluster deletion mutants. We assayed the susceptibility of the P69D single mutant and found a slight yet significant increase in susceptibility, probably caused by an increased growth of the pathogen in its root system. The potential role of P69D in defense and other aspects regarding P69 subtilases are addressed., [spa] El fitopatogen Ralstonia solanacearum és el bacteri causant de la devastadora malaltia coneguda com a marciment bacterià. Aquesta malaltia afecta més de 200 espècies de plantes de 50 famílies diferents, incloent importants plantes de cultiu com la tomaquera o la patatera. R. solanacearum entra als seus hostes a través de petites ferides en les arrels o en els punts en que emergeixen les arrels laterals. Un cop ha penetrat l’arrel, R. solanacearum es mou seguint una via apoplastica –a través dels espais intercel·lulars– fins que arriba als feixos vasculars de la planta, on colonitza el xilema. En el xilema, aquest bacteri es multiplica en grans quantitats i comença a secretar una substància mucosa que acaba col·lapsant el feix vascular. La falta d’un flux hidràulic continu provoca l’aparició dels clàssics símptomes de marciment, que desemboquen en la mort de la planta. Ara per ara, la millor estratègia per controlar R. solanacearum és l’ús de varietats de cultius resistents. En aquesta Tesi Doctoral hem utilitzat noves tècniques d’anàlisi de proteïnes per caracteritzar la resposta de les plantes resistents a R. solanacearum, utilitzant la planta de la tomaquera com a model. Primerament hem caracteritzat quins són els principals colls d’ampolla que restringeixen la colonització del patogen en tomaqueres resistents, identificant els espais intercel·lulars –o apoplast– i el xilema com els camps de batalla més importants entre la planta i R. solanacearum (Capítol 1). Posteriorment (Capítols 2 i 3), hem analitzat el context proteic d’aquests dos ambients en resposta al patogen comparant una varietat susceptible (Marmande) amb una de resistent (H7996). Finalment, en el Capítol 4, hem aprofundit en la caracterització d’una família de proteases concreta, conegudes com a P69, ja que certs membres de la família s’acumulaven tant en l’apoplast com el xilema en resposta a R. solanacearum. En el Capítol 1 hem identificat quins teixits i òrgans restringeixen el moviment de R. solanacearum dins la planta. Utilitzant soques reporteres del bacteri –emissores de llum o fluorescents– i la tècnica de l’empelt, hem observat com l’arrel és la primera i més important barrera contra el patogen. Així, les arrels de plantes resistents eren capaces de limitar l’entrada del patogen a la planta o bé reduir-ne la multiplicació. Empeltant arrels susceptibles en tiges resistents, hem aconseguit infectar les tiges de plantes resistents amb un inòcul bacterià molt més gran del normal –similar al que es troben les plantes susceptibles–. En fer-ho hem observat una disminució en la colonització dins dels vasos xilemàtics de la part resistent, indicant que en la tija actuarien uns mecanismes de defensa independents de l’arrel. A més, hem pogut observar com, la varietat resistent impedeix que el bacteri s’escapi dels feixos vasculars infectats. Aquest fet no es dona en plantes susceptibles, on el bacteri envaeix els espais intercel·lulars de les cèl·lules parenquimàtiques circumdants. Finalment, hem definit un llindar de massa bacteriana en l’hipocòtil per sobre de la qual les plantes es marceixen. Un cop identificats l’apoplast i el xilema com a principals camps de batalla entre la planta i R. solanacearum, en els Capítols 2 i 3 hem caracteritzat la resposta proteica de la planta en aquests dos compartiments. En el Capítol 2 s’ha investigat l’activitat de cisteïna i serina proteases (PLCP i SH, respectivament, de les seves sigles en anglès) de l’apoplast. En resposta a la infecció, la varietat resistent mostra un increment en l’activitat d’aquestes famílies de proteases. Mitjançant espectrometria de masses hem identificat canvis en l’acumulació de 4 PLCPs i 27 SHs en les dues varietats davant la infecció. Entre les PLCPs, hem identificat les ja caracteritzades Rcr3 i Pip1. A més, moltes SHs encara no han estat caracteritzades. Addicionalment, amb la construcció d’una xarxa d’interaccions proteiques hem pogut denotar com la varietat resistent, al contrari de la susceptible, experimenta menys canvis topològics davant la infecció. Aquests fets indicarien que aquesta varietat està més preparada per respondre al patogen en el seu estat basal. En el Capítol 3 ens hem endinsat en l’estudi proteòmica del xilema. Com en l’apoplast, també s’ha observat que moltes proteïnes s’acumulen de forma diferent en resposta a la infecció. És més, s’ha observat una clara disminució de proteïnes secretades en les plantes susceptibles, que pot ser deguda a una inhibició deliberada de la resposta immunològica per part de R. solanacearum, o bé al mateix dany tissular causat pel seu creixement. Per altra banda, davant la infecció s’observa una clara acumulació xilemàtica de peroxidases, glicosil hidrolases i proteases, cosa que reforça la importància de l’enfortiment dels vasos i les parets cel·lulars com un dels mecanismes claus per combatre la infecció. Finalment, tant en l’apoplast com el xilema hi hem identificant membres de la família de subtilases coneguda com a P69. Aquestes proteases s’acumulen en els dos compartiments davant la infecció. La seva caracterització es presenta al Capítol 4. Es tracta d’una família de 10 gens (en tomaquera) altament conservats, ja que les seqüències de les proteïnes comparteixen més d’un 71% d’identitat. S’ha constatat que les P69s són específiques de solanàcies, tot i que no totes es troben conservades en les espècies que hem analitzat (patatera, alberginiera, pebrotera i tabac). No només s’acumulen per la infecció de R. solanacearum, sinó que altres patògens també en provoquen la inducció. A més, analitzant l’especificitat de tall, s’ha observat que poden tallar seqüències similars, tot i que P69A, P69B, P69D presenten certes especificitats. Un anàlisis in silico ha determinat que aquestes proteïnes pateixen diferents modificacions post-traduccionals en forma de N-glicosilació, cosa que podria afectar la seva especificat de substrat en la mateixa planta. Finalment, s’ha utilitzat la tecnologia CRISPR/Cas9 per crear delecions en un gen concret (P69D, ja que només es va detectar actiu en plantes resistents), o bé eliminar per deleció el màxim nombre de gens de la família. S’ha caracteritzat una lleugera susceptibilitat a R. solanacearum per part del mutant per P69D, que probablement s’atribueix a una facilitat per part del patogen de colonitzar el sistema radicular.

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

Las enzimas proteolíticas vegetales desempeñan un papel decisivo en muchos procesos, tanto biológicos, como fisiológicos a lo largo del ciclo de vida de la planta. Una de las especies rica en este tipo de enzimas es Ananas comosus, proporcionándole numerosas propiedades a la especie botánica que favorecen su uso a nivel culinario, medicinal y en la industria farmacéutica. Estas enzimas se encuentran en un extracto acuoso presente en el tallo y/o fruto de la piña conocido como Bromelina y concretamente el extracto acuoso extraído del tallo presenta propiedades muy interesantes para el ámbito farmacéutico, donde destacamos su aplicación tópica en quemaduras, donde ejerce un papel como desbridante, eliminando el tejido dañado o necrótico. La aprobación en 2014 por la AEMPS de un medicamento huérfano llamado NexoBrid®, basado en un concentrado de enzimas proteolíticas enriquecido en Bromelina, demuestra el potencial clínico de Bromelina en quemaduras, ya que actualmente se está empezando a emplear como posible alternativa al desbridamiento quirúrgico en el tratamiento de ciertas quemaduras. El objetivo general de este trabajo es investigar el uso tópico de Bromelina en quemaduras, analizando su eficacia y seguridad como agente desbridante, así como las ventajas e inconvenientes frente a otros tratamientos similares, y de manera más específica, recopilar y analizar la información existente, sobre la utilidad clínica y la seguridad de NexoBrid®. Para ello, se ha llevado a cabo una búsqueda bibliográfica en las principales bases de datos biomédicas sobre los estudios más destacados que evaluasen la efectividad y seguridad de NexoBrid® en quemaduras, acotando dicha búsqueda a los últimos 5 años. Tras introducir las palabras clave en las bases de datos y aplicar los criterios de inclusión y de exclusión, se seleccionaron finalmente 10 artículos para su estudio. Según los artículos revisados, se concluye que NexoBrid® ha demostrado ser un medicamento seguro y eficaz en quemaduras de origen térmico, profundas, de segundo (espesor parcial), de tercer grado (espesor total) y mixtas, presentando una serie de ventajas potenciales frente al desbridamiento quirúrgico, confirmando los beneficios de este producto natural. De esta manera, demuestra ser una posible alternativa al desbridamiento convencional empleado en este tipo de quemaduras. Universidad de Sevilla. Grado en Farmacia

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Ginger (Zingiber officinale Roscoe) is a medicinal plant known for its anti-inflammatory, antitumoral, antioxidant and antimicrobial properties. The objective of this study was to optimize the extraction of sesquiterpenes (α-curcumene, α-zingiberene, α-farnesene, β-sesquifelandrene and β-bisabolene), to evaluate their antimicrobial and antibiotic potential against Staphylococcus aureus, Streptococcus mutans, S. agalactiae, Escherichia coli and Pseudomonas aeruginosa, in addition to their antiproteic activity. Analyzes of the extracted compounds were performed by GC-MS. Antimicrobial and biofilm formation assays were performed by the 96 well plate microdilution technique. In addition to assessing biofilm inhibition, the hydrophobicity index was also determined as an indirect way of assessing the ability of microorganisms to adhere to host tissues and abiotic surfaces. The best conditions for obtaining the sesquiterpene-enriched fraction (SEF) were: extraction time: 4 min, plant / solvent ratio: 1:20 (m / v) and microwave power: 400 W. The SEF presented a MIC of 2000 μg / mL compared to S. mutans and was ineffective in inhibiting the growth of other bacteria. Nevertheless, SEF (250 μg / mL) inhibited the biofilm formation of S. aureus, S. agalactiae and E. coli by 50%, 80% and 80%, respectively. In addition, and E. coli bacteria by 61.86%, 73.44% and 49.10%, respectively. FES (500 μg / mL) was able to reduce papain enzymatic activity by 71.5%, with an IC50 of 386.8 μg / mL. The results showed that SEF had potential to combat the production of bacterial biofilms. Thus, these substances are good candidates for drug prototypes used to treat infections caused by multi-resistant bacteria. To our knowledge, this is the first time that the SEF antimicrobial and antibiofilm activity has been studied. O gengibre (Zingiber officinale Roscoe) é uma planta medicinal conhecida por suas propriedades anti-inflamatórias, antitumorais, antioxidantes e antimicrobianas. O objetivo deste trabalho foi otimizar a extração de sesquiterpenos (α-curcumeno, α-zingibereno, α-farneseno, β-sesquifelandreno e β-bisaboleno), avaliar seu potencial antimicrobiano e antibiofilme contra Staphylococcus aureus, Streptococcus mutans, S. agalactiae, Escherichia coli e Pseudomonas aeruginosa, além da sua atividade antiproteásica. As análises dos compostos extraídos foram realizadas por CG-ES. Os ensaios antimicrobianos e formação de biofilmes foram realizados pela técnica de microdiluição em placas de 96 poços. Além de avaliar a inibição do biofilme, o índice de hidrofobicidade também foi determinado como uma forma indireta de avaliar a capacidade de adesão dos micro-organismos aos tecidos hospedeiros e superfícies abióticas. As melhores condições para obtenção da fração enriquecida em sesquiterpenos (FES) foram: tempo de extração: 4 min, razão planta/solvente: 1:20 (m/v) e potência de micro-ondas: 400 W. A FES apresentou CIM de 2000 μg/mL frente a S. mutans e foi inefetivo na inibição do crescimento das outras bactérias. Apesar disso a FES (250 g/mL) inibiu a formação de biofilme de S. aureus, S. agalactiae E. coli em 50%, 80% e 80%, respectivamente. Ademais, a FES (250 g/mL) reduziu o índice de hidrofobicidade das bactérias S. aureus, S. agalactiae e E. coli em 61,86%, 73,44% e 49,10%, respectivamente. A FES (500 μg/mL) foi capaz de reduzir em 71,5% a atividade enzimática da papaína, apresentando IC50 386,8 μg/mL. Os resultados mostraram que a FES apresentou potencial no combate à produção de biofilmes bacterianos. Desse modo, essas substâncias representam-se como bons candidatos a protótipos de medicamentos utilizados no tratamento de infecções causadas por bactérias multirresistentes. Até onde sabemos, esta é a primeira vez que a atividade antimicrobiana e antibiofilme da FES foi estudada. São Cristóvão, SE

Fish viscera are part of the solid waste discarded after consumption and processing of these animals. However, they can be utilized as a rich resource of enzymes. The traíra, Hoplias malabaricus (Bloch, 1794), little studied fish and having scarce of information about the enzymes produced by it, is a fish with great potential to be explored. In addition, the enzyme trypsin can be isolated from the traíra and present application in the scientific and technological field. However, it is necessary to know its structural and biological characteristics. With this information, this work pursued the purification and characterization of trypsin from traíra. With the traíra’s pyloric cecum was made a crude extract, which was fractionated by the precipitation with organic solvent and the precipitation with ammonium sulphate. Enzymatic hydrolysis tests were performed with the substrate N-α-benzoyl-L-arginine-4-nitroanilide (BApNA) in all fractions of the precipitations and it was observed that the fraction 20-40% of the saline precipitation had higher activity. Gel filtration chromatography (Sephacryl S-100 – 60 x 1 cm) of the 20-40% fraction showed a peak with high trypsin hydrolytic activity and low protein concentration, in others words, high specific activity. SDS-PAGE demonstrated the isolation of trypsin after saline precipitation and gel filtration on the Sephacryl S-100 column. . The enzyme isolated was characterized and showed high activity at pH 9.0 and temperature of 50 ºC, besides having inhibition of 98,95% by phenylmethylsulfonyl fluoride (PMSF), 94.04% by the soybean trypsin inhibitor (SBTI) and 94,74% by benzamidine, showing to be a serine protease The kinetic constant for this enzyme had Km with 1.62 mM and Vmax with 0.69 mM s-1. It is concluded in this work that trypsin from traira was partially isolated by two purification methods and that the enzyme showed catalytic efficiency in temperature and pH conditions distinct from others trypsins isolated from fish, thus demonstrating great potential for possible biotechnological applications. CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As vísceras de peixes são parte dos resíduos sólidos descartados após o consumo e processamento desses animais. No entanto, elas podem ser aproveitadas como recurso rico de enzimas. A traíra, Hoplias malabaricus (Bloch, 1794), peixe pouco estudado e com poucas informações sobre enzimas produzidas, representa um grande potencial a ser explorado. Além disso, a enzima tripsina pode ser isolada da traíra e apresentar aplicação no âmbito científico e tecnológico. Porém, é necessário antes conhecer as características estruturais e biológicas da enzima de interesse. Diante dessas informações, esse estudo buscou a purificação e caracterização a tripsina da traíra. Com o ceco pilórico da traíra foi feito o extrato bruto, que foi fracionado pela precipitação com etanol e a precipitação com sulfato de amônia. Foram realizados testes de hidrólise enzimática com o substrato N-α-benzoil-L-arginina-4-nitroanilida (BApNA) em todas as frações das precipitações e foi observado que a fração 20-40% da precipitação salina teve maior atividade. A cromatografia de gel filtração Sephacryl S-100 (60x1 cm) da fração 20-40%, mostrou um pico com alta atividade hidrolítica de tripsina e baixa concentração de proteína, ou seja, alta atividade específica. O SDS-PAGE demonstrou o isolamento da tripsina após precipitação salina e gel filtração na coluna Sephacryl S-100. A enzima parcialmente isolada foi caracterizada e apresentou pH ótimo 9,0 e temperatura ótima de 50 ºC, além de ter inibição de 98,95% por fluoreto de fenilmetilsulfonil (PMSF), 94,04% pelo inibidor da tripsina da soja (SBTI) e 94,74% pela benzamidina, mostrando ser uma serinoprotease. A constante cinética para essa enzima teve Km de 1,62 mM e Vmax de 0,69 mM s-1. Conclui-se nesse trabalho que a tripsina da traíra foi parcialmente isolada por dois métodos de purificação e que a enzima mostrou eficiência catalítica em condições de temperatura e pH distintas das demais tripsinas isoladas de peixes, assim demostrando ter grande potencial para possíveis aplicações biotecnológicas.

Conselho Nacional de Desenvolvimento Científico e Tecnológico Os experimentos foram conduzidos no Departamento de Zootecnia da Universidade Federal de Viçosa, UFV, campus de Viçosa, gerando informações apresentadas em dois capítulos. No primeiro capítulo, objetivou-se isolar e identificar bactérias do ácido lático em silagem de sorgo, quantificar por qPCR a população do L. buchneri, bem como, avaliar mudanças no perfil fermentativo e valor nutritivo de silagens de sorgo inoculadas com cepas autóctones. Os tratamentos foram dispostos em esquema fatorial 4 × 5, sendo quatro inoculantes (I), controle (CON), L. buchneri estirpe 50.1 (LB.1), L. buchneri estirpe 50.4 (LB.4) e LALSIL AS, L. buchneri CNCM I-4323, Lallemand (LAS) e cinco períodos de fermentação (7, 14, 28, 45 e 90 dias) no delineamento inteiramente casualizado, com três repetições. Foram obtidos quarenta e sete isolados, com predominância de L. buchneri após 45 e 90 dias de fermentação. Os dados do qPCR demonstraram que L. buchneri predominou durante todos períodos de fermentação (P) nas silagens inoculadas. Foi verificado efeito da interação I × P (P0,05), exceto nas doses de 1,2 e 1,5% da enzima, nas quais silagens de GS apresentaram maiores concentrações (P0,05) entre silagens de GM e GS. Silagens de GS apresentaram populações de LEV superiores (P0.05) LAB count between CG and SG silages at 60 and 90 d of fermentation. Sorghum grain silages showed greater yeast population (P

The growth of fish production over the years has contributed to obtain a large quantity of byproducts, among which we highlight the residue of the fish posting from the band saw machine, usually discarded in the environment, which contributes to environmental pollution and economic losses. The objective of this study was to obtain peptides from the by-product of fish posting in the band saw machine, characterizing the final product as regards nutritional and technological parameters. In the first step of the experiment, the physical and chemical characteristics of the by-products of three fish species were evaluated: “dourada” (Brachyplathystoma flavicans), "serra" (Scomberomorus brasiliensis) and "pescada-cambucu" (Cynoscion virescens) produced after machine sawing tape. After the characterization analyzes and considering the relation between the protein and lipid concentration, the "Serra" fish residue was selected for the preparation of the protein hydrolysate. By means of previous selection of the proteolytic enzyme, alcalaseÓ was used to determine the optimal condition to obtain the protein hydrolysate. According to the enzyme: substrate (w / w) and hydrolysis time (min) using a Central Compound Design Rotational (DCCR) 2². Then, hydrolysates with higher and lower hydrolysis (GH) were evaluated for nutritional and technological aspects. The chemical and physical characteristics of the byproducts suggest that the fish waste of the band saw machine consists mainly of the edible fraction of the fish, possessing proteins of high biological value. The optimum conditions for obtaining fish protein hydrolysate were 5.0% of the enzyme: substrate (w / w) ratio and 240 minutes of enzymatic hydrolysis. The hydrolysates (+ GH and - GH) of sawn fish showed volatile compounds with a characteristic fish odor, and can be used for flavoring. The hydrolysate -GH presented better performance in the technological properties of stabilizing emulsions and capacity to retain oil, due to its ability of fat biding capacity, and could be added in emulsified products to improve the technological and sensorial characteristics. With respect to the antioxidant capacity, the hydrolysate + GH showed greater efficiency due to the shorter chain length of its peptides, being indicated for use in food products with the objective of prolonging shelf life through the capacity to stabilize lipids and proteins of the food, guaranteeing the quality of the product throughout the storage. Nenhuma O crescimento da produção de pescado ao longo dos anos tem contribuído para obtenção de grande quantidade de subprodutos, dentre os quais destaca-se o resíduo do postejamento de peixes oriundo da máquina serra-fita, usualmente descartado no ambiente, o que contribui para poluição ambiental e perdas econômicas. Objetivou-se obter peptídeos a partir do subproduto do postejamento de peixes na máquina serra fita, caracterizando o produto final quanto aos parâmetros nutricionais e tecnológicos. Na primeira etapa do experimento, avaliou-se as características físicas e químicas dos subprodutos de três espécies de peixes: “dourada” (Brachyplathystoma flavicans), “serra” (Scomberomorus brasiliensis) e “pescada-cambucu” (Cynoscion virescens) produzidos após o postejamento em máquina serra-fita. Após as análises de caracterização e considerando-se a relação entre a concentração de proteínas e lipídeos, o resíduo do peixe “serra” foi selecionado para elaboração do hidrolisado proteico. Mediante seleção prévia da enzima proteolítica, a alcalase foi utilizada para determinar a condição ótima para obtenção do hidrolisado proteico, de acordo com a relação enzima:substrato (p/p) e o tempo de hidrólise (min) utilizando-se um Delineamento Composto Central Rotacional (DCCR) 2². Em seguida, os hidrolisados com maior e menor grau de hidrólise (GH) foram avaliados quanto aos aspectos nutricionais e tecnológicos. As características químicas e físicas dos subprodutos sugerem que os resíduos de peixes da máquina serra fita são constituídos majoritariamente pela fração comestível do pescado, possuindo proteínas de alto valor biológico. As condições ótimas para obtenção do hidrolisado proteico de peixe serra foram 5,0% da relação enzima:substrato (p/p) e 240 minutos de hidrólise enzimática. Os hidrolisados (+GH e -GH) de peixe serra apresentaram compostos voláteis com odor característico de peixe, podendo ser utilizados para elaboração de flavorizantes. O hidrolisado -GH apresentou melhor desempenho nas propriedades tecnológicas de estabilizar emulsões e capacidade de reter óleo, devido a sua habilidade em ligar-se a moléculas de gorduras, podendo ser inserido em produtos emulsionados para melhorar as características tecnológicas e sensoriais. Com relação a capacidade antioxidante, o hidrolisado +GH demonstrou maior eficiência advinda do menor comprimento das cadeias de seus peptídeos, sendo indicado para utilização em produtos alimentícios com o objetivo de prolongar sua vida útil através da capacidade de estabilizar lipídeos e proteínas do alimento, garantindo a qualidade do produto ao longo do armazenamento.

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251–259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo , when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome β5i subunit and to increase the proliferation of CD8 + T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity. Biological significance The functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.

Made available in DSpace on 2018-12-11T17:07:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-16 Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168 h with 760 U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72 h with 460 U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5–8 and at 55–60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu2+ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1 h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum. Department of Pharmaceutical Sciences School of Pharmaceutical Sciences of Ribeirao Preto University of Sao Paulo Department of Biology Institute of Biosciences Letters and Exact Sciences Sao Paulo State University UNESP/IBILCE Department of Biochemistry and Microbiology Sao Paulo State University UNESP Department of Biology Institute of Biosciences Letters and Exact Sciences Sao Paulo State University UNESP/IBILCE Department of Biochemistry and Microbiology Sao Paulo State University UNESP

Objective Although it is well established that a-calcitonin gene-related peptide (CGRP) stabilizes muscle-type cholinergic receptors nicotinic subunits (AChR), the underlying mechanism by which this neuropeptide regulates muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear. Methods To elucidate the mechanisms how CGRP controls NMJ stability in denervated mice skeletal muscles, we carried out physiological, pharmacological, and molecular analyses of atrophic muscles induced by sciatic nerve transection. Results Here, we report that CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca2+-dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP. Conclusion Taken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions., Highlights • CGRP exerts antiproteolytic effects in atrophic denervated muscles and maintains the stability of NMJ. • CGRP blocks the endocytosis of AChRs and decreases the colocalization of autophagosome to NMJ in denervated muscles. • CGRP inhibits the Ca2+-dependent proteolysis, a degradative system that destabilizes NMJ during muscle atrophy. • The inhibition of autophagy and calpain induced by CGRP represents an important mechanism for the preservation of synapse.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015. Proteases constituem um dos grupos de enzimas industriais mais importantes, com participação na indústria de detergentes, couro, alimentos e medicamentos. A indústria de detergentes é uma grande consumidora de enzimas hidrolíticas. Novas metodologias que possibilitem a purificação de biomoléculas são de grande interesse para a indústria pelas suas potenciais aplicações. O objetivo principal deste trabalho foi produzir, purificar e caracterizar proteases extracelulares de Penicillium restrictum cultivado sob fermentação submersa. A cepa de Penicillium restrictum isolada do solo do Cerrado foi capaz de produzir proteases extracelulares após 7 dias de cultivo a 28°C e 150 rpm em meio contendo farelo de trigo. Após os 7 dias de cultivo o filtrado do meio fermentado foi submetido ao processo de ultrafiltração em membrana de 100 kDa e a fração ultrafiltrada foi adicionada ao SMDFA em diferentes condições, sendo avaliada a purificação através de um planejamento fatorial 2³ com três repetições no ponto central. Os resultados mostraram que o SMDFA foi eficaz na partição das proteases presentes na fração < 100 kDa para a fase pobre em micelas, apresentando um coeficiente de partição (k) menor que 1 para todos os sistemas testados. O sistema 3 apresentou 80 de balanço de massas e 138,24% de rendimento. Baseado nos valores de k e nos valores do balanço de massa e do rendimento, o sistema contendo 10% Triton X114, 10% de caldo e separado a 31°C foi escolhido para dar continuidade ao processo de purificação da enzima de interesse. A fração pobre em micelas, foi ultrafiltrada em membrana de 30 kDa e o fator de purificação desta fração foi igual a 10,4. A purificação parcial de duas proteases extracelulares foi confirmada por SDS-PAGE seguido de zimograma. As bandas com atividade proteolítica revelaram pesos moleculares de aproximadamente 19,2 e 30,5 kDa. As proteases parcialmente purificadas apresentaram atividade máxima em pH 8,0 e a temperatura ótima foi estimada em 55°C. A enzima foi levemente inibida por PMSF e bastante inibida pelos íons metálicos Fe e Zn e o íon metálico Mg+2 induziu um aumento da atividade enzimática. As enzimas parcialmente purificadas foram aplicadas junto a detergentes comerciais apresentando potencial para possível aplicação na indústria de detergentes. Proteases are one of the largest groups of industrial enzymes, participating in the detergent industry, leather, food and medicine. The detergent industry is a large consumer of hydrolytic enzymes. New methodologies for the purification of biomolecules are of great interest to the industry for its potential applications. The aim of this work was to produce and purify extracellular proteases from Penicillium restrictum under submerged fermentation. The strain of Penicillium restrictum isolated from cerrado soil was able to produce extracellular protease after 7 days of cultivation at 28°C and 150 rpm in medium with wheat bran. After 7 days of fermentation, broth culture was submitted to ultrafiltration process through a membrane of 100 kDa. Ultrafiltered fraction was added to SMDFA under different conditions, purification being evaluated through a factorial design 2³ with three replications at center point. The results showed that the SMDFA was effective in partitioning of proteases present in the fraction

CAPES Lectinas, são proteínas hemaglutinantes que ligam reversivelmente a carboidratos enquanto que as proteases constituem um grupo de enzimas que catalisam a hidrólise de ligações peptídicas. Estas duas classes de proteínas apresentam diversas propriedades biológicas, incluindo as atividades inseticida e antimicrobiana. Insetos-praga provocam prejuízos a economia dos países e dentre estes destacam-se as espécies Tetranychus bastosi (Tetranychidae) e Nasutitermes corniger (Termitidae). Bactérias e fungos provocam doenças em plantas e animais e a busca por novos agentes antimicrobianos amplia as alternativas para tratamento ou prevenção. Este trabalho investigou atividades biológicas de duas preparações de sementes de Cratylia mollis, uma contendo as lectinas 1, 2 e 3 (denominada Cramoll 1,2,3) e outra contendo atividade proteolítica (denominada CmSP, do inglês C. mollis seed protease). Foi avaliada a atividade acaricida de Cramoll 1,2,3 (0,02, 0,05, 0,1 e 0,2 mg/mL) contra T. Bastosi e para a atividade termiticida foi avaliado a resistência á degradação proteolítica da Cramoll 1,2,3, às proteases de cupins operários, bem como o efeito na sobrevivência de soldados e operários de N. corniger; além disso foi avaliado também os efeitos na atividade de enzimas digestivas de operários. A atividade antimicrobiana de Cramoll 1,2,3 e CmSP foi investigada contra bactérias e fungos. Cramoll 1,2,3 induziu a mortalidade dos ácaros promovendo mortalidade maior que 50% nas concentrações de 0,1 e 0,2 mg/mL. Cramoll 1,2,3 foi um agente ovicida para T. bastosi diminuindo a taxa de eclosão. A população de T. bastosi esteve em estado ascendente (ri positiva) em todas as concentrações testadas. Cramoll 1,2,3 não foi resistente á degradação proteolítica do intestino de N. coringer, no entanto os peptídeos resultantes promoveram mortalidade de operários (CL50 de 78,72 mg/mL) e soldados (CL50199,27 mg/mL); não apresentou atividade repelente. Peptídeos resultantes da hidrólise de Cramoll 1,2,3 modulou atividades de enzimas de digestivas de operários. Cramoll 1,2,3 inibiu o crescimento de C. albicans, C. tropicallis e C. krusei com concentração mínima inibitória (CMI) de 100, 200 e 400μg/mL, respectivamente, mas não foi ativa sobre C. Glabrata e todas as bactérias testadas. CmSP apresentou atividade antifúngica contra todos os fungos testados com valores de CMI variando de 55 a 400 μg/mL e concentração mínima fungicida (CMF) variando de 110 a 1.760 μg/mL. CmSP apresentou atividade bacteriostática para todas as espécies testadas com valores de CMI variando de 55 a 1.760 μg/mL. Em conclusão, as sementes de C. mollis são fontes de lectinas e proteases ativas contra insetos e microrganismos. Lectins are hemagglutinating proteins that reversibly bind to carbohydrates; proteases are a group of enzymes that catalyze the hydrolysis of peptide bonds. These two classes of proteins have several biological properties, including insecticide and antimicrobial activities. Pest insects damage the economies of the countries, and among these the species Tetranychus bastosi (Tetranychidae) and Nasutitermes corniger (Termitidae). Bacteria and fungi cause diseases in plants and animals. The search for new antimicrobial agents increases the alternatives for treatment or prevention. This work investigated the biological activities of two preparations from Cratylia mollis seeds, the one containing lectins 1, 2 and 3 (known as Cramoll 1,2,3) and another containing proteolytic activity (called C. mollis seed protease). It was evaluated acaricidal activity of Cramoll 1,2,3 (0.02, 0.05, 0.1 and 0.2 mg/mL) against T. bastosi ans for the termiticidal activity the resistance to proteolytic degradation of Cramoll 1,2,3 was evaluated, to the termite proteases workers, as well as the effect on the survival of soldiers and workers of N. corniger; In addition, the effects on the digestive enzymes activity of workers were also evaluated. The antimicrobial activity of Cramoll 1,2,3 and CmSP was investigated against the bacteria and fungi. Cramoll 1,2,3 induced mortality of mites by promoting mortality greater than 50% at concentrations of 0.1 and 0.2 mg/mL. Cramoll 1,2,3 was an ovicidal agent for T. bastosi decreasing the hatch rate. The population of T. bastosi was in an ascending state at all concentrations tested. Cramoll 1,2,3 was not resistant to proteolytic degradation of N. coringer's gut; however, the resulting peptides promoted mortality of workers (LC50 of 78.72 mg/mL) and solids (LC50 199.27 mg/mL); did not present repellent activity. Peptides resulting from the hydrolysis of Cramoll 1,2,3 modulated activities of digestive enzymes of workers. Cramoll 1,2,3 inhibited the growth of C. albicans, C. tropicallis and C. krusei with minimum inhibitory concentration (MIC) of 100, 200 and 400 μg/mL, respectively, but was not active on C. glabrata and all bacteria tested. CmSP showed antifungal activity against all fungi tested with MIC values ranging from 55 to 400 μg / mL and minimal fungicidal concentration (CMF) ranging from 110 to 1760 μg / mL. CmSP presented bacteriostatic activity for all species tested with MIC values varying from 55 to 1760 μg/mL. In conclusion, C. mollis seeds are sources of active lectins and proteases against insects and microorganisms.

Premio a la mejor comunicación del XVI Congreso de la Sociedad Andaluza de Enfermedades Infecciosas, Desde el año 2012 en que se aprueban los primeros Antivirales de Acción Directa (AADs), el tratamiento de la hepatitis C (VHC) ha experimentado un cambio histórico y se está viviendo un momento muy esperanzador para erradicar esta enfermedad. Los resultados de los ensayos clínicos realizados con estos nuevos tratamientos alcanzan tasas de respuestas virales del 90 al 100% dependiendo de la combinación del fármaco y del genotipo del virus de la hepatitis C (VHC). Y esto es así, las guías nacionales e internacionales (AASLD/IDSA., 2015) (EASL, 2015) (GEHEP 2015) nos van a recomendar emplear una u otra combinación de AADs según la condición clínica del paciente (en especial grado de fibrosis), y según el genotipo del VHC. Desde luego hay mas variables que manejar en la consulta clínica como puede ser la valoración clínica de las manifestaciones extrahepáticas, grado de replicación del virus, necesidad de trasplante, embarazo y un largo etc.…. En este trabajo de tesis uno de los aspectos en los que hemos incidido ha sido la importancia de la correcta determinación del genotipo del VHC., Tesis Univ. Granada.Tesis. Departamento de Medicina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A soja é o quarto grão mais consumido e produzido em todo o mundo. Está entre as atividades econômicas que cresceram significativamente nas últimas décadas devido às múltiplas aplicações alimentares e industriais. A lagarta da soja, Anticarsia gemmatalis Hiuúbner, 1818 (Lepidoptera: Noctuidae), é uma praga que causa grande prejuizo na produtividade dessa cultura, mesmo em baixas densidades populacionais, devido ao desfolhamento severo durante sua fase larval. As plantas têm mecanismos de defesa ao ataque de insetos que levam à inibição da atividade proteolítica desses herbívoros, retardando seu desenvolvimento. Por outro lado, os insetos mostram uma importante diversidade e plasticidade de suas proteases digestivas. Além do papel de digerir proteinas da dieta, essas proteases também desempenham um papel importante na defesa contra inibidores de proteases (IP) de plantas. Essa complexa relação protease- inibidor é importante para a elaboração de métodos alternativos de controle. Baseado nisso, a busca por IP potentes depende do conhecimento acerca das proteases intestinais do inseto fitófago. Portanto, o trabalho teve como objetivo avaliar os perfis enzimáticos das proteases intestinais da lagarta da soja em diferentes instares e identificar as proteases encontradas no intestino. Os resultados mostraram que há uma plasticidade no perfil proteolítico do intestino da A. gemmatalis ao longo do desenvolvimento larval. As atividades de enzimas do tipo cisteino proteases foram mais importantes nos primeiros instares. Por outro lado, as enzimas do tipo sermno proteases tiveram maiores atividades nas etapas mais tardias da fase larval. Além disso, três proteases foram identificadas por espectrometria de massas, através de homologia do banco de dados para Lepidoptera. O comportamento proteolítico do extrato bruto do intestino da lagarta da soja seguiu a cinética de Michaelis-Menten. Foi demonstrado pela primeira vez que o perfil de proteases do intestino de A. gemmatalis se altera ao longo do seu desenvolvimento. Estes resultados podem mudar o foco do desenvolvimento racional de IP para a lagartada soja e outras Lepidoptera, uma vez que a expressão das principais proteases de insetos não é constante. Soybean 1s the fourth most consumed and produced gram in the world. It is among the economic activities that have grown significantly in recent decades due to multiple food and industrial applications. The soybean caterpillar, Anticarsia gemmatalis Húbner, 1818 (Lepidoptera: Noctuidae), 1s a pest that causes great damage to the productivity of this crop, even at low population densities, due to severe defoliation during 1ts larval phase. The plants have mechanisms of defense to the attack of insects that lead to the inhibition of the proteolytic activity of these herbivores, retarding their development. On the other hand, insects show an important diversity and plasticity of their digestive proteases. In addition to the role of digesting dietary proteins, these proteases also play an important role in the defense against plant protease inhibitors (PIs). This complex protease-inhibitor relationship 1s important for the development of alternative methods of control. Based on this, the search for potent PI depends on the knowledge about the intestinal proteases of the phytophagous insect. Therefore, the objective of this work was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars and to identify the proteases found 1n the intestine. The results showed that there 1s a plasticity in the proteolytic profile of the intestine of 4. gemmatalis throughout larval development. The activities of cysteme protease type enzymes were more important in the first instars. On the other hand, the serine protease enzymes had greater activities mn the later stages of the larval phase. In addition, three proteases were identified by mass spectrometry, through homology of the Lepidoptera database. The proteolytic behavior of the crude intestinal extract of the soybean caterpillar followed the Michaelis-Menten kimmetics. It has been demonstrated for the first time that the protease profile of the gut of A. gemmatalis changes throughout its development. These results may shift the focus of rational PI development to the soybean caterpillar and other Lepidoptera, since the expression of major insect proteases Is not constant.

Conselho Nacional de Desenvolvimento Científico e Tecnológico As cigarrinhas-das-pastagens, Mahanarva spectabilis causam danos severos em pastagens tropicais, comprometendo a produção bovina. O controle-químico de cigarrinha-das-pastagens é economicamente inviável. Sendo assim, a seleção de cultivares forrageiras gramíneas resistentes é uma alternativa de controle muito importante. Estudos relatando a bioquímica da interação entre M. spectabilis e diferentes forrageiras gramíneas são raros. O objetivo foi avaliar o impacto do ataque de M. spectabilis na resposta defensiva de plantas de capim-elefante (Pennisetum purpureum), cv. Roxo Botucatu (RX) e cv. Pioneiro (PI), Brachiaria brizantha cv. Marandú (MA) e Brachiaria decumbens cv. Basilisk (DE). O impacto da alimentação em diferentes cultivares de forrageiras nas proteases digestivas do inseto também foi avaliado. O experimento foi realizado com os quatro genótipos distintos, infestados ou não por M. spectabilis. A atividade de lipoxigenases (LOX), inibidores de proteases e as concentrações de fitohormônios foram avaliadas nas folhas das forrageiras. A atividade de proteases totais e tripsina-like foram avaliadas na região do intestino de M. spectabilis. Os genótipos RX, MA e DE tiveram aumento na atividade de LOX após injúrias por M. spectabilis. A ativação da via da LOX está relacionada com o aumento nos níveis do hormônio jasmonato. O aumento na concentração de ácido abscisico aconteceu nos quatro cultivares. O ácido abscisico pode atuar sinergicamente com o jasmonato na transcrição de genes de defesa. Os cultivares MA e DE tiveram maiores níveis basais de ácido salicílico, mas apenas o cultivares DE foi capaz de aumentar os níveis do hormônio em resposta à herbivoria. A atividade de proteases totais foi maior em M. spectabilis que se alimentaram de DE, indicando secreção de enzimas. Há varialibidade na indução de LOX dentro da espécie Pennisetum purpureum. Todos os cultivares analisados apresentaram aumento nas concentrações de ABA e JA, quando atacados por M. spetabilis, indicando ações de defesa em comum. A indução nas concentrações de SA apenas em DE, pode indicar aumento nos níveis de radicais livres, que causam danos aos herbívoros. O aumento na atividade de proteases totais de M. spectabilis alimentadas com DE indica mecanismos de secreção de proteases para compensar àquelas inibidas, que não estão relacionado com enzimas tripsina-like. Mahanarva spectabilis are spittlebugs that cause several damages in tropical pastures, compromising cattle production. The chemical control of spittlebugs is economically unfeasible. Therefore, the selection of resistant forage grasses cultivars is an important control alternative. Studies reporting the biochemistry of the interaction between M. spectabilis and different forage grasses are rare. Thus, the goal was to assess the impact of M. spectabilis injury on the defensive response of elephant grass (Pennisetum purpureum), cv. Roxo Botucatu (RX) and cv. Pioneiro (PI), Brachiaria brizantha cv. Marandú (MA) and Brachiaria decumbens (DE). The impact of feeding on different cultivars of forages in the digestive proteases of the insect was also assessed. The experiment was carried out with four distinct cultivars, infested or not by M. spectabilis. The lipoxygenases activity, protease inhibitors and concentrations of phytohormones were evaluated in leaves of forage grasses. Total proteases and trypsin-like activities were evaluated in the gut region of M. spectabilis. RX, MA and DE genotypes showed increased LOX activity after injury by M. spectabilis. The LOX pathway activation is related to the increase in levels of the hormone jasmonate. The increase in abscisic acid concentration after injury occurred in the four genotypes. Abscisic acid acts synergistically with jasmonate in the transcription of defense genes. MA and DE cultivars had higher basal salicylic acid levels, but only DE was able to increase concentration in response to herbivory. There is variability in the induction of LOX within the species Pennisetum purpureum. All cultivares showed increased concentrations of ABA and JA when attacked by M. spectabilis, indicating common defense actions. Total proteases activity was higher in M. spectabilis fed with DE. The induction of SA concentration only in DE, may indicate an increase in free radical levels, which cause damage to herbivores. The increased activity of total proteases of M. spectabilis fed with DE indicates protease secretion mechanisms to compensate for those inhibited, which are not related to trypsin- like proteases.

CNPq Proteases desempenham papeis cruciais em diversos processos metabólicos e são o grupo de enzimas de maior importância industrial no mercado mundial. Através do processo de imobilização de proteínas é possível aumentar a produtividade numa escala industrial, incluindo processos de catálise de reações e purificação de biomoléculas. A caracterização da enzima fornece informações que ajudam na compreensão do mecanismo de ação e regulação da sua atividade catalítica. Além disso, através da ação dessas proteases é possível obtenção de peptídeos bioativos que vem sendo utilizados para promoção da saúde e prevenção de doenças. A presente tese relata a produção da protease de Penicillium aurantiogriseum e sua imobilização em nanopartículas magnéticas revestidas com polianilina, purificação da enzima utilizando seu substrato imobilizado nas mesmas partículas e caracterização da protease, além da obtenção de peptídeos antioxidantes pela hidrólise da caseína bovina. A partir dos resultados, foram publicados dois artigos principais: o primeiro intitulado “Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides’ obtainment”, na revista Preparative Biochemistry and Biotechnology; e o segundo “Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum”, na revista Protein Expression and Purification. Além das publicações principais, foram publicados mais três artigos em colaboração ao decorrer do doutorado, dispostos em anexo. Tais estudos foram essenciais para a elucidação das características dessa nova enzima e sua aplicabilidade industrial, além de contribuir com o conhecimento sobre o potencial biotecnológico de espécimes da microbiota nacional. Proteases play crucial roles in various metabolic processes and constitute the group of enzymes of major industrial importance in the world market. Through the process of protein immobilization, it is possible to increase productivity on an industrial scale, including processes of reactions catalysis and biomolecules purification. The enzyme characterization provides information that helps understanding the mechanism of action and regulation of its catalytic activity. In addition, through the action of these proteases it is possible to obtain bioactive peptides that have been used for health promotion and disease prevention. The present thesis reports the Penicillium aurantiogriseum protease production and its immobilization in polyaniline coated magnetic nanoparticles, purification of the enzyme using its substrate immobilized on the same particles and characterization of the protease, besides the obtainment of antioxidant peptides by bovine casein hydrolysis. From these results, two main articles were published: the first entitled "Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides’ obtainment”, on Preparative Biochemistry and Biotechnology journal; and the second article entitled “Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum”, on Protein Expression and Purification journal. In addition to the main publications, three more articles were published in collaboration, during the doctorate, supplied in annex. The main studies were essential for elucidating this new enzyme characteristics and its industrial applicability, besides contributing with the knowledge about the biotechnological potential of specimens from national microbiota.

FACEPE A utilização de enzimas proteolíticas em aplicações terapêuticas tem sido um dos objetivos da indústria farmacêutica nos últimos anos. Entre as várias proteases utilizadas em terapias, destacam-se as proteases fibrinolíticas, que são enzimas capazes de degradar à fibrina, principal componente do coágulo sanguíneo. Há uma crescente busca por metodologias eficazes e de baixo custo para a purificação dessas enzimas, uma vez que qualquer fármaco necessita de um alto grau de pureza e especificidade para evitar reações imunológicas. Nesse sentido, o objetivo desse trabalho foi purificar proteases fibrinolíticas produzidas por Mucor Subtilissimus UCP 1262 através de sistema de duas fases aquosas e métodos cromatográficos, além de caracterizá-las bioquímica e estruturalmente. Um planejamento fatorial completo do tipo 2 ³ e a metodologia de superfície de resposta foram usados para identificar as condições ótimas para a extração de uma protease fibrinolítica por sistema de duas fases aquosas, enquanto métodos cromatográficos tradicionais foram utilizados para purificar outra protease fibrinolítica. A primeira enzima foi recuperada e purificada parcialmente por sistemas de duas fases aquosas constituídos por polietilenoglicol (PEG) e sulfato de sódio, onde a melhor condição a do sistema formado por 30,0% (m/m) de PEG 6000g/mol e 13,2% (m/m) de sulfato de sódio, que assegurou um fator de purificação de 10,um coeficiente de partição de 0,2 e uma recuperação de 102,0%. A SDS-PAGE e o zimograma de fibrina revelaram que essa protease fibrinolítica purificada tem uma massa molecular de 97 kDa e um ponto isoelétrico de 5,4 e é uma serino protease semelhante a quimotripsina. A temperatura ótima da enzima foi de 37ºC e sua atividade foi estável por 150 minutos. A outra protease fibrinolítica foi pré-purificada inicialmente através de precipitação com sulfato de amônio (40 – 60% de saturação) e posteriormente através de cromatografia de troca iônica (DEAE-Sephadex A50) e gel filtração (Superdex 75 HR10/300) e igualmente caracterizada como serino protease do tipo quimiotripsina, com uma massa molecular de 20 kDa, um ponto isoelétrico de 4,94,e temperatura e pH ótimos de 40°C e 8,0 respectivamente. Sua atividade proteásica foi aumentada na presença de Cu²⁺, Mg²⁺ e Fe²⁺ e sua estrutura secundária mostrou alta composição de α-hélice e nenhuma mudança significativa após exposição à presença de polímeros ou da maioria de agentes desnaturantes. Este estudo demonstra a possibilidade de purificar proteases fibrinolíticas com baixo custo e de considerá-las como potenciais candidatos a agentes trombolíticos. The use of proteolytic enzymes in therapeutic applications has been one of the goals of the pharmaceutical industry in recent years. Among the various proteases used in therapies are fibrinolytic proteases, which are enzymes capable of degrading fibrin, the main component of the blood clot. There is a growing search for efficient and low cost methodologies for the purification of these enzymes, since any drug requires a high degree of purity and specificity to avoid immunological reactions. In this sense, the objective of this work was the purification of fibrinolytic proteases produced by Mucor subtilissimus UCP 1262 through aqueous two-phase system and chromatographic methods, as well as their biochemical and structural characterization. A full 2 ³ type factorial design and response surface methodology were used to identify optimal conditions for the extraction of a fibrinolytic protease by aqueous two-phase system, while traditional chromatographic methods were used to purify another fibrinolytic protease. The former enzyme was recovered and partially purified by aqueous two phase systems consisting of polyethylene glycol (PEG) and sodium sulfate, the best condition being that of the system consisting of 30.0% (w/w) PEG 6000 g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10, a partition coefficient of 0.2 and a recovery of 102.0%. SDS-PAGE and fibrin zymogram revealed that this purified fibrinolytic protease has a molecular mass of 97 kDa and an isoelectric point of 5.4, and is a chymotrypsin-like serine protease. The enzyme optimum temperature was 37ºC and its activity was stable for 150 minutes. The other fibrinolytic protease was initially pre-purified by ammonium sulfate precipitation (40-60% saturation) and subsequently by ion exchange chromatography (DEAE-Sephadex A50) and gel filtration (Superdex 75 HR10/300), and equally characterized as chymotrypsin like serine protease, with a molecular mass of 20 kDa, an isoelectric point of 4.94, and optimum temperature and pH of 40°C and 8.0, respectively. Its protease activity was increased in the presence of Cu ²⁺ , Mg ²⁺and Fe ²⁺, and its secondary structure showed high α-helix composition and no significant change after exposition to the presence of polymers or of most of the denaturing agents. This study demonstrates the possibility of purifying fibrinolytic proteases at low cost and considering them as potential candidates as thrombolytic agents.

En la presente investigación se evaluó la composición química (CQ) y digestibilidad in situ de la materia seca (DISMS) materia orgánica (DISMO), fibra detergente neutra (FDN) y (FDA) del pasto estrella africana en cuatro edades (21, 28, 35, 43 días), en la unidad de pastos y forrajes de la ESPAM MFL, ubicada en el cantón Bolívar, provincia de Manabí. Para la (CQ) se tomó las muestras de cada edad y se llevó al laboratorio para realizar el análisis proximal. Para la degradabilidad in situ de la MS, MO, FDN y FDA se utilizaron 4 toretes fistulado, a quienes se le incubo las bolsas con 10 g de muestra al rumen por siete tiempos, 0, 3, 6, 12, 24, 48, 72 horas. Los datos obtenidos fueron procesados en el software estadístico SAS. En los resultados se evidenció mayor contenido de MS a los 35 días, (P0.05), en cuanto a la proteína fue mayor a los 21 días (P0.05); de la misma manera la DISMS y DISMO fue mayor a los 21 días al igual que la fracción soluble (P

[eng] Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix. They make a major contribution to the progression of a vast number of diseases, particularly cancer and some central nervous system (CNS) disorders, such as epilepsy and Alzheimer’s disease. Although a number of MMP inhibitors (MMPi) have been developed for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and/or, most importantly, severe side effects. The latter can be explained by their lack of selectivity. Elevated levels of gelatinases (MMP-2 and MMP-9) have been described in various animal models and in clinical studies in humans with epilepsy after seizures, being MMP-9 the most overexpressed in all these cases. Thus, a selective inhibitor for MMP- 9, and to some extent MMP-2, might be beneficial for the treatment of this disease. In this thesis, computer-aided drug design was used to design novel MMP-9 inhibitors which were synthesised and evaluated using in vitro enzyme assays. Following this approach, we obtained several molecules that show selectivity for gelatinases over other MMP family members (MMP-1, MMP-3 and MMP-7). These compounds may be of particular interest for the treatment of epileptogenesis, the process by which a normal brain is functionally altered and leads to the development of epilepsy, because of the prominent role of MMP-9 in this process. The permeability across the Blood-brain barrier (BBB) is the main obstacle and a prerequisite for drugs targeting the central nervous system. In this thesis, I worked in the refinement of the structure of the molecules to improve the BBB permeability of the compounds under development. One of the developed compounds, was selected for further studies because its enzymatic and selectivity for Gelatinases and high proteolytic stability. Once the lead candidate was selected, this compound was studied in vivo. First, the pharmacokinetics of the compound were studied in mice and rats, indicating that the molecule was absorbed in the body and distributed through the BBB. Later, I evaluated the effects of the lead compound on the progression of epilepsy in three animal models (the pentylenetetrazole (PTZ) acute injection mouse model, the intrahippocampal kainic acid (KA) mouse model and the rapid kindling rat model) of epilepsy. Results from these experiments indicate that the selected compound has the capacity to inhibit MMP-9 in vivo, has anticonvulsive effects and is able to attenuate the progression of epilepsy in the tested epilepsy models, without any side effects. Moreover, the effects of increased MMP-9 activity in memory and learning were also reduced by using the tested compound. The last part of this thesis was focused on the lead compound optimization addressing metabolic deficiencies through structural modification. Based on this approach, two optimized lead candidates were obtained showing higher potency and selectivity for gelatinases and improved drug-like properties (in vitro BBB permeability, cytotoxicity and stability in serum, intestinal fluids and liver microsomes). As a conclusion, this thesis followed the general structure of the drug discovery phase in the drug research process. MMP-9 was identified and selected as a potential therapeutic target for the treatment of epileptogenesis. The hit identification and hit- to-lead process were achieved using in silico calculations in combination with in vitro tools to design MMPi that selectively target gelatinases and have the ability to cross the BBB. The pharmacokinetic study, the efficacy and toxicity of the lead compounds were demonstrated in different models of epilepsy. Finally, the lead compound was optimized for oral administration., [cat] Les metal·loproteinases de matriu (MMPs) constitueixen una família de proteases dependents del zinc i involucrades en la degradació de la matriu extracel·lular. Participen en la progressió d’un gran nombre de malalties, principalment en càncer i en malalties del sistema nerviós central, com ara l’Alzheimer i l’epilèpsia. En les últimes dècades, s’han desenvolupat una serie d’inhibidors de MMPs (MMPi) pel tractament del càncer, però aquests han fallat en estudis clínics degut a la seva ineficàcia o efectes secundaris adversos, principalment deguts a la falta de selectivitat del inhibidors. En la literatura s’ha descrit un increment en els nivells d’expressió i activitat de gelatinases (MMP-2 i MMP-9) en diferents models animals d’epilèpsia, així com també en humans amb aquesta condició, sent la MMP-9 les més sobreexpressada. Per això, un inhibidor selectiu de la MMP-9, i en part també de la MMP-2, podria ser beneficial pel tractament d’aquesta malaltia. En aquesta tesis, el disseny de fàrmacs assistit per ordinador s’ha utilitzat per a dissenyar inhibidors de la MMP-9 que han estat sintetitzats i avaluats in vitro mitjançant assajos enzimàtics. Els compostos més potents i selectius obtinguts per la inhibició de gelatinases s’han seleccionat i optimitzat per creuar la barrera hematoencefàlica. Per la molècula més prometedora, s’han dut a terme en ratolins i rates. A més, la seva eficàcia i potencial toxicitat s’ha estudiat en 3 models animals d’epilèpsia, demostrant que aquest candidat a fàrmac inhibeix la MMP-9 in vivo i pot atenuar els efectes convulsius i epileptogènics presents en els models animals, a més a més no s’han observat símptomes clínics adversos durant els experiments amb animals. Finalment, aquest compost s’ha optimitzat amb l’objectiu de millorar la seva estabilitat metabòlica i poder ser administrat per via oral. Després d’aquest procés d’optimització s’han obtingut dos compostos potents i selectius amb bones propietats ADME .

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2018. Alternativas para melhorar a estabilidade e atividade enzimática têm importância crescente, visto que a aplicação industrial de enzimas aumentou nos últimos anos. No entanto, as características da enzima em termos de estabilidade e eficiência catalítica podem reduzir, em alguns casos, devido às condições reacionais. Uma vez que há uma falta na literatura relacionada à enzima Eversa® Transform 2.0 (NS-40116), este trabalho avaliou a influência do campo magnético na atividade da protease de Bacillus sp. (2 g.L-1) e da lipase NS-40116 (5 g.L-1) quando expostas ao campo de 0,7 e 1,34 T de modo estático (2 h) e com recirculação (1, 2 e 4 h) a 0,06 L.min-1. O efeito do pH da solução enzimática (pH 5, 7 e 9) associado à exposição ao campo magnético também foi avaliada. Os melhores resultados em termos de atividade enzimática residual foram para a lipase NS-40116 em pH 7,0, 1,34 T no sistema de recirculação por 4 h. A atividade da protease de Bacillus sp. não sofreu influência do campo magnético nas condições estudadas. Então, a análise de espectrometria de massa foi realizada para confirmar a estrutura da enzima após diálise e liofilização. Além da atividade enzimática, espectrometria de fluorescência, dicroísmo circular e estabilidade térmica foram realizadas afim de avaliar o efeito do campo magnético na estrutura da NS-40116, e foi observada alterações na estrutura terciária da enzima. Assim, a aplicação de campo magnético como tratamento enzimático é uma técnica promissora e viável para incremento da atividade lipásica, apresenta baixo custo, baixo impacto ambiental e facilidade de operação quando comparado com outras alternativas de ativação enzimática. Abstract : Alternatives to improve the enzyme stability and activity have been rising in the last years due to the importance of the industrial application. However, the enzyme characteristics in terms of stability and catalytic efficiency can reduce, in some cases, due to the reaction conditions. Once there is a lack in the literature related to the structural Eversa® Transform 2.0 enzyme (NS-40116), this work evaluated the influence of magnetic density flux on the Bacillus sp. (2 g.L-1) and lipase NS-40116 (5 g.L-1) activity exposed to a magnetic flux (0.7 and 1.34 T) by static (2 h) and recirculation mode (1, 2 and 4 h) at 0.06 L.min-1. The effect of pH in the enzymatic solution (pH 5, 7 and 9) associated to the magnetic field was also evaluated. The best results in terms of residual enzymatic activity were for NS-40116 at pH 7.0, 1.34 T after 4 h in a recirculation mode. The enzymatic activity for protease from Bacillus sp. was not influenced by conditions studied. Then, mass spectrometry analysis was performed to confirm the enzyme structural after dialysis and lyophilization. In addition to the enzymatic activity, fluorescence spectrometry, circular dichroism and thermal stability analyzes were performed to verify the effect of the magnetic field on the NS-40116 enzyme structure and changes in the tertiary structure of this enzyme was observed. Thus, the application of magnetic fields as enzymatic treatment is a promising and viable technique to increase lipase activity, since it has low cost, low environmental impact and easy operation when compared with other alternatives enzyme activation.

Submitted by Clariana Zanutto Paulino da Cruz null (clarianadacruz@gmail.com) on 2018-01-17T13:23:25Z No. of bitstreams: 1 Tese Clariana COMPLETA.pdf: 3282828 bytes, checksum: 66774fa4ce11cc7a992231649f8d7320 (MD5) Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-01-23T10:58:03Z (GMT) No. of bitstreams: 1 cruz_czp_dr_araiq_par.pdf: 1938716 bytes, checksum: 3b78ccfcd5f87e17af178eb5e6333fdb (MD5) Made available in DSpace on 2018-01-23T10:58:03Z (GMT). No. of bitstreams: 1 cruz_czp_dr_araiq_par.pdf: 1938716 bytes, checksum: 3b78ccfcd5f87e17af178eb5e6333fdb (MD5) Previous issue date: 2017-12-21 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) O atual trabalho teve como objetivo realizar a hidrólise das proteínas do soro do queijo bovino (SQB) utilizando a alcalase® imobilizada em pó de sabugo de milho, para obtenção, purificação parcial e avaliação das propriedades biológicas de peptídeos bioativos. O sabugo de milho (SM) é o principal resíduo formado da indústria de processamento de milho. Alcalase® foi usada para a imobilização em SM e agarose. O desempenho dos dois derivados foi estudado em relação aos parâmetros cinéticos de imobilização. A hidrólise das proteínas do SQB em sistema descontínuo foi realizada em reator de batelada (pH 9,0; 50ºC/48h) usando a alcalase® livre (AL) e derivados alcalase®-glioxil-agarose (AGA); alcalase®-glioxil-SM (AGSM) para fins de comparação. O grau de hidrólise (GH) para AL, AGA e AGSM foi de 59,63, 43,88 e 26,59% respectivamente. O derivado estável AGSM apresentou capacidade de cinco ciclos de reuso de 24 h consecutivos mantendo atividade maior que 70% e exibiu estabilidade térmica (65ºC/pH 7,0) 62 vezes maior do que a AL, retendo 12% de sua atividade inicial após 48 horas, enquanto que a AL e AGA foram totalmente inativados. Os hidrolisados proteicos do soro (HPS) obtidos com AGSM foram fracionados por RP-HPLC em três frações (F1, F2 e F3) de acordo com a hidrofilicidade dos peptídeos (F1: hidrofílicos, F2: hidrofilicidade intermediária e F3: hidrofóbicos). As frações foram analisadas por MALDI-TOF que identificou peptídeos de massa molecular (F2C>F3) e a atividade quelante de ferro para as frações (F1, F2 e F3) foram nulas. As espécies E. coli (ATCC 43895) e Listeria monocytogenes (ATCC 7644) foram susceptíveis à ação dos peptídeos obtidos neste presente estudo. Os peptídeos de hidrofilicidade intermediária (F2) apresentaram maior atividade antimicrobiana, tanto para E. coli quanto para L. monocytogenes, sendo mais efetivos contra a segunda (pF2C>F3) was observed while the chelating activity of iron for the all fractions (F1, F2, and F3) was null. The obtained peptides showed high antimicrobial activity (87.75-100%) against the species E. coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC=10 mg/mL). A packed bed reactor (PBR) containing 25 g of the AGCCP derivative (8.66 U/g) was used for the hydrolysis of whey proteins in a continuous system process (50°C/pH 9.0). The hydrodynamic characterization of the reactor was performed using red phenol dye (0.6 g.L-1) as a tracer. The PBR operated at a flow rate of 6 mL.h-1, which represented a residence time of 12.9 h, approximately 19% higher than the spatial time value (10.8 h). Therefore, the formation of preferential paths was minimal and the system presented a high degree of hydrolysis (58.98-70.70%) during 180 hours of reaction. The half-life of the AGSM derivative was very high (246 h) and bioactive peptides of molecular weight (

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O imunoproteassoma é uma isoforma de proteassoma constituído por três subunidades denominadas LMP2, LMP7 e MECL-1. Sua atividade proteolítica aumenta a quantidade e a qualidade dos peptídeos a serem apresentados pelas moléculas de MHC de classe I às células T CD8+. Por esta razão, esta isoforma especializada de proteassoma desempenha um importante papel no controle de infecções em hospedeiros. Neste trabalho, foi abordado o papel do imunoproteassoma durante a infecção por Brucella abortus, uma bactéria Gram-negativa, intracelular facultativa, patogênica para humanos e animais, cuja resposta imune protetora é dependente dos linfócitos T CD4+ e T CD8+. Vários trabalhos demonstraram a importância do imunoproteassoma no controle de infecções por diferentes patógenos por meio da utilização de camundongos knockout para uma ou duas subunidades do imunoproteassoma. Nós buscamos investigar, pela primeira vez, o impacto da ausência das três subunidades desta estrutura durante a infecção por uma bactéria intracelular. Para isto, a infecção por B. abortus foi induzida em camundongos triplo knockout para o imunoproteassoma, que se mostraram mais susceptíveis à infecção bacteriana, devido a uma diminuição na produção de IFN-γ por linfócitos TCD4+ e TCD8+. Além disso, a ausência do imunoproteassoma teve um impacto sobre a expressão de moléculas de MHC-I na superfície de células dendríticas e na geração e apresentação de antígenos por essas células. A função dos linfócitos T, que desempenha um papel fundamental na imunidade adaptativa contra B. abortus, também foi comprometida, tendo sido observada uma redução na produção de granzima-B e consequentemente na atividade citotóxica dos linfócitos T CD8+. Em conclusão, esses resultados sugerem fortemente que o imunoproteassoma é importante na resistência do hospedeiro à infecção por B. abortus, impactando tanto a magnitude quanto a qualidade das respostas das células T CD8+ The immunoproteasome is a specific proteasome isoform constituted of three subunits termed β1i, β2i and β5i. Its proteolytic activity enhances the quantity and quality of peptides to be presented by MHC class I molecules to CD8+ T cells. However, the role of combined deficiency of the three immunoproteasome subunits in protective immunity against bacterial pathogens have not been investigated. In this study, we addressed the role of immunoproteasome during Brucella abortus infection, an intracellular bacterium that requires CD8+ T cell responses for control of infection. Here, we demonstrate that immunoproteasome triple knockout mice (TKO) were more susceptible to the Brucella infection. This observed susceptibility was accompanied with reduced IFN-γ production by mouse CD4+ and CD8+ T lymphocytes. Moreover, the absence of the immunoproteasome had an impact on dendritic cells MHC-I surface expression and antigen presentation by these cells. CD8+ T cell function, which plays a pivotal role in B. abortus immunity, also presented partial impairment of granzyme-B expression and consequently reduced cytotoxic activity. In conclusion, these results strongly suggest that immunoproteasome subunits are important components in host resistance to B. abortus infection by impacting both the magnitude and quality of CD8+ T cell responses.

[eng] Chloroplasts provide plants with metabolic pathways that are unique among eu- karoytes, including the methylerythritol 4-phosphate pathway (MEP) for the pro- duction of isoprenoids essential for photosynthesis and plant growth. The first re- action of the MEP pathway involves the synthesis of deoxyxylulose 5-phosphate (DXP) from the central metabolic intermediates glyceraldehyde 3-phosphate (GAP) and pyruvate catalyzed by DXP synthase (DXS). DXS has a major role in regulating the MEP pathway flux, but little is known about how its levels and activity are regu- lated. It has been shown that DXS stability and enzymatic activity can be modulated by interaction with other plastidial DXS-interacting (DXI) proteins. The goal of this thesis work has been to characterize the physiological role of DXS-DXI interactions and the molecular pathways leading from the interactions to the eventual biological effects in the model plant Arabidopsis thaliana. To investigate whether loss of DXI function in mutants impacted DXS activity, we analyzed their resistance to clomazone (CLM), a DXS-specific inhibitor. Most of the loss-of-function mutants tested did not show resistance or sensitivity to this in- hibitor. However, two mutant alleles for the gene SBP, which encodes the Calvin cycle enzyme sedoheptulose 1,7-bisphosphatase (SBPase), showed an increased re- sistance to CLM. In contrast, overproduction of SBPase in transgenic Arabidopsis plants resulted in reduced CLM resistance. Strikingly, we found that DXS protein levels or activity did not change when SBPase levels are altered in plants. Although co-immunoprecipitation assays were unable to confirm the interaction DXS-SBPase, our results do show a functional relationship between the Calvin cycle and the MEP pathway. We propose that excess GAP in the sbp mutant is diverted into the MEP pathway, preventing CLM binding to DXS. The second part of the thesis continued previous work with DXI1, a DXS-interacting J-protein that facilitates the recognition of inactive DXS forms to deliver them to eventual reactivation or degradation pathways. In particular, we focused on inves- tigating the molecular components involved in these two opposite pathways. By bioinformatic and experimental approaches, we confirmed that DXS is prone to ag- gregate in the chloroplast and associate to insoluble (membrane) fractions in an in- active form. These inactive forms of the enzyme were found to overaccumulate in plants defective in DXI1 (renamed J20). J20 is an adaptor of the Hsp70 chaperone. We demonstrated that the DXS-Hsp70 complex interacts with the Hsp100/ClpC1 vi chaperone to unfold DXS for delivery into the proteolytic chamber of the Clp pro- teolytic complex. On the other hand, correct folding of DXS is achieved with the contribution of Hsp100/ClpB3. Our work suggests that degradation or activation of DXS might depend mostly on changes in ClpB3 levels. This disaggregase accumulates when the MEP pathway flux is decreased and in situations causing protein folding stress. Through molecular, genetic and pharmacological approaches, we demonstrated that this accumulation depends on a mechanism called chloroplast Unfolded Protein Response (cpUPR). Elicitation of this cpUPR by inhibition of protein synthesis in the chloroplast led to increased expression of nuclear genes encoding ClpB3 and other chloroplast chap- erones, eventually causing a stress acclimation response. We further demonstrated that cpUPR is independent of GUN1, an integrator of retrograde signaling, since we observed that chaperones accumulate in both wild-type and gun1 mutant plants. However, GUN1-defective plants were unable to develop the acclimation response. Our data therefore confirm that GUN1 is a central integrator of different pathways controlling chloroplast protein homeostasis beyond the control of nuclear gene ex- pression. Our results will contribute to taking more informed decisions on future approaches to manipulate levels of chloroplast isoprenoids of interest (such as vitamins, biofuels or drugs against cancer and malaria) in crop plants., [spa] Los cloroplastos tienen vías metabólicas únicas entre los eucariontes, incluyendo la vía del metileritritol 4-fosfato (vía MEP) para la producción de isoprenoides. La primera reacción de la vía MEP es la síntesis de desoxixilulosa 5-fosfato (DXP) a partir de gliceraldehído 3-fostato (GAP) y piruvato, catalizada por la enzima DXP sintasa (DXS). DXS tiene un papel central en regular el flujo de la vía MEP, pero aún se sabe relativamente poco acerca de cómo se regulan sus niveles y actividad enz- imática. En esta tesis se ha estudiado cómo la interacción de DXS con otras proteínas regula su función en Arabidopsis thaliana. De entre estas proteínas interactoras, solo la pérdida de función de la enzima sedoheptulosa 1,7-bifosfatasa generó un fenotipo de resistencia a la inhibición de DXS. Los resultados de la tesis permiten concluir que existe un vínculo funcional entre el ciclo de Calvin y la vía MEP, seguramente medi- ado por la disponibilidad de GAP. Por otro lado, se ha observado que DXS es un en- zima propenso a agregarse. Estas formas inactivas interaccionan con la proteína J20, un adaptador de la chaperona Hsp70. El complejo DXS-Hsp70 a su vez interactúa con ClpC1 para degradar DXS mediante la proteasa Clp. Por otro lado, la interacción del complejo con ClpB3 pliega correctamente y activa DXS. El destino de DXS podría depender mayoritariamente de los cambios en los niveles de ClpB3. Un menor flujo de vía MEP, la pérdida de la homeostasis proteica en el cloroplasto, o la expresión defectuosa del plastoma causan la acumulación de ClpB3 y de otras chaperonas me- diante la activación de la expresión de los correspondientes genes nucleares. Esta respuesta no requiere de la actividad de GUN1, un nodo central en la comunicación cloroplasto-núcleo. Sin embargo, GUN1 es esencial para la posterior respuesta de aclimatación que permite a las plantas soportar mejor otros tipos de estrés como los causados por la inhibición de la síntesis de isoprenoides. Nuestros resultados pueden contribuir a tomar decisiones más informadas para manipular los niveles de isoprenoides cloroplastídicos de interés (como vitaminas, biocombustibles o fárma- cos contra el cáncer y la malaria) en plantas de cultivo.

Submitted by Milena Novais da Costa null (miuli_nc@hotmail.com) on 2017-12-01T20:56:50Z No. of bitstreams: 1 dissertacao com carta de aprovacao.pdf: 1961789 bytes, checksum: 1c01b1ab10c4730f3b81e006c1ef5d88 (MD5) Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2017-12-04T11:44:58Z (GMT) No. of bitstreams: 1 costa_mn_me_araiq_par.pdf.pdf: 772561 bytes, checksum: b22698e65315d11eef43806a1ca9c92b (MD5) Made available in DSpace on 2017-12-04T11:44:58Z (GMT). No. of bitstreams: 1 costa_mn_me_araiq_par.pdf.pdf: 772561 bytes, checksum: b22698e65315d11eef43806a1ca9c92b (MD5) Previous issue date: 2017-09-13 Devido ao avanço científico e tecnológico, notável sucesso tem sido alcançado na identificação de novos compostos antitumorais. Entretanto, problemas associados à baixa estabilidade e seletividade tem limitado o sucesso da grande maioria destes compostos. Dentre as estratégias para aumentar a estabilidade de moléculas bioativas, a bioconjugação em domínios de ligação a albumina (DLA) tem se mostrado promissor para ampliar o tempo de vida médio de moléculas susceptíveis à degradação proteolítica. Neste trabalho, a estrutura e a atividade de um composto contendo um peptídeo citotóxico, um DLA e um sítio de clivagem específico foram avaliadas. Motivado pela perspectiva do peptídeo melitina apresentar atividade antitumoral, o nosso grupo de pesquisa avaliou a síntese e atividade biológica deste composto com o peptídeo RQKRSLGG-WQRPSSW. O peptídeo obtido foi testado contra tipos de celulas tumorais e não tumorais (linhagem MCF-7 e HaCaT, respectivamente), mostrando-se potente, porém tóxico. Nos estudos de dicroísmo circular, os peptídeos não apresentaram estrutura secundária em solução aquosa. Em presença de miméticos de membrana, os peptídeos adquiriram uma estrutura em α-hélice exceto o peptídeo sítio de clivagem-DLA. Estudos de vazamento de carboxifluoresceína em LUVs (POPC:POPS), através da técnica de espectroscopia de fluorescência, mostraram que o peptídeo completo tem capacidade de permeabilização similar ao da melitina e que é dependente da concentração. Os resultados de fluorescência confirmaram a interação do peptídeo com a proteína HSA. A intensidade da fluorescência do Trp presente nos peptídeos aumenta após a ligação com a HSA e o comprimento de onda de emissão é deslocado para o azul (blue-shift), indicando a mudança de ambiente do Trp para um meio mais hidrofóbico. A ligação dos peptídeos com a HSA não foi suficiente para proteger os compostos antitumorais das enzimas proteolíticas presente no plasma sanguíneo, mostrando baixa estabilidade do peptídeo. Os dados obtidos mostraram que, a estratégia utilizada para o aumento da seletividade e estabilidade da melitina não é adequada. Due to the scientific and technological advanced permitted remarkable successful in the field of identification of new antitumour compounds. However, problems associated with low stability and selectivity have been a restriction in the successf ul of majority of this compounds. Inside the strategic for increase the stability of bioactive molecules, bioconjugation with albumin - binding domain (ABD) have been promising for increasing t he lifetime of molecules susceptible for the proteolytic degradat ion. Herein, structure and activity of a compound containing a cytotoxic peptide, ABD, and cleavage site were evaluated. The ABD (WQRPSSW) can give more stability for molecules, while the cle avage site RQKRSLGG can give more selectivity to the peptide. Thi s sequence is degraded for the protease Kallikrein 4 (KLK4), which occurs in elevated quantity in the tumours cells, releasing the cytotoxic compound, especially in the microenvironments of t hese cells, promoting the higher selectivity. Inspired for the pe rspective of Melittin peptide holds a promising antitumour activity, our research group evaluated the synthesis and biological activity with this peptide containing the sequence RQKRSLGG - WQRP SSW. The peptide was evaluated against diversity of tumours and n o tumours cells (MCF - 7 and HaCaT respectively), presenting effective activity but toxic. In circular dichroism studies the peptides did not show second structure in aqueous solution. In the p resence of membrane mimetic, the peptides acquired an α - helix structure, excluding the peptide cleavage site - ABD. Studies of carboxyfluorescein release in LUVS (POPC:POPS), by fluorescence spectroscopy, showed that the complete peptide has a similar capac ity of Melittin for permeability, also, the Melittin and the complete peptide activities were concentration - dependent. Nevertheless, for the complete peptide this effect was less manifested, which means that the electrostatic interaction between the positi ve charges of peptides and the negative charges of membranes play a key role in the process. The fluorescence assays confirmed the peptide interaction with HSA protein. The Trp fluorescence quantum yield of the peptides increased after the HSA attachement , while, Trp emission wavelength resulted in blue - shift. Indicating that the Trp activity change between a polar solvent for a hidrophobic environment. Probably, conformational changes occurred in the molecule, which not enough were for protect the antitu mour compounds from proteolytic enzymes current in blood serum, endorsing the fewer stability of the antitumour peptides.

Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná A maciez é um dos atributos mais exigidos pelos consumidores com relação à carne bovina. Assim, técnicas de amaciamento aplicadas em cortes cárneos de baixa maciez possibilitam agregar valor de forma direta à estes cortes. A aplicação do ultrassom, uma tecnologia emergente, tem sido uma alternativa proposta para o aumento da maciez em cortes cárneos de maciez reduzida, como é o caso do músculo Bicep femoris. Este estudo teve como objetivo avaliar os efeitos do ultrassom sobre a qualidade do músculo Biceps femoris de bovinos da raça Nelore utilizando o banho ultrassônico por meio da metodologia de superfície de resposta. Um delineamento composto central rotacional (DCCR) 22 com triplicata no ponto central (11 ensaios) foi utilizado para a aplicação do ultrassom no músculo Biceps femoris 24 h post-mortem. A potência (x1) e o tempo de ultrassom (x2) foram as variáveis independentes. As variáveis dependentes () foram a medida instrumental de força de cisalhamento (FC), perda de peso por exsudação, pH, cor (L*, a* e b*) e capacidade de retenção de água (CRA). A redução da medida instrumental de FC foi utilizada como parâmetro para identificação da condição ótima. As amostras submetidas ao tratamento ultrassônico na condição ótima foram comparada às amostras controle quanto a oxidação lipídica (TBARS), teor de cálcio sarcoplasmático, índice de fragmentação miofibrilar e perfil de ácidos graxos. Todas as determinações analíticas foram realizadas 24, 72 e 144 h após o tratamento. No DCCR, o termo linear de x2 foi estatisticamente significativo (p ≤ 0,05) para redução dos valores de FC nas amostras do músculo Biceps femoris. A metodologia de superfície de resposta possibilitou observar que a aplicação de ultrassom com potência de 22,6 W cm-2 e tempo de 120 s resultou no menor valor de FC, sendo esta a condição ótima. Os resultados de pH, cor (L*, a* e b*), CRA, perda de peso por exsudação e TBARS das amostras tratadas na condição ótima não diferiram (p  0,05) das amostras controle. Estes resultados apontaram que a aplicação de ultrassom não afetou a oxidação lipídica da carne, mesmo após 144 h. Amostras tratadas apresentaram maior teor de cálcio sarcoplasmático (p ≤ 0,05) do que as controle. Contudo, o IFM—medida indireta da atividade proteolítica do sistema calpaína— não diferiu (p > 0,05) entre as amostras tratadas e controle, sugerindo que a redução da FC seja oriunda de modificações do colágeno e não da degradação de proteínas da linha z. No perfil de ácidos graxos, 24 h após aplicação do ultrassom, as somatórias dos ácidos graxos monoinsaturados e poli-insaturados foram superiores (p ≤ 0,05) nas amostras tratadas do que nas controle, e o inverso foi observado para a somatória de ácidos graxos saturados. Estes resultados demonstram que a aplicação de ultrassom contribuiu para a ruptura da membrana celular fosfolipídica e consequente liberação de ácidos graxos insaturados. A aplicação do ultrassom não afetou negativamente os parâmetros de qualidade da carne bovina e contribuiu para a melhoria da maciez com redução de 24% na FC em relação à amostra controle. In relation to beef, tenderness is one of the most required attributes by consumers. Therefore, tenderness techniques applied in low tenderness meat cuts allow to aggregate value of the directlyto these cuts. Ultrasound treatment, an emergent technology, has been an alternative proposed to tenderness increase in low tenderness meat cuts, as Biceps femoris bovine muscle. In this research, the aim was to evaluate the ultrasound effects on quality of Biceps femoris muscle from Nelore bovines using ultrasound-bath by response surface methodology (RSM). A central composite rotational design (CCRD) 22 with triplicate in central point (11 assays) was used to ultrasound treatment in Biceps femoris 24 h post-mortem. Ultrasound power (x1) and time (x2) were the independents variables. Dependents variables () were Warner-Bratzler shear-force (SF), water loss, pH, color (L*, a* and b*), and water holding capacity (WHC). SF decrease was used as parameter to identify the optimum condition. Samples sonicated in optimum condition were compared with control samples by analysis of lipid oxidation (TBARS), sarcoplasmic calcium content, myofibril fragmentation index (MFI), and fatty acid profile. All analytical determinations were performed in 24, 72 and 144 h after ultrasound treatment. In CCRD, x2 linear term was significant statistically (p ≤ 0.05) to SF decrease in Biceps femoris bovine muscle. RSM allowed noting that ultrasound application with 22.6 w cm-2 ultrasound power and 120 s resulted in lower SF value, characterized as optimum experimental condition. The results of pH, color (L*, a* and b*), WHC, water loss and TBARS in sonicated samples in optimum condition were not different (p > 0.05) of control samples. These results pointed that ultrasound treatment did not affect lipid oxidation of beef until 144 h. Sonicated samples exhibited higher sarcoplasmic calcium content (p ≤ 0.05) than control. However, MFI—indirect measure of calpain proteolytic activity— was not different (p > 0.05) between sonicated samples and control. This result suggests that SF decrease could have been result from collagen changes and did not from z-line protein degradation. In fatty acid profile, the sums of monounsaturated fatty acids and polyunsaturated fatty acids were higher (p ≤ 0.05) in sonicated samples than control, and the inverse was observed to saturated fatty acids sum. These results demonstrated that ultrasound treatment contributed to phospholipid cellular membrane rupture and consequent release of unsaturated fatty acids. Ultrasound treatment did not affect negatively the beef quality parameters and contributed to tenderness increase as 24% of SF decrease in relation to control samples.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A lagarta-da-soja, Anticarsia gemmatalis, é considerada uma das principais pragas da cultura da soja. Os danos causados pelo ataque deste inseto associado à relevância econômica do cultivo da soja para o Brasil e para o mundo fomentam a busca por alternativas no controle deste inseto. Estratégias de controle de insetos-pragas baseadas no uso de inibidores de proteases têm sido estudadas, o conhecimento das enzimas digestivas e metabolismo do inseto tem se mostrado fundamental. Neste contexto, este trabalho teve como objetivo identificar proteínas com abundância diferencial no intestino de A. gemmatalis em resposta a inibidor de protease, visando obter dados que poderão contribuir para esclarecer o mecanismo adaptativo de A. gemmatalis em resposta a inibidores de proteases na interação planta-inseto. Foram dissecados intestinos de lagartas alimentadas com os inibidores de proteases berenil e o Kunitz, nas horas 6 h, 12 h, 24 h e 48 h e as proteínas extraídas por maceração. Os spots com abundância diferencial foram digeridos e os peptídeos analisados no espectrômetro de massa MALDI/TOF-TOF. Os espectros (MS/MS) foram pesquisados em bancos de dados usando a ferramenta MASCOT e as proteínas identificadas foram validadas usando o software Scaffold. Foram 44 spots de proteínas identificados, nos tratamentos com os inibidores berenil e Kunitz, nas diferentes horas, que correspondeu a 33 proteínas distintas. As proteínas identificadas pertenciam a diferentes categorias funcionais envolvidas no metabolismo energético, do carbono, de aminoácido, de carboidratos, de lipídeos, no estresse oxidativo e no turnover proteico e chaperonas moleculares. Algumas isoformas da proteína ATPase apresentaram menor abundância em relação ao controle, em ambos tratamentos. Enquanto a maioria das proteínas de choque térmico 70 apresentaram maior abundância que o controle. As proteínas piruvato desidrogenase, fosfoglicerato mutase e a ATP sintase aumentaram abundância e as proteínas aldeído desidrogenase e carbonil redutase diminuíram abundância no tratamento com berenil. No tratamento com Kunitz as proteínas arginina quinase e a choque térmico Hsp 90 aumentaram a abundância enquanto as proteínas enoil-CoA hidratase, ATP sintase e a aspartato aminotransferase diminuíram a abundância. A presença dos inibidores berenil e Kunitz na lagarta A. gemmatalis alterou o perfil de proteínas no intestino em ambos tratamentos. The soybean caterpillar, Anticarsia gemmatalis, is considered one of the main pests of soybean crop. The damage caused by the attack of this insect associated to the economic relevance of soybean cultivation to Brazil and to the world foments the search for alternatives in the control of this insect. Strategies for control of insect pests based on the use of protease inhibitors have been studied, knowledge of digestive enzymes and insect metabolism has been shown to be fundamental. In this context, this work aimed to identify proteins with differential abundance in the midgut of A. gemmatalis in response to protease inhibitor, aiming to obtain data that could contribute to scientifically elucidate the adaptive mechanism of A. gemmatalis in response to protease inhibitors in the interaction plant and insect. Midgut of caterpillars fed with a berenyl protease inhibitor and Kunitz type soybean inhibitor were dissected at 6h, 12 h, 24 h and 48 h and the proteins extracted. Protein extracts were submitted to two-dimensional gel electrophoresis. The differential abundance spots were digested and the peptides analyzed in the MALDI / TOF-TOF mass spectrometer. Fragmentation spectra (MS / MS) were searched in databases using the MASCOT tool, and the identified proteins were validated using the Scaffold software. A total of 44 protein spots were identified in the berenyl and Kunitz treatments at different times, which corresponded to 33 distinct proteins. The proteins identified belong to different functional categories involved in the energy, amino acids, carbohydrates, lipids metabolism, oxidative stress, protein turnover and chaperones. Some ATPase protein isoforms presented lower abundance than control in both treatments. While most heat shock 70 proteins were more abundant than control. Pyruvate dehydrogenase, phosphoglycerate mutase and ATP synthase proteins increased abundance, and aldehyde dehydrogenase and carbonyl reductase proteins decreased abundance in berenyl treatment. In Kunitz treatment arginine kinase and heat shock Hsp 90 proteins increased abundance, while enoyl-CoA hydratase, ATP synthase and aspartate aminotransferase proteins decreased abundance. Berenyl and Kunitz inhibitors in caterpillar A. gemmatalis altered the protein profile in the midgut in both treatments.

FACEPE Proteases microbianas são muito utilizadas na indústria, tendo papel chave no desenvolvimento de diversos processos biotecnológicos. As colagenases são um grupo de proteases que degradam a tripla hélice do colágeno e são utilizadas na indústria farmacêutica, do couro e no amaciamento de carnes. O objetivo do presente trabalho foi identificar a linhagem Streptomyces sp. através de características morfológicas, bioquímicas e técnicas moleculares, avaliar a produção de proteases em diversos substratos (gelatina, resíduo de café, farinha de soja, farelo de trigo e penas de galinha), produzir e purificar colagenase obtida a partir de fermentação submersa, caracterizar a colagenase quanto à influência da temperatura, pH, íons metálicos, surfactantes e inibidores enzimáticos, produzir peptídeos a partir da degradação de colágeno tipo I e tipo V, verificar o potencial anticoagulante dos peptídeos obtidos, e avaliar a cinética e termodinâmica da hidrólise de azocoll pela colagenase purificada. A actinobactéria foi identificada como Streptomyces antibioticus UFPEDA3421, e apresentou produção de enzimas proteolíticas quando cultivada em fermentação submersa em todos os substratos testados. As maiores atividades enzimáticas obtidas foram: protease (46,62 U/mL) em meio contendo farinha de soja, queratinase (32,96 U/mL) em meio contendo penas de galinha e protease fibrinolítica (9,3 U/mL) em meio com farinha de soja. Essas proteases não apresentaram afinidade pela resina de troca iônica e foram coletadas na fração não adsorvida. A colagenase foi produzida nos cinco substratos, apresentando maior atividade em meio contendo resíduo de café (377,50 U/mL), a partir do qual foi purificada por cromatografias de troca iônica e exclusão molecular, com atividade colagenolítica de 150,50 U/mL, atividade específica de 16.723,00 U/mg, fator de purificação de 136,7 e massa molar de 41,28 kDa. A caracterização da colagenase demonstrou que a enzima é uma metaloprotease neutra, com máxima atividade a 60 °C, pH 7.0, e possui afinidade por íons metálicos mono- e di- valentes, além de ser potencializada na presença dos surfactantes Tween 20, Tween 80 e Triton X-100. A degradação de colágeno tipo V produziu peptídeos com atividade anticoagulante que retardaram o tempo de coagulação sanguínea em 88s em comparação ao controle. Na hidrólise do azocoll, os parâmetros cinéticos obtidos da colagenase foram: Km = 27,14 mg/mol; Vmax = 714,29 mg/mol/min; Kcat = 79,9 s⁻¹, e; Kcat / Km = 2,95 ml/mg.s. Os parâmetros termodinâmicos apresentaram os seguintes valores (a 25 °C): Energia de Ativação (Ea) = 65,224 KJ/mol; Entalpia (ΔH) = 62,75 KJ/mol; Entropia (ΔS) = 1,96 J/mol; Energia Livre de Gibbs (ΔG) = 62,16 KJ/mol; Energia Livre na Ligação ao Substrato (ΔGᴇ₋s) = 8,18 KJ/mol, e; Energia Livre no Estado de Transição (ΔGᴇ₋ᴛ) = -2,64 KJ/mol. Os dados mostram potencial na produção de proteases, especificamente de colagenase, em diversos substratos pela linhagem Streptomyces antibioticus UFPEDA3421, em especial no cultivo em meio de cultura contendo resíduo de café, um resíduo abundante no Brasil. A colagenase purificada apresenta características que a qualificam para uso na produção de peptídeos bioativos. Microbial proteases are widely used in industry, playing a key role in the development of various biotechnological processes. Collagenases are a group of proteases that degrade the triple helix structure of collagen and are used in pharmaceutical, leather and meat industry. The aim of the present work was to identify the strain Streptomyces sp. using morphological, biochemical and molecular techniques, to evaluate the production of proteases in various substrates (gelatin, coffee powder residue, soybean meal, wheat bran, and chicken feathers), to produce and purify collagenase obtained from submerged fermentation, to characterize the influence of temperature, pH, metallic ions, surfactants and enzymatic inhibitors on collagenase activity, produce peptides from degradation of type I and type V collagen, to verify the anticoagulant potential of the peptides obtained, and to evaluate the kinetics and thermodynamics of the azocoll hydrolysis by the purified collagenase. The actinobacteria was identified as Streptomyces antibioticus UFPEDA3421, and showed proteolytic enzyme production when grown in submerged fermentation in all substrates tested. The highest enzymatic activities were: protease (46.62 U/mL) in medium containing soybean meal, keratinase (32.96 U/mL) in medium containing chicken feathers and fibrinolytic protease (9.3 U/mL) in medium with soybean meal. These proteases had no affinity for the ion exchange resin and were collected in the non-adsorbed fraction. The collagenase was produced in the five substrates analyzed, showing higher activity in medium containing coffee powder residue (377.50 U/mL), from which it was purified by ion exchange and molecular exclusion chromatography, with a collagenolytic activity 150.50 U/mL, specific activity 16,723.00 U/mg, purification factor 136.7 and molar mass 41.28 kDa. The characterization of collagenase demonstrated that the enzyme is a neutral metalloprotease, with maximum activity at 60 °C, pH 7.0, has affinity for mono- and di-valent metal ions, and is potentiated in the presence of Tween 20, Tween 80 and Triton X-100 surfactants. Degradation of type V collagen produced peptides with anticoagulant activity that delayed blood coagulation time in 88s when compared to control. In the azocoll hydrolysis, the kinetic parameters obtained from collagenase were: Km = 27,14 mg/mol; Vmax = 714,29 mg/mol/min; Kcat = 79,9 s⁻¹, e; Kcat/ Km = 2,95 ml/mg.s. The thermodynamic parameters showed the following values (at 25 °C): Activation energy (Ea) = 65,224 KJ/mol; Enthalpy (ΔH) = 62,75 KJ/mol; Entropy (ΔS) = 1,96 J/mol; Gibbs free energy of activation (ΔG) = 62,16 KJ/mol; Free energy of substrate binding (ΔGᴇ₋s) = 8,18 KJ/mol, and; Free energy for transition state formation (ΔGᴇ₋ᴛ) = -2,64 KJ/mol. The data show potential in the production of proteases, specifically collagenase, in several substrates by the strain Streptomyces antibioticus UFPEDA3421, especially in culture medium containing coffee residue, a residue abundant in Brazil. Purified collagenase has characteristics that qualify it for use in the production of bioactive peptides.

CAPES Proteases estão entre as mais produzidas no mundo. Dentre elas a tripsina é a mais utilizada em diversas áreas da biotecnologia. A purificação de biomoléculas por partículas magnéticas baseia-se na interação dos ligantes, componentes químicos presentes na superfície das partículas, com a molécula alvo. Neste trabalho, foram preparados dois compósitos magnéticos: o primeiro a partir de azocaseína e óxido de ferro (mAzo) e o segundo, a partir de partículas de óxido de ferro revestidas com polianilina tratadas com azocaseína (mPANI-Azo). Os novos compósitos foram avaliados como matriz para a purificação de tripsina de extrato bruto de Tilapia (Oreochromis niloticus). Uma caracterização físico-química do compósito magnético (mAzo) foi também realizada para descrever as principais características do material sintetizado. Os resultados da purificação mostraram que as partículas mAzo e mPANI-Azo permitiram purificar o extrato bruto de peixe, sem nenhum pré tratamento, 61 vezes e 43 vezes, respectivamente. A eletroforese (SDS-PAGE) da alíquota purificada revelou uma banda a 24 kDa, valor compatível com tripsina obtida de vísceras de tilapia. As analises das técnicas de caracterização (MEV-EDX, DRX, FTIR e medidas de magnetização) evidenciaram o sucesso da síntese do compósito magnético (mAzo). Além disso, foi observado que as partículas mAzo são heterogêneas e apresentam rápida resposta magnética. Por fim, ao purificar a tripsina com precipitação, dialise e mAzo obteve-se um aumento da purificação de até 220 vezes. Concluímos que o compósito magnético mAzo mostrou ser uma matriz eficiente para a purificação de e o reuso do manteve a qualidade da purificação. Nesta pesquisa desenvolvemos uma técnica de purificação simples, eficaz e econômica que pode ser utilizada para a purificação de proteases utilizando as partículas mAzo como matriz. Proteases are among the most produced in the world. Among them trypsin is the most used in several areas of biotechnology. The purification of biomolecules by magnetic particles is based on the interaction of the binders, chemical components present on the surface of the particles, with the target molecule. In this work, two magnetic composites were prepared: the first from azocasein and iron oxide (mAzo) and the second from iron oxide particles coated with azacasein-treated polyaniline (mPANI-Azo). The new composites were evaluated as a matrix for the purification of trypsin from Tilapia (Oreochromis niloticus) crude extract. A physical-chemical characterization of the magnetic composite (mAzo) was also performed to describe the main characteristics of the synthesized material. The results of the purification showed that the mAzo and mPANI-Azo particles allowed to purify the fish crude extract, without any pretreatment, 61 times and 43 times, respectively. Electrophoresis (SDS-PAGE) of the purified aliquot revealed a band at 24 kDa, trypsin compatible value obtained from tilapia viscera. The analysis of the characterization techniques (SEM-EDX, XRD, FTIR and measurements of magnetization) evidenced the success of the synthesis of the magnetic composite (mAzo). In addition, it was observed that the mAzo particles are heterogeneous and have a fast magnetic response. Finally, after precipitation, dialysis and mAzo treatment, a purification increase up to 220 times was obtained. We conclude that the magnetic composite mAzo has proved to be an efficient matrix for the purification and reuse of the quality of the purification. In this research we have developed a simple, efficient and economical purification technique that can be used for the purification of proteases using the mAzo particles as matrix.

Tese (doutorado)—Universidade de Brasília, Faculdade de Ceilândia, Programa de Pós-Graduação Stricto Sensu em Ciências e Tecnologias e Saúde, 2017. A doença de Chagas é endêmica da América Latina, tem como agente etiológico o parasito Trypanosoma cruzi. O tratamento da doença durante a fase crônica gera diversos efeitos colaterais. Nesse sentido, as proteases de tripanossomatídeos têm sido investigadas na busca de novos alvos terapêuticos. A dipeptidil peptidase 8 de Trypanosoma cruzi (DPP8Tc) é uma enzima que faz parte das prolyloligopeptidases, essa família possui membros com conhecida relevância médica, dentre elas está a enzima ortóloga DPPIV humana, cuja inibição é utilizada como uma das formas de tratamento da diabetes tipo II. A DPP8Tc, foi investigada neste trabalho por meio da caracterização bioquímica e por nocaute físico dos alelos do gene dpp8tc do parasito T. cruzi da cepa CL Brener. Os resultados obtidos indicam que a DPP8Tc é uma enzima dimérica, solúvel, ativa em tampão tris 50mM em pH situado entre 7,5 e 8,0 e melhora sua atividade com aditivos. A temperatura ótima para atividade enzimática situa-se em torno dos 28ºC e substrato específico, Gly-Pro-AMC. Com o uso deste substrato, foram obtidos os parâmetros KM de 10,78 μM, Kcat de 1,67x10-3 s-1, Vmax 4,6x10-5 μmol de AMC liberado/min e Kcat/KM:1,55x10μM-1s-1. Os inibidores clássicos de proteases exercem baixa inibição enzimática da DPP8Tc. Em contrapartida, os inibidores de DPPIV exercem maior taxa de inibição entre eles o IB-7, o mais potente, inibe em 96,59% da DPP8Tc, com constante de inibição (Ki) de 8,2 nM. Outros inibidores da DPPIV apresentaram os seguintes ICs-50: KS (IV) com 95,47 nM, IB-4 com 484,9 nM e por último, KAII-23 com 2,7μM. A inibição enzimática ocorre principalmente por inibidores que possuem o grupo adamantil na sua composição química, semelhante à composição do inibidor vidaglipitina da DPPIV humana. A atividade ótima ocorre em torno dos 28ºC, temperatura requerida à sobrevivência da forma epimastigota, indicando potencial função no barbeiro. O nocaute simples do gene dpp8tc, diminuiu em 50% a atividade enzimática, em 20% a fluorescência e aumentou 2,9% a taxa de infecção do parasito DPP8Tc+. As diferenças existentes nas regiões UTRs 5’ e 3’ dos alelos do gene DPP8Tc, impediu o sucesso do nocaute duplo, sendo necessária a construção específica de um cassete direcionado para o nocaute do segundo alelo, visando conhecer por completo a função da DPP8Tc de Trypanosoma cruzi. Chagas disease is endemic in Latin America, and has the parasite Trypanosoma cruzi as its etiological agent. Treatment of the disease during the chronic phase generates several side effects. In this sense, trypanosomatid proteases have been investigated in the search for new therapeutic targets. The dipeptidyl peptidase 8 of Trypanosoma cruzi (DPP8Tc) is an enzyme that is part of the prolyloligopeptidases, this family has members with known medical relevance, among them is the human orthological enzyme DPPIV, whose inhibition is used as one of the forms of treatment of type diabetes II. DPP8Tc was investigated in this work through the biochemical characterization and physical knockout of the dpp8tc gene alleles of the T. cruzi parasite of the CL Brener strain. The results indicate that DPP8Tc is a soluble dimeric enzyme active in 50mM tris buffer at pH between 7.5 and 8.0 and improves its activity with additives. The optimal temperature for enzymatic activity is around 28ºC and specific substrate, Gly-Pro-AMC. With the use of this substrate, KM parameters of 10.78 μM, Kcat of 1.67x10-3 s-1, Vmax 4.6x10-5 μmol of released AMC / min and Kcat / KM were obtained: 1.55x10μM-1s -1. Classical protease inhibitors exert low enzymatic inhibition of DPP8Tc. In contrast, DPPIV inhibitors exert a greater inhibition rate among them, the more potent IB-7 inhibits in 96.59% of DPP8Tc, with inhibition constant (Ki) of 8.2 nM. Other DPPIV inhibitors showed the following ICs-50: KS (IV) with 95.47 nM, IB-4 with 484.9 nM and, finally, KAII-23 with 2.7 μM. Enzymatic inhibition occurs primarily by inhibitors that possess the adamantyl group in their chemical composition, similar to the composition of the human DPPIV vidagliptin inhibitor. The optimal activity occurs around 28ºC, temperature required to the survival of the epimastigote form, indicating potential function in the barber. The simple knockout of the dpp8tc gene, decreased enzyme activity by 50%, fluorescence by 20% and the infection rate of the parasite DPP8Tc+- increased by 2.9%. Differences in the 5 'and 3' UTRs regions of the DPP8Tc gene alleles prevented the success of double knockout, necessitating the specific construction of a cassette directed to the knockout of the second allele, in order to fully understand the function of DPP8Tc from Trypanosoma cruzi.