Memoria para optar al Título Profesional de Médico Veterinario / El agente causal de la enfermedad de Chagas o tripanosomiasis americana, es el parásito hemoflagelado Trypanosoma cruzi. Esta enfermedad es de carácter endémico en América Latina; se estima un promedio de 10-15 millones de personas infectadas y 75-90 millones en riesgo de contraer la enfermedad. T. cruzi posee un ciclo de vida indirecto y se presenta en cuatro formas celulares; epimastigote, forma extracelular replicativa y no infectiva, tripomastigote metacíclico y tripomastigote sanguíneo, formas no replicativas e infectivas y amastigote, forma intracelular replicativa. T. cruzi es capaz de sobrevivir al daño oxidativo del DNA generado por especies reactivas de oxígeno y nitrógeno (ROS/RNS) producidas por su propio metabolismo, así como aquellas producidas en el intestino del insecto vector y en células del hospedero mamífero. Este daño sería reparado mediante la actividad de las endonucleasas apurínicas/apirimidínicas (endonucleasas AP) de la vía de escisión de bases (BER). T. cruzi presenta en su genoma secuencias que codificarían para endonucleasas AP, entre ellas TcAP1, homóloga de APE1 humana y TcAP2, homóloga de APE2 humana y de Apn2 de Schizosaccaromyces pombe. La expresión de estas proteínas podría ser fundamental para la sobrevida del parásito, tanto en el vector triatomino como en el hospedero mamífero. Para un estudio sistemático de las características de estas enzimas, se requiere obtenerlas en el más alto grado de pureza desde el parásito o por técnicas de ingeniería genética. En esta Memoria de Título se clonaron las secuencias génicas que codifican para TcAP1 y TcAP2 en vectores de expresión para células eucariontes, lo que se confirmó mediante secuenciación automática de DNA. Con estos constructos se transfectaron células S2 de Drosophila melanogaster, levaduras Pichia pastoris y epimastigotes de T. cruzi cepa Dm28c. Sorprendentemente, sólo fue posible expresar ambas endonucleasas en este último modelo. La imposibilidad de expresar ambas endonucleasas AP en los modelos P. pastoris y D. melanogaster podría relacionarse a una variedad insuficiente o limitada de ciertos tRNAs, necesarios para la expresión de TcAP1 y TcAP2, con la consecuente disminución de la traducción de los mRNAs codificantes para ambos genes. Probablemente, los tRNAs que presentan anticodones específicos para la traducción de las endonucleasas AP de T. cruzi sólo se encuentran en concentraciones adecuadas en modelos celulares de expresión filogenéticamente cercanos a este protozoario. Mediante cromatografía de afinidad se intentó purificar TcAP1-GFP a partir de homogeneizados de proteínas totales de epimastigotes transfectados que expresaban esta proteína recombinante. Sin embargo no se logró obtener la proteína recombinante en condiciones nativas de forma pura. Se concluye que la utilización de GFP asociado como proteína de fusión a TcAP1 sólo permite la identificación de la proteína recombinante y no una purificación adecuada de la misma / Proyecto Bicentenario Anillo ACT 112, CONICYT, Chile. Proyecto FONDECYT 1090124.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2017. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). / A Família Triponosomatidae compreende diversas espécies que infectam mamíferos, répteis peixes e plantas. As espécies que acometem cães no Distrito Federal são Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi e Leishmania spp. Sendo parasitas com abrangência epidemiológica por toda a América, a correta identificação das espécies mostra-se fundamental para o diagnóstico preciso e consequente abordagem terapêutica assim como estabelecimento de epidemiologia ecológica e genética populacional das diferentes linhagens de parasitas. Objetivou-se com esse estudo avaliar a ocorrência da infecção por Trypanosoma spp. em cães residentes no Distrito Federal (DF), Brasil e sua distribuição por região administrativa analisada e habitat. Para isso, foram analisadas por meio da PCR 206 amostras de sangue de cães residentes no DF. Dessas amostras, 16 (7,7%) foram positivas para a infecção por Trypanosoma spp. em diferentes habitats (domiciliar, peridomiciliar e rural ou silvestre) e regiões administrativas do DF, não demonstrando diferença (p>0,05) entre os habitats ou regiões estudadas. A ocorrência da infecção nas amostras estudadas demonstrou uma baixa taxa de infecção por Trypanosoma spp. na região do Distrito Federal. / The Family Tripanosomatidae comprises several species who infects mammals, reptiles, fishes and plants. The species who infect dogs in Federal District are Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi and Leishmania spp. As parasites with epidemiological scope for all America continent, the right species’ identification show itself fundamental for precise diagnosis as to provide the right therapeutic approach, ecological epidemiology and genetics population of the different parasites lineage. The research’s objectives was to evaluate the occurrence of infection by Trypanosoma spp. in resident dogs on Federal District, Brazil for habitat and administrative region distribuition. Were analyzed by PCR 206 dogs’ blood samples residents in Federal District. These samples, 16 (7,7%) were positive for infection by Trypanosoma spp. in different habitats (domicile, peridomiciliary, and rural or wild) and administrative regions of Federal District, doesn’t showing difference (p>0,05) between the habitats or administrative regions. The occurrence of infection in studied samples reveal results bellow expectation for infection by Trypanosoma spp. in Federal District area.

No description available.

Hexokinase is the first enzyme in glycolysis, a major pathway for the generation of energy in all eukaryotes. Mammalian cells have four isoforms (I, II, III, IV) that have different tissue distribution and kinetic properties. Among all isoforms, human hexokinase II (hHKII) has been found to be implicated in many cancers with an increased expression which serves a dual role. First, it maintains the high glycolytic rate of malignant cells (Warburg effect) and second it prevents apoptosis when is bound to mitochondria. Trypanosoma brucei is a parasite that causes Human African Trypanosomiasis (HAT) and has two isoforms with extensive sequence similarity (98%), TbHKI (active form) and TbHK2 (inactive form). The bloodstream-form parasites (BSF) depend exclusively on glycolysis for their survival. The enzyme from both organisms is a validated target for drug-discovery against both cancer and HAT. The aim of the present study is the discovery of novel and specific inhibitors of the enzymes based on their structure. Structure-based drug discovery is commonly used in pharmaceutical companies to aid in the discovery of potent lead compounds. In silico studies were performed in this project using the known crystal structure of human hexokinase I and a model of TbHKI generated by the protein modelling tool Phyre2. The docking programs, AutoDock (AD) and AutoDock Vina (Vina), were chosen to perform the docking of ~3 million compounds to the target molecules and scoring functions calculated the predicted binding affinities of each compound. In total, 28 compounds were purchased to test on the target molecules. In the experimental part of the project, the two enzymes were cloned, expressed and purified. hHKII was successfully purified giving a high yield of active and pure protein. The protein was characterised using many biophysical methods to establish the oligomeric state, the homogeneity and the secondary structure. Crystallisation trials failed and for this reason, N and C domains of the hHKII were purified separately. Unfortunately, the domains also failed to crystallise thus SAXS data were collected and analysed to gain information of their shape at low resolution. A novel inhibition assay was developed and used to identify four weak inhibitors against full length hHKII. TbHKI was difficult to express in a soluble form as most of the protein was expressed in inclusion bodies. The purification resulted in a small amount of active protein that was used entirely for biochemical assays. Four compounds were purchased from the docking of the TbHKI model and one was found to inhibit the enzyme over 65% at 100 μM. Because the active site of both enzymes (hHKII, TbHKI) is well conserved the compounds from hHKII docking were also screened against the TbHKI. Four compounds were found to inhibit this enzyme while one of them was also an inhibitor for human isoform. The remaining three were specific for inhibition of TbHKI.

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Tesis presentada a la Universidad de Chile para optar al grado académico de Doctor en Bioquímica / Trypanosoma cruzi (T. cruzi) es un protozoo parásito, agente causal de la enfermedad de Chagas, endémica en el continente americano. Presenta un ciclo de vida complejo, infectando tanto hospederos mamíferos como insectos triatominos (hematófagos) y presentando cuatro formas celulares: tripomastigotes sanguíneos, epimastigotes, amastigotes y tripomastigotes metacíclicos. En humanos, la infección por T. cruzi en etapas avanzadas puede provocar serios trastornos cardíacos y digestivos, con un alto costo médico, y que pueden ser mortales de no ser tratados adecuadamente. Hasta el momento, no existe una cura definitiva para la infección crónica por T. cruzi. Este parásito sobrevive al efecto de especies reactivas de oxígeno y nitrógeno (ROS/RNS) generadas tanto por células del hospedero mamífero como por la digestión de la sangre en el insecto hematófago. Se ha propuesto que estas especies reactivas dañan el DNA del parásito y que éste sobrevive activando el mecanismo de reparación del DNA por escisión de bases (vía BER). Este es un proceso muy conservado, que presenta dos sub-vías: la vía corta, en la que se reemplaza un nucleótido, o la vía larga, donde se insertan dos o más nucleótidos. En esta vía de reparación del DNA participan distintas enzimas, como DNA glicosilasas, AP endonucleasas (APE), DNA polimerasas, flap endonucleasas (FEN1) y DNA ligasas. Hasta el momento, se ha demostrado que: 1) el DNA del parásito es dañado por ROS/RNS; 2) T. cruzi tiene la capacidad de reparar el daño producido por estas especies reactivas; 3) metoxiamina, un inhibidor de AP endonucleasas, aumenta la muerte celular provocada por ROS/RNS; 4) el parásito tiene dos DNA glicosilasas y dos AP endonucleasas activas. A la fecha de realización de esta Tesis no se había definido la presencia de flap endonucleasas así como de las diferentes vías (corta y/o larga) de reparación del DNA por escisión de bases en T. cruzi. Las enzimas flap endonucleasas (FEN1) se caracterizan por reconocer sustratos de DNA con estructuras específicas, que se generan en diversos procesos celulares, como la replicación del DNA o su reparación mediante la vía larga del mecanismo BER. Estudios en diversos eucariontes han demostrado su importancia en la reparación del daño oxidativo al DNA, así como en la mantención de la integridad genómica. En esta Tesis se investigó la presencia y actividad de una enzima flap endonucleasa de T. cruzi (TcFEN1), su expresión, su localización celular y su rol en la proliferación y en la sobrevida del parásito frente a condiciones de estrés oxidativo. Se demostró la presencia de actividad flap endonucleasa en distintas formas celulares de T. cruzi y se describieron algunas de sus propiedades. Paralelamente se encontró una secuencia correspondiente a una flap endonucleasa en el genoma publicado de T. cruzi, a partir de la cual se crearon plásmidos que permitieron la expresión en epimastigotes de T. cruzi de proteínas de fusión TcFEN1-GFP que también presentaron actividad flap endonucleasa y que se localizaron en el núcleo de epimastigotes del parásito, según estudios de microscopia de fluorescencia. Por otro lado, se observó un incremento en la proliferación de los epimastigotes que expresaban TcFEN1-GFP, así como una mayor sobrevida frente a la exposición sostenida a H2O2 respecto a lo observado en parásitos control. En base a los resultados obtenidos, TcFEN1 se presenta como protagonista en procesos claves para la infección por T. cruzi como son la proliferación y resistencia frente a daño oxidativo, por lo que desarrollar inhibidores específicos de TcFEN1 podría potenciar el efecto citotóxico del daño oxidativo al DNA generado por las células del hospedero, así como potenciar el tratamiento de la infección por T. cruzi por drogas convencionales, actualmente muy poco eficientes en el tratamiento de la enfermedad de Chagas crónica / Trypanosoma cruzi (T. cruzi) is a protozoan parasite, the etiological agent of Chagas´s disease, endemic in the American continent. It presents a complex life cycle, infecting both mammalian and triatomine insects and presenting four cellular forms: blood trypomastigote, epimastigote, amastigote and metacyclic trypomastigote. In humans, T. cruzi infection in advanced stages can cause serious cardiac and digestive disorders, with a high medical cost, and can be fatal if not treated properly. To date, there is no definitive cure for chronic T. cruzi infection. This parasite survives the effect of reactive oxygen and nitrogen species (ROS / RNS) generated by both mammalian host cells and by the digestion of blood in the hematophagous insect. It has been proposed that those reactive species damage the DNA of the parasite but it survives by activating the mechanism of DNA repair by base excision (BER pathway). This is a highly conserved process, which has two sub-pathways: the short one, where a single nucleotide is replaced, or the long one, where two or more nucleotides are inserted. Different enzymes, such as DNA glycosylases, AP endonucleases (APE), DNA polymerases, flap endonucleases (FEN1) and ligases are involved in that DNA repair pathway. To date, it has been shown that: 1) the DNA of the parasite is damaged by ROS / RNS; 2) T. cruzi repairs the damage produced by these reactive species; 3) methoxyamine, an inhibitor of AP endonucleases, increases ROS/RNS-induced parasite cell death and 4) T. cruzi presents two DNA glycosylases and two active AP endonucleases. At the date of this thesis, the presence of the different pathways (short and / or long) of DNA repair by base cleavage in T. cruzi had not been defined. Flap endonuclease enzymes (FEN1) are characterized by recognizing DNA substrates with specific structures, which are generated in various cellular processes, such as DNA replication or repair by the long path of the BER mechanism. Studies in various eukaryotes have demonstrated their importance in the repair of oxidative damage to DNA, as well as in the maintenance of genomic integrity. In this thesis the presence and activity of a T. cruzi flap endonuclease enzyme (TcFEN1), its expression, its cellular localization and its role in the proliferation and survival of the parasite against oxidative stress conditions were investigated. The presence of TcFEN1 was demonstrated in different cellular forms of T. cruzi and some of its properties were described. In parallel, a sequence corresponding to a flap endonuclease was found in the published T. cruzi genome, from which plasmids were created that allowed expression in T. cruzi epimastigotes of TcFEN1-GFP fusion proteins that also had flap endonuclease activity and were located in the epimastigotes nucleus, according to fluorescence microscopy. On the other hand, there was an increase in the proliferation of epimastigotes expressing TcFEN1-GFP, as well as a higher survival to H2O2 sustained exposure than observed in control parasites. Based on the results obtained, TcFEN1 is presented as a protagonist in key processes for infection by T. cruzi, such as proliferation and resistance to oxidative damage. Therefore, the development of specific inhibitors of TcFEN1 may enhance the cytotoxic effect of oxidative DNA damage generated by the host cells, as well as potentiate the treatment of T. cruzi infection by conventional drugs, currently very ineffective in the treatment of chronic Chagas´disease / Conicyt; Fondecyt

Thesis (doctoral)--Eberhard-Karls-Universität zu Tübingen, 1994.

No description available.

No description available.