Transsulfuration is an active pathway for cysteine biosynthesis in Trypanosoma rangeli

BACKGROUND: Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. METHODS: We cloned and characterised genes coding for a cystathionine β-synthase (CβS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. RESULTS: Our results show that T. rangeli CβS (TrCβS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CβS. Moreover, CβS biochemical assays revealed that the T. rangeli CβS enzyme also has serine sulfhydrylase activity. CONCLUSION: These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite

Caracterización de las excusas del servicio del personal uniformado que conforma la región No 7 de la Policía Nacional.

Caracterizar las excusas del servicio del personal que conforma la Región Nº 7 de la Policía Nacional.El presente proyecto de grado para ostentar el título de especialista gerencia del riesgo Laboral y SGSST, está encaminado a abordar de manera lógica y concluyente el problema de investigación, el cual tiene como línea de investigación, la gestión de los actores en la seguridad y salud en el trabajo, describiendo puntualmente la necesidad que tiene la Policía Nacional de establecer estudios que orienten al nivel directivo y administrativo para establecer las estrategias que mitiguen problemáticas que afectan el desarrollo misional. El estudio está fundamentado en la aplicación de métodos de análisis de la problemática definida referente a la generación de las excusas laborales por motivos relacionadas a las actividades propias del servicio o fuera de él , apoyada de la metodología de investigación como referente técnico y científico para validar los resultados

Consecuencias sociojurídicas de la prestación informal del transporte público

La prestación informal del servicio público de transporte es de relevancia nacional en razón a la proliferación de este servicio y a la correlativa demanda de parte de la ciudadanía, hecho que ha desencadenado problemáticas en diversos campos. En tanto, se pretendió establecer las posibles consecuencias sociales y jurídicas que se presentan con ocasión a esa actividad a través de un análisis cualitativo y cuantitativo, como analítico descriptivo de los actores involucrados, incidencia del marco legal colombiano y el desarrollo jurisprudencial de las altas cortes colombianas. Producto del cual se determinó la responsabilidad extracontractual del Estado por omisión al deber de control sobre esta actividad; la responsabilidad civil de los transportadores informales por los posibles daños que causen a los usuarios, como los delitos en que se pueden ver inmersos; a su vez se identificó la inexistencia de una política pública eficiente que contrarreste este fenómeno social. Permitiendo concluir que los municipios objeto de estudio pueden ser acreedores de responsabilidad administrativa por el irrisorio control que realizan a esta actividad informal, y en unos municipios aun siendo viable la formalización, por carencia de voluntad no se realiza; correlativamente, quienes ejecutan esta actividad no son conscientes de las consecuencias jurídicas que acarrean sus prácticas; todo esto por factores sociales como el desempleo y el bajo nivel educativo de quienes en esta labor encuentran el sustento para sus hogares

Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

Understanding of the dynamics and distribution of Leishmania in the human host is fundamental to the targeting of control measures and their evaluation. Amplification of parasite gene sequences in clinical samples from cutaneous leishmaniasis patients has provided evidence of Leishmania in blood, other tissues and sites distinct from the lesion and of persistence of infection after clinical resolution of disease. However, there is uncertainty about the interpretation of the presence of Leishmania DNA as indicative of viable parasites. Because RNA is short-lived and labile, its presence provides an indicator of viability. We amplified Leishmania 7SLRNA, a molecule involved in intracellular protein translocation, to establish viability and estimate parasite load in blood monocytes, tonsil swab samples, and tissue fluid from healthy skin of patients with dermal leishmaniasis. Results showed that during active dermal leishmaniasis, viable Leishmania are present in blood monocytes, tonsils and normal skin in quantities similar to that in lesions, demonstrating widespread dissemination of infection and subclinical involvement of tissues beyond the lesion site. Leishmania 7SLRNA will be useful in deciphering the role of human infection in transmission

Análise funcional de genes associados ao transporte e ao estresse oxidativo em macrófagos humanos infectados com Leishmania braziliensis e tratados com glucantime

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014As Leishmanioses afetam cerca de 15 milhões de pessoas e constiuem um importante problema de saúde pública mundial. O controle da parasitose baseia-se principalmente na quimioterapia, usando como medicamento de primeira escolha o antimonial pentavalente SbV (Glucantime®). O surgimento de resistência aos fármacos de uso clínico, a ausência de vacinas eficazes, a propagação de vetores resistentes aos inseticidas tornam o controle das Leishmanioses difícil e complexo. Para entender melhor o papel dos macrófagos na Leishmaniose humana e estabelecer estratégias para prevenir os efeitos nocivos da Leishmania, dados sobre os padrões de expressão de genes e proteínas, em Macrófagos Derivados de Monócitos de Humanos (MDM) infectados com Leishmania sob condições de tratamento, são essenciais. Neste estudo, foram analisadas as alterações nos níveis de expressão de genes e proteínas de macrófagos humanos, durante a infecção in vitro por Leishmania braziliensis e tratamento com Glucantime usando qPCR-arrays, ensaios de Western blot, microscopia confocal e ensaios de inibição usando siRNA. Os resultados indicam que a infecção com L. braziliensis e o tratamento SbV conjuntamente, modulam a expressão de genes em macrófagos, em particular aqueles envolvidos na via de biossíntese da glutationa. Análises de correlação permitiram determinar superexpressão dos marcadores GSTP1, GCLM, GSR, GSS, TRX e ABCB5 tanto nos níveis do mRNA quanto das proteínas, em MDM de humanos infectados e tratados quando comparados ao grupo controle. O estudo da localização subcelular mostrou que o transportador ABCB5 apresenta uma localização predominantemente fagolisossomal, indicando que essa proteína poderia estar envolvida no transporte de Glucantime. Nos ensaios de inibição selectiva o silenciamento dos genes gstp1, gss e abcb5 aumentaram o efeito leishmanicida in vitro do SbV induzindo uma redução da sobrevivência intracelular de L. brazilienisis em macrófagos THP-1. Essa resposta foi igualmente confirmada para o gene gstp1 onde os resultados de tempo e concentração dependente mostraram que a redução da sobrevivência intracelular era observada 24h após exposição ao SbV e a uma dose menor. Nossos resultados sugerem que há uma ativação de genes envolvidos na defesa antioxidante em macrófagos humanos como resposta a infecção com L. braziliensis e o tratamento com Glucantime, potencializando a detoxificação do fármaco. Conclui-se que as proteínas GSTP1, GSS e ABCB5 seriam potenciais alvos candidatos para intervenção terapêutica mediante a inibição seletiva destes marcadores. Abstract: Leishmaniasis affects millions of people worldwide and represents a major public health problem. Leishmaniasis control relies primarily on chemotherapy, with the mainstay being pentavalent antimony (SbV) complexed to carbohydrates in the form of sodium stibogluconate (Pentostam®) or meglumine antimoniate (Glucantime®). Due to the emergence of drug resistance, the absence of effective vaccines and the spread of insecticide-resistant vectors, Leishmaniasis control is becoming extremely difficult and complex. To better understand the role of macrophages in human Leishmaniasis and to advance in new strategies to prevent the harmful effects of Leishmania parasites, information about the protein expression patterns in the context of Leishmania-infected human monocyte-derived macrophages (MDM) under drug treatment conditions are essential. In this study, we analyzed the changes in the expression levels of human MDM proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime using qPCR arrays, Western blot, confocal microscopy and siRNA inhibition assays. The results indicate that L. braziliensis infection/SbV treatment modulated the host gene expression, particularly in the glutathione biosynthesis pathway. Comparing results from gene transcription and protein expression analysis, allowed to determine that GSTP1, GCLM, GSR, GSS, TXN, and ABCB5 were strongly up-regulated at both mRNA and protein levels when compared to the control group. Subcellular localization studies showed mostly fagolissosomal location of the ABCB5 transportes, indicating that this protein may be involved in the transport of Glucantime. Selective inhibition assays by gene silencing siRNA assays of gstp1, gss and abcb5 showed an increased in vitro leishmanicidal effect to Sb exposure inducing a decrease in intracellular survival of L. brazilienisis in THP-1 macrophages. This response was also confirmed for gstp1 gene in which the results showed a time and dose-dependent effect in the reduction of intracellular survival where even at 24 hours of exposure to SbV and a lower dose of 8 mg/mL significant intracellular leishmanicidal effects were found. These analyses suggest that, modulations of human MDM by L. braziliensis infection and Glucantime treatment raise the activation of genes participating in antioxidant defense enhancing the drug detoxification. Therefore we concluded that GSTP1, GSS and ABCB5 proteins could be potential targets for therapeutic interventions by selective inhibition of these markers

Knockdown of host antioxidant defense genes enhances the effect of Glucantime on intracellular Leishmania braziliensis in Human Macrophages

Submitted by Manoel Barata ([email protected]) on 2018-02-09T14:16:48Z No. of bitstreams: 1 SoaresKnockdow.pdf: 3136068 bytes, checksum: ebb592618e20a841cc69559f52f383bf (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2018-05-04T14:45:03Z (GMT) No. of bitstreams: 1 SoaresKnockdow.pdf: 3136068 bytes, checksum: ebb592618e20a841cc69559f52f383bf (MD5)Made available in DSpace on 2018-05-04T14:45:03Z (GMT). No. of bitstreams: 1 SoaresKnockdow.pdf: 3136068 bytes, checksum: ebb592618e20a841cc69559f52f383bf (MD5) Previous issue date: 2017Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Departamento de Microbiologia, Imunologia e Parasitologia. Laboratório de Protozoologia. Florianópolis, SC, Brasil. / Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia e Biociências. Florianópolis, SC, Brasil.Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Departamento de Microbiologia, Imunologia e Parasitologia. Laboratório de Protozoologia. Florianópolis, SC, Brasil. / Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia e Biociências. Florianópolis, SC, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Departamento de Microbiologia, Imunologia e Parasitologia. Laboratório de Protozoologia. Florianópolis, SC, Brasil. / Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia e Biociências. Florianópolis, SC, Brasil.Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Departamento de Microbiologia, Imunologia e Parasitologia. Laboratório de Protozoologia. Florianópolis, SC, Brasil. / Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia e Biociências. Florianópolis, SC, Brasil.Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime