Um gene que codifica uma piruvato/indolpiruvato descarboxilase (PDC/IPDC) está presente no tripanossomatídeo de plantas Phytomonas serpens. A PDC atua na fermentação alcoólica, enquanto que a IPDC atua na biossíntese do fitormônio ácido indol-3-acético (AIA). Análises filogenéticas indicam que a PDC/IPDC de P. serpens é monofilética com IPDCs de gama-proteobactérias, sugerindo um evento de transferência horizontal gênica. A análise de meios de cultura de P. serpens confirma a produção de etanol e AIA. A funcionalidade do fitormônio foi confirmada em ensaios de alongamento de hipocótilos de tomateiros. Tomates inoculados com P. serpens mostraram aumento no teor de AIA-amida e -éster conjugados. A atividade PDC foi mostrada em extratos de P. serpens. Concluímos que a PDC/IPDC seria uma 2-cetoácido descaboxilase com atividade catalítica variável para diferentes substratos. A atividade PDC parece ser predominante em P. serpens, representando um mecanismo para oxidar parte do NADH formado na glicólise, principal responsável pela produção de ATP neste organismo. / A gene codifying a pyruvate/indolepyruvate decarboxylase (PDC/IPDC) is present in the plant trypanosomatid Phytomonas serpens. PDC acts in the alcoholic fermentation, whyle IPDC acts in the biosynthesis of the phytohormone indole-3-acetic acid (IAA). Phylogenetic analysis indicate that P. serpens PDC/IPDC is monophyletic with gamma-proteobacteria IPDCs, suggesting a horizontal gene transfer event. Analysis of P. serpens culture media confirms production of ethanol and IAA. The functionality of the phytohormone was confirmed by tomato hypocotyl elongation tests. Tomatoes inoculated with P. serpens showed an increase in the concentration of IAA amide and ester conjugated. PDC activity was shown in P. serpens extracts. We conclude that the PDC/IPDC would be a 2-keto acid decaboxylase with variable catalytic activity for different substrates. The PDC activity appears to be prevalent in P. serpens representing a mechanism to oxidize part of NADH formed in glycolysis, responsible for ATP production in this organism.

Um gene que codifica uma piruvato/indolpiruvato descarboxilase (PDC/IPDC) está presente no tripanossomatídeo de plantas Phytomonas serpens. A PDC atua na fermentação alcoólica, enquanto que a IPDC atua na biossíntese do fitormônio ácido indol-3-acético (AIA). Análises filogenéticas indicam que a PDC/IPDC de P. serpens é monofilética com IPDCs de gama-proteobactérias, sugerindo um evento de transferência horizontal gênica. A análise de meios de cultura de P. serpens confirma a produção de etanol e AIA. A funcionalidade do fitormônio foi confirmada em ensaios de alongamento de hipocótilos de tomateiros. Tomates inoculados com P. serpens mostraram aumento no teor de AIA-amida e -éster conjugados. A atividade PDC foi mostrada em extratos de P. serpens. Concluímos que a PDC/IPDC seria uma 2-cetoácido descaboxilase com atividade catalítica variável para diferentes substratos. A atividade PDC parece ser predominante em P. serpens, representando um mecanismo para oxidar parte do NADH formado na glicólise, principal responsável pela produção de ATP neste organismo. / A gene codifying a pyruvate/indolepyruvate decarboxylase (PDC/IPDC) is present in the plant trypanosomatid Phytomonas serpens. PDC acts in the alcoholic fermentation, whyle IPDC acts in the biosynthesis of the phytohormone indole-3-acetic acid (IAA). Phylogenetic analysis indicate that P. serpens PDC/IPDC is monophyletic with gamma-proteobacteria IPDCs, suggesting a horizontal gene transfer event. Analysis of P. serpens culture media confirms production of ethanol and IAA. The functionality of the phytohormone was confirmed by tomato hypocotyl elongation tests. Tomatoes inoculated with P. serpens showed an increase in the concentration of IAA amide and ester conjugated. PDC activity was shown in P. serpens extracts. We conclude that the PDC/IPDC would be a 2-keto acid decaboxylase with variable catalytic activity for different substrates. The PDC activity appears to be prevalent in P. serpens representing a mechanism to oxidize part of NADH formed in glycolysis, responsible for ATP production in this organism.

La plus grande sensibilité des souris TLR9-/- a révélé le rôle de ce récepteur dans l'infection par Leishmania major. Les cellules dendritiques (DCs) sont activées de manière TLR9-dépendante par l'ADN du L. major et d'autres Trypanosomatidae et non par l'ADN de vertébré. La nature de l'ADN capable d'activer le TLR9 reste controversée quant à la séquence/charpente de l'ADN et l'implication de cofacteurs se liant avec le TLR9 ou l'ADN. Nous avons démontré l'importance de la séquence d'ADN. Contrairement aux génomes de parasites, l'ADN de vertébré présente une contre-sélection des motifs activateurs du TLR9 au profit des motifs inhibiteurs. De plus, l'activation du TLR9 par l'ADN du parasite est augmentée en présence de la protéine HMGB1, qui se fixe mieux sur l'ADN de parasite que de vertébré. La maturation du TLR9 requiert un clivage protéolytique par des protéases endosomales, dont les cathepsines (Cat) B, S, L et l'asparagine endopeptidase (AEP) qui interviennent différemment dans les macrophages et les DCs. Après infection par L. major, nous avons montré que les souris AEP-/-, CatS-/- et CatL-/- ont une pathologie identique aux souris WT, ce qui peut être dû à la redondance de leur fonction. Etonnamment, les souris CatB-/- sont plus résistantes. Leurs lésions et la charge parasitaire dans les ganglions se résolvent plus rapidement, reflétant une réponse immune plus précoce et un contrôle plus rapide de la réaction inflammatoire.En conclusion, ces résultats contribuent à une meilleure compréhension des mécanismes permettant au TLR9 de discriminer entre l'ADN de pathogène et de vertébré et soulèvent le rôle non protecteur de la cathepsine B dans l'infection par L. major.

Parasitic protists of genera Trypanosoma and Leishmania are members of Trypanosomatidae family. In our studies, we investigated genetic influence on infections caused by these parasites in a mouse model. These diseases are on genetic level controlled by quantitative trait loci (QTLs), when the resulting phenotype is controlled by set of genes with small individual effect. As a mouse model for mapping of QTLs controlling these infections, we used recombinant congenic strains (RCS). Each RCS carry unique set of 12.5% of the genome from donor parental strain on genetic background of other parental strain. For mapping of QTLs controlling infections caused by Trypanosoma brucei brucei (T. b. brucei) and Leishmania tropica (L. tropica) and eosinophil infiltration into inguinal lymph nodes after Leishmania major (L. major) infection, we used RCS from CcS/Dem series, where STS is donor strain and BALB/cHeA is strain of genetic background. First, it was necessary to find suitable model strains for mapping. In all three studies, we selected RCS, which exceeded range of monitored phenotype parameters in comparison with any other tested RCS or parental strains. Mice of RCS CcS-11 showed shorter survival after T. b. brucei infection and strain CcS-9 exhibited higher eosinophil infiltration after L. major infection. For...

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1992, härtill 8 uppsatser</p> / digitalisering@umu

This work expands the knowledge on phospholipid metabolism in the kinetoplastid parasites: T. brucei, T. cruzi, Leishmania spp. that cause neglected tropical diseases and the related non-human pathogenic Crithidia fasiculata. As a close relative of parasitic kinetoplasts, specifically Leishmania, it is hypothesised that Crithidia fasiculata possesses a similar lipid biosynthetic capability and therefore represent an attractive model organism. Database mining the Crithidia genome revealed the ability to biosynthesise all of the main phospholipid species. Utilising various lipidomic techniques, a high level of an ω-6 18:3 fatty acid was observed, alongside an uncommon Δ19:0 fatty acid that was later identified to be exclusive attributed to PE species. Sphingolipid metabolism was shown to resemble that of Leishmania and T. cruzi, given the exclusive production of inositol-phosphoceramide species and no sphingomyelin species being observed. Using labelled precursors, Crithidia were seen to uptake and incorporate extracellular inositol into both phosphatidylinositol and inositol-phosphoceramide species. Crithidia were also shown to utilise both the Kennedy pathway and methylation of phosphatidylethanolamine to form phosphatidylcholine. The phospholipidome of T. cruzi revealed several phosphatidylserine species for the first time, suggesting a functional phosphatidylserine synthase. Current knowledge of T.cruzi sphingolipid biosynthesis was also confirmed as only inositol xxxi phosphoceramide species were observed. The identification and subsequent characterisation of novel phosphonolipid species are reported for the first time. Utilising lipidomic methodologies and labelled precursors, the relative contribution of the intracellular inositol pools within bloodstream and procyclic T. brucei towards PI biosynthesis was examined. This highlighted that the synthesis/turnover rates for specific phosphatidylinositol and inositol-phosphoceramide species are unequal. Efforts to optimise media conditions highlighted that under reduced levels of serum/glucose/inositol, bloodstream T. brucei unexpectedly adjusts its inositol metabolism. The procyclic parasite exemplifies this fact, as under inositol/glucose deficient media conditions they appear to have adapted to utilising glucogenesis and inositol de-novo synthesis. This work highlights that these parasites are rapidly dividing, their unique features of lipid metabolism may be exploitable for drug discovery purposes.

Orientador: Marlene Tiduko Ueta / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T04:30:52Z (GMT). No. of bitstreams: 1 Molina_JuliaPereira_M.pdf: 1442066 bytes, checksum: e8ad2a162950fbe267341b74c08ad0f7 (MD5) Previous issue date: 2010 / Resumo: Os tripanossomas já foram reportados em diversas espécies de peixes de água salgada e doce, sendo a maioria das espécies descritas com base nas características morfológicas. Tripanossomas parasitos de peixes são heteroxenos e são transmitidos por hirudíneos. Este trabalho tem como objetivos caracterizar a prevalência e densidade da infecção por Trypanosoma sp. presentes no sangue de três espécies de cascudos do rio Mogi Guaçu, Cachoeira de Emas, município de Pirassununga, SP e caracterizar morfologicamente e por técnicas moleculares os exemplares de Trypanosoma encontrados no sangue dos cascudos. Entre fevereiro de 2008 e fevereiro de 2009 foram coletados 256 exemplares de cascudos sendo 60 Hypostomus albopunctatus, 100 Hypostomus regani e 96 Hypostomus strigaticeps. O sangue foi coletado por punção cardíaca e alíquotas de 8?l foram retiradas para confecção de esfregaço em camada delgada, que foram fixados em metanol e corados por Giemsa, para a determinação da positividade e densidade dos tripanossomas. Após a biometria e coleta do sangue os peixes foram marcados e metade foi acondicionada em um tanque de criação do CEPTA/ ICMBIO para verificação da manutenção da infecção e o restante foi devolvido ao rio. A prevalência geral de tripanossomas foi de 47,6 % e densidade média geral de 0,75 parasitas/ µl de sangue. A prevalência ao longo do ano apresentou diferenças significativas apenas para Hypostomus regani (p=0,0054) e Hypostomus strigaticeps (p=0,0007). A densidade média de parasitas não apresentou diferenças significativas entre as três espécies de peixes analisados, assim como a variação mensal da densidade por espécie de peixes. Hirudíneos do gênero Placobdella sp. foram coletados da superfície do corpo e da boca de H regani e H. strigaticeps para extração do DNA, os oligonucleotídeos utilizados (S-1842 e S-1843) amplificaram um fragmento de 1000pb do gene 18S rRNA dos tripanossomas isolados de hirudíneos coletados em H regani e H. strigaticeps. Dos 19 peixes recapturados após dois meses apenas três se mantiveram positivos. Foram medidos 255 tripanossomas sendo 66 de H. albopunctatus, 64 de H. regani e 95 de H. strigaticeps e os valores foram comparados com os de outras espécies de tripanossomas encontradas em peixes do gênero Hypostomus. Os parasitas encontrados nas três espécies de peixes apresentaram características morfológicas semelhantes. Foi estabelecida a frequência de ocorrência de acordo com a variação do comprimento do corpo para evidência de pleomorfismo. Os dados morfológicos mostram que Trypanosoma sp.1 e Trypanosoma sp.2 podem ser considerados de espécies diferentes; as análises evidenciaram a presença de pleomorfismo nos tripanossomas encontrados nas três espécies de peixes. / Abstract: Trypanosomes have been reported in several species of fish that inhabit salt and fresh waters. The majority of these protozoan species are described based on morphologic features. The parasites are heteroxenic and are transmitted by leeches. This work aims to characterize the prevalence and density of infection by Trypanosoma sp. present in the blood of three species of armored catfish collected from Mogi Guaçu river (Cachoeira das Emas, Pirassununga city, SP) and characterize morphologically and by molecular techniques the trypanosomes found in the blood of catfish. Between february 2008 and february 2009, 256 specimens of armored catfish were collected, being 60 Hypostomus albopunctatus, 100 Hypostomus regain, and 96 Hypostomus strigaticeps. The blood was collected by cardiac puncture and a sample of 8µl was removed to realize blood smears on microscope slides. The slides were fixed in methanol and stained with giemsa for posterior analysis on light microscopy for determination of positive presence and density of trypanosomas. After biometric analysis and blood collection fish were marked half of the fish marked were maintened in a tank of CEPTA/ ICMBIO for future verification of the infeccion maintence and the other half were retorned to the river. It was found a prevalence of 47,6% and the average density of 0,75 parasitas/µl of blood. The prevalence during the year presented significative differences only for Hypostomus regani (p=0,0054) e Hypostomus strigaticeps (p=0,0007). The average density of parasites showed no significant differences among the three fish species analyzed and the monthly variation of density per species of fish. Also, leeches of the genus Placobdella sp. were collected from the surface of the body and mouth of armored catfish for DNA extraction. The primers used (S-1842 and S- 1843) amplified a 1000bp fragment of the 18S rRNA gene of trypanosomes isolated from leeches collected in H regani and H. strigaticeps. Of the 19 fish recaptured after two months only three remained positive. Therein, 255 trypanosomes were measured being 66 H. albopunctatus, 64 H. regani and 95 H. strigaticeps and values were compared with those of other parasite species found in different species of fish. The parasites found in the three species of fish presented similar morphological characteristics. The frequency of occurrence was established according to the variation in body length as evidence of pleomorphism. Nevertheless morphological data show that Trypanosoma sp.1 and Trypanosoma p.2 can be considered distinct species, the analysis revealed the presence of pleomorphism in trypanosomes found in the three species of fish. / Mestrado / Parasitologia / Mestre em Parasitologia