This study was undertaken primarily to examine the skeletal and muscular anatomy of a small species of field mouse, Microtus oregoni serpens Merriam, which inhabits the Puget Sound area of British Columbia and Washington. This species is largely subterranean in its habits and in an attempt to discern the degree of specialization for its habitat comparison was made between serpens and Microtus townsendi townsendi, (Backman) and Microtus longicauda vellerosa (Allen) species which burrow to a limited degree only. A detailed comparison of the external features, skeletal and muscular anatomy of the three species was made and a juvenile specimen of Microtus richardsoni richardsoni, (De Kay) was used for some comparisons. Certain parts of Microtus oregoni serpens were found to be strongly modified in a direction which seems to better adapt the animal to its subterranean mode of life. The external features which are of adaptive significance are the soft plush-like pelage, the short tail and the arrangement of the vibrissae the longest ones being furthest from the snout rather than scattered. The eyes are only about one-half the size of those of the larger species and associated with them is an interesting modification of the orbicularis oculi muscle whereby it is strongly developed doubtless as an aid in preventing dirt from entering the eyes. Microtus oregoni serpens does not have conspicuously enlarged or elongated fore-feet but among the modifications for digging which do occur are the shortening of the limbs and the inclusion of a larger part of them within the body skin. Almost the entire musculature of the anterior segment of the body is more strongly developed in serpens including the muscles of the shoulder, chest, and fore-limbs and to some extent the masticatory musculature and the musculature of the spinal column. The muscles attached to the pectoral girdle show the most marked modifications they are almost universally better-developed in serpens than in townsendi or longicauda. Correlated with a greater development of the pectoralis muscles is the development of a heel on the manubrium sterni and the sternebras in serpens, providing a larger area for muscle attachments. Microtus oregoni serpens seems to represent one of the earliest stages of adaptive specialization of a mammal for subterranean life externally approaching the condition found in specialized burrowers such as the mole but having no radical skeletal modifications. / Science, Faculty of / Zoology, Department of / Graduate

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.

El objetivo de esta tesis fue estudiar tres especies vegetales empleadas por la medicina tradicional: Euphorbia serpens, E. schickendantzii y E. collina, para evaluar la presencia de actividad, diurética, antiparasitaria, citotóxica y antimicrobiana, e identificar y aislar el/los principio/s activo/s. Del material vegetal desecado se realizaron extracciones seriadas con solventes de polaridad creciente, hexano, acetato de etilo y metanol, obteniéndose extractos hexánicos, de acetato de etilo y metanólicos respectivamente. Además, a partir del material desecado se obtuvieron extractos hidroalcólicos por maceración y extractos acuosos por infusión. En el Capítulo 1 se evaluó la actividad diurética de E. serpens, debido a su uso como diurética en la medicina tradicional. Utilizando modelos in vivo se ensayaron los extractos hexánico, metanólico y de acetato de etilo de E. serpens, y acuosos e hidroalcólicos de las tres especies vegetales estudiadas. No se encontró actividad diurética, para ninguna de las plantas. En el Capítulo 2 se evaluó la actividad antihelmíntica de las tres especies vegetales, exponiendo larvas de Ancylostoma caninum a extractos hexánicos, de acetato de etilo y metanólicos; y huevos de Haemonchus contortus a extractos hidroalcólicos. Los extractos metanólicos obtenidos de las tres especies tuvieron efecto inhibitorio sobre la motilidad larval. El extracto metanólico de E. collina, además, inhibió la eclosión de huevos. Los ensayos confirmaron el efecto antihelmíntico de E. collina. En el Capítulo 3 se evaluó la actividad citotóxica de las tres especies de Euphorbia utilizando el test de letalidad de Artemia salina, exponiendo larvas a diferentes concentraciones de extractos hexánicos, de acetato de etilo y metanólicos. Todos los extractos mostraron actividad citotóxica al menos en una de las dosis empleadas. El extracto hexánico de E. collina fue el que presentó la mayor actividad. Además, se realizaron ensayos de toxicidad in vivo de extractos metanólico, hidroalcohólico y hexánico de E. collina, los cuales no mostraron toxicidad en ratones. En el Capítulo 4 se evaluó la actividad antimicrobiana de los extractos hexánicos, de acetato de etilo y metanólicos de E. serpens, E. schickendantzii y E. collina, contra Escherichia coli, Pseudomona aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis y Fusarium verticillioides, en métodos in vitro. Se determinó que los extractos de acetato de etilo y metanólico de E. collina, y metanólico de E. schickendantzii, tuvieron actividad antimicrobiana. Los extractos hexánico y de acetato de etilo de E. collina crudos, se reunieron y se fraccionaron mediante una cromatografía en columna. De las fracciones obtenidas, una de ellas, denominada G10-11 inhibió el 50 % del crecimiento de Fusarium verticillioides. En el Capítulo 5 se realizó una marcha fitoquímica con extractos etanólicos de E. serpens, E. schickendantzii y E. collina, determinando la presencia de compuestos esteroidales, triterpenos, flavonoides, taninos, fenoles, saponinas, lípidos, proteínas e hidratos de carbono. De la fracción G10-11 se aisló una mezcla de 51 % de cicloartenol y un 49 % de 24-metilenocicloartenol. / The aim of this work was to study three species used by traditional medicine: E. serpens, E. schickendantzii and E. collina, to evaluate the presence of diuretic, antiparasitic, cytotoxic and antimicrobial activity; identify and isolate their active principles. Sequential extractions were carried out on dry plant with solvents of growing polarity, hexane, ethyl acetate and methanol, obtaining hexane, ethyl acetate and methanolic extracts, respectively. Also, hydroalcoholic extracts by maceration and aqueous extracts by infusion from the dry material were obtained. In Chapter 1, the diuretic activity of E. serpens was evaluated due to its use as a diuretic in traditional medicine. Using in vivo models, the hexane, methanlic and ethyl acetate extracts of E. serpens and aqueous and hydroalcoholic extracts of the three species were tested. No diuretic activity was found for any of the plants. In Chapter 2, the anthelmintic activity of the three plant species was evaluated, exposing Ancylostoma caninum larvae to hexanic, ethyl acetate and methanolic extracts; and Haemonchus contortus eggs to hydroalcoholic extracts. The methanolic extracts obtained from the three species had an inhibitory effect on larval motility. The methanolic extract of E. collina also inhibited the hatching of eggs. The tests confirmed the anthelmintic effect of E. collina. In Chapter 3, the cytotoxic activity of the three Euphorbia species was evaluated using the Artemia salina lethality test, exposing larvae to different concentrations of hexane, ethyl acetate and methanolic extracts. All the extracts showed cytotoxic activity at least in one of the doses used. The hexane extract of E. collina was the one that presented the highest activity. In addition, in vivo toxicity tests of methanolic, hydroalcoholic and hexanic extracts of E. collina were carried out, which did not show toxicity in mice. In Chapter 4, the in vitro antimicrobial activity of the hexane, ethyl acetate and methanolic extracts of E. serpens, E. schickendantzii and E. collina was evaluated against Escherichia coli, Pseudomona aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis and Fusarium verticillioides. It was determined that the ethyl acetate and methanolic extracts from E. collina, and methanolic extracts from E. schickendantzii, had antimicrobial activity. The crude E. collina hexane and ethyl acetate extracts were combined and fractionated by column chromatography. Of the fractions obtained, one of them, called G10-11, inhibited 50 % of the growth of Fusarium verticillioides. In Chapter 5 a phytochemical screening was carried out with ethanolic extracts of E. serpens, E. schickendantzii and E. collina, determining the presence of steroidal compounds, triterpenes, flavonoids, tannins, phenols, saponins, lipids, proteins and carbohydrates. In the fraction G10-11, a mixture of 51 % cycloartenol and 49 % 24-methylenecycloartenol was isolated.

The thesis summarizes data gathered on various components of respiratory chain of Trypanosoma brucei. Namely, NADH:ubiquinone oxidoreductase (complex I), alternative NADH:ubiquinone oxidoreductase (NDH2) and mitochondrial glycerol-3-phosphate dehydrogenase are discussed themselves and in broader context of energy metabolism. Also, a work done using RNA interference library is described.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-07-20T17:10:28Z No. of bitstreams: 1 prisicila_mo_costa_ioc_bp_0037_2006.pdf: 2669544 bytes, checksum: 9b2e6aaf65027a066f8ad3713540db99 (MD5) / Made available in DSpace on 2012-07-20T17:10:28Z (GMT). No. of bitstreams: 1 prisicila_mo_costa_ioc_bp_0037_2006.pdf: 2669544 bytes, checksum: 9b2e6aaf65027a066f8ad3713540db99 (MD5) Previous issue date: 2006 / Cnpq / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O estudo de tripanosomatídeos de plantas teve início em 1909, quando foram encontradas formas flageladas em látex, sendo depois encontradas em floema, frutas e flores. Desde a refutada tentativa de Donovan de criar o gênero Phytomonas, temos atualmente, várias espécies classificadas dentro do gênero Phytomonas, incluíndo Phytomonas serpens, organismo alvo de nosso estudo. O ciclo de vida de P. serpens compreende planta e inseto. A transmissão é feita do tomate para inseto, do inseto para tomate. O vetor natural é o hemíptero Phthia picta (Hemiptera: Coriidae) que quando se alimenta de tomates infectados, se torna infectado após 10 a 15 dias, apresentando parasitos nas fezes, urina, tubo digestivo e glândulas salivares. Pouco se sabe sobre P. serpens e o conhecimento é ainda mais escasso se levarmos em conta todos os organismos dentro do gênero Phytomonas. O objetivo deste projeto é explorar o genoma de P. serpens gerando e analisando seqüências de seu genoma, aumentando o conhecimento deste organismo e comparando seu genoma com outros organismos. A cepa 9T de P. serpens (CT–IOC-174) foi cultivada a 27oC em meio Schneiders ́insect medium suplementado com 10% de soro fetal bovino. O DNA genômico foi extraído por lise alcalina para a construção da biblioteca genômica. O DNA foi parcialmente digerido com a enzima de restrição Sau3A. Os fragmentos de DNA com tamanhos entre 1-3 kb foram recuperados do gel de agarose e purificados. Os fragmentos de DNA foram clonados no sítio BamHI do vetor de clonagem pUC18. A reação de sequenciamento foi realizada na plataforma de sequenciamento ABI3730 – 48 capilar PDTIS/FIOCRUZ e na plataforma MegaBase na UERJ. Todas as seqüências foram armazenadas em banco de dados “open source” nos servidores Linux no Laboratório de Biologia Molecular de Tripanosomatídeos e Flebotomíneos (DBBM/IOC/FIOCRUZ). Foram seqüenciados 829 clones da biblioteca de DNA genômico obtendo-se um total de 379 seqüências GSS (Genomic Sequence Survey) de alta qualidade. Foram utilizadas para a complementação deste estudo, as seqüências do projeto EST de P. serpens que estavam disponíveis no GenBank. Os 221 clusters GSS e os 697 clusters de EST (Expressed Sequence Tag) foram comparados com o programa de busca de similaridade Blast a diferentes bancos de dados, utilizando como parâmetro evalue 10 -5, gerando 599 clusters com entradas positivas (Hits) e 303 clusters com nenhum hit, representando possíveis genes espécie específicos. Os clusters foram anotados de acordo com sua função quando comparados ao banco de dados Gene Ontology (GO) representando uma análise inédita no genoma de P.serpens. Inferências filogenéticas e de similaridade com o banco “Taxonomy” do NCBI foram realizados, usando inclusive seqüências do genoma ambiental, para verificar se as mesmas pertencem a Kinetoplastida, o que não se comprovou. As inferências filogenéticas realizadas com genes concatenados mostraram que P. serpens é mais próximo de T. cruzi, enquanto que inferências com genes individuais apontaram para L. major. Com base na literatura, inferimos que a análise da árvore de genes concatenados é correta. Foram encontradas com o GLIMMER 20 genes hipotéticos nas seqüências de GSS. Encontramos 22 cluters em GSS que não foram encontrados anteriormente em EST, como por exemplo, Piroglutamil-peptidase I, antígeno nuclear de proliferação celular (PCNA) e Peptidase_M8. / The study of trypanosomatids of plants began in 1909, when flagellate forms were found in latex from Euphorbiaceae, being later found in phloem, fruits/seeds and flowers in a wide variety of plants species. Since the refuted attempt of Donovan to create the genus Phytomonas, a great number of species were classified in the genus Phytomonas, including Phytomonas serpens, the major organism of our study. The described life cycle of P. serpens involves plant (tomato) and insect. The natural vector is the Coreid bug, Phthia picta (Hemiptera: Coriidae), that becomes infected 10 a 15 days after feeding on infected tomatoes, presenting the parasite in excrements, urine, digestive tube and salivary glands. Very little is known about these organisms and even less is known about the genus Phytomonas. The goal of this project is to explore the P. serpens genome by generating and analyzing sequences of its genome aiming to increase the knowledge of this organism and to analyze comparatively with genomes of other organisms. P. serpens infects tomatoes but we still have doubts about its pathogenicity. A strain of P. serpens 9T (CT-IOC-174) was cultured at 27oC in Schneiders ́insect medium supplemented with 10% heat-inactivated fetal bovine serum. The DNA was extracted by alkaline lyses for the construction of a genomic library. Genomic DNA was partially digested with the restriction enzyme Sau3A. DNA fragments with an average size of 1.0-3.0 kb were recovered and purified from agarose gel. DNA inserts were cloned into the BamHI restriction site of pUC18 cloning vector. The sequencing reaction was made at the Sequencing Platform ABI3730 - PDTIS/FIOCRUZ and MegaBase platform at UERJ. All the sequences were stored in an open source database in the Linux server in the Laboratory of Molecular Biology of Trypanosomatids and Phlebotomines (DBBM/IOC/FIOCRUZ). 829 clones of the P. serpens genomic DNA library were sequenced obtaining a total of 379 high quality sequences GSS (Genome Sequence Survey). Sequences from the EST (Expressed Sequence Tag) project of P. serpens available at GenBank were used for the complementation of this study. 221 GSS clusters and 697 EST clusters were compared with different databases with the similarity search program Blast, using evalue 10 -5 as default, obtaining 599 clusters with positive hits and 303 clusters without hit, indicating possible species-specific genes. 357 clusters that were identified when compared with Gene Ontology (GO) had their function classified, representing the first analysis of P. serpens genome using GO. Phylogenetics and similarity studies with Taxonomy database from NCBI were carried out confirming the position of P. serpens in relations to the others trypanosomatids and disclosing similarity with T. cruzi genome. The phylogenetic study included sequences of the enviromental genome, in order to verify if these sequences belong to the Kinetoplastid, what was not proven to be true. Phylogenetic studies using concatenated genes showed that P. serpens is phylogenetically closer to T. cruzi, but studies with individuals genes pointed to L. major. Based on literature, we infer that the analysis of the tree of concatenated genes is correct. 20 hypothetical genes in the GSS sequences were found with the GLIMMER. We found 22 clusters in GSS sequences that were not found in EST before, among others, Pyroglutamil-peptidase I, proliferating cell nuclear antigen (PCNA) and Peptidase_M8.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-05-27T16:22:16Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ComposicaoQuimicaEfeitos.pdf: 965292 bytes, checksum: 050ded50f966f6a7bb38266bba436513 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-09-09T12:34:46Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ComposicaoQuimicaEfeitos.pdf: 965292 bytes, checksum: 050ded50f966f6a7bb38266bba436513 (MD5) / Made available in DSpace on 2014-09-09T12:34:46Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ComposicaoQuimicaEfeitos.pdf: 965292 bytes, checksum: 050ded50f966f6a7bb38266bba436513 (MD5) Previous issue date: 2011 / A Peperomia serpens (Piperaceae) é uma liana herbácea e epifíta popularmente chamada de “carrapatinho’’. Esta planta cresce na Floresta Amazônica de maneira selvagem em diferentes árvores. As folhas são usadas na medicina tradicional brasileira para dor, inflamação e asma. Neste estudo investigaram-se os efeitos do óleo essencial de P.serpens (OEPs) em roedores através de testes para dor e inflamação. A atividade antinociceptiva foi avaliada usando-se modelos nociceptivos químicos (ácido acético e formalina) e térmicos (placa quente) em camundongos, enquanto a atividade antiinflamatória foi avaliada por testes de edema de pata induzidos por carragenina (Cg) e dextrana em ratos, edema de orelha induzido por óleo de cróton, bem como migração celular, rolamento, e adesão induzida por Cg em camundongos. Além disso, a análise fitoquímica do OEPs foi realizada. A composição química do OEPs foi analisada por cromatografia gasosa acoplada a espectrometria de massa. 25 constituintes, representando 89,51% do total do óleo, foram identificados. (E)-Nerolidol (38.0%), ledol (27.1%), α-humulene (11.5%), (E)-caryophyllene (4.0%) and α-eudesmol (2.7%) foram encontrados como principais constituintes. O pré-tratamento oral com o OEPs (62,5- 500mg/kg) reduziu de maneira significante o número de contorções, com um valor de DE50 de 188,8mg/kg que foi ulitizado em todos os testes. Não houve efeito no teste da placa quente mas reduziu o tempo de lambida em ambas as fases do teste de formalina, efeito que não foi significativamente alterado pela naloxona (0,4 mg/kg). OEPs impediu o desenvolvimento do edema induzido por Cg e dextrana em ratos. Em camundongos, o OEPs inibiu o edema induzido por óleo de cróton bem como a migração de leucócitos e neutrófilos, e rolamento e adesão. Estes resultados sugerem que o OEPs possui atividade antinociceptiva periférica sem interação com receptores opióides e atividade antiinflamatória em diferentes modelos de inflamação aguda. / The Peperomia serpens (Piperaceae), popularly known as “carrapatinho”, is an epiphyte and herbaceous liana grown wild on different host trees in the Amazon rainforest. Its leaves are largely used in Brazilian folk medicine to treat inflammation, pain and asthma. This study investigated the effects of essential oil of P. serpens (EOPs) in standard rodent models of pain and inflammation. The antinociceptive activity was evaluated using chemical (acetic acid and formalin) and thermal (hot plate) models of nociception in mice whereas the anti-inflammatory activity was evaluated by carrageenan (Cg) - and dextraninduced paw edema tests in rats croton oil-induced ear edema, as well as cell migration, rolling and adhesion induced by Cg in mice. Additionally, phytochemical analysis of the EOPs has been also performed. Chemical composition of the EOPs was analyzed by gas chromatography and mass spectrometry (GC/MS). Twenty five compounds, representing 89.6% of total oil, were identified. (E)-Nerolidol (38.0%), ledol (27.1%), α-humulene (11.5%), (E)- caryophyllene (4.0%) and α-eudesmol (2.7%) were found to be the major constituents of the oil. Oral pretreatment with EOPs (62.5-500 mg/kg) significantly reduced the writhing number, with an ED₅₀ value of 188.8 mg/kg that was used thereafter in all tests. EOPs had no significant effect on hot plate test but reduced the licking time in both phases of the formalin test, an effect that was not significantly altered by naloxone (0.4 mg/kg, s.c.). EOPs inhibited the edema formation induced by Cg and dextran in rats. In mice, EOPs inhibited the edema formation by croton oil as well as the leukocyte and neutrophil migration, the rolling and the adhesion of leukocytes. These data show for the first time that EOPs has a peripheral antinociceptive effect that seems unrelated to interaction with the opioid system and a significant anti-inflammatory effect in acute inflammation models.