Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-12-15T15:47:47Z No. of bitstreams: 1 texto completo.pdf: 838499 bytes, checksum: 205879d615c3eb53836a006b577f876e (MD5) / Made available in DSpace on 2016-12-15T15:47:47Z (GMT). No. of bitstreams: 1 texto completo.pdf: 838499 bytes, checksum: 205879d615c3eb53836a006b577f876e (MD5) Previous issue date: 2016-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A matriz peritrófica é formada por camadas não celulares de fibrilas de quitina associadas à glicoproteínas e proteoglicanas revestindo o alimento no intestino médio dos artrópodes, servindo como uma barreira entre o conteúdo alimentar e o epitélio do intestino médio. A permeabilidade da matriz peritrófica depende da presença de poros, que é uma das características funcionais. O objetivo foi determinar se a permeabilidade da matriz peritrófica altera ao longo do intestino médio e na presença do anticorpo antiperitrofina 55 em abelhas Melipona quadrifasciata (Apidae: Meliponini) e Apis mellifera (Apidae: Apini). A matriz peritrófica tem espessura média variável entre as regiões anterior e posterior do intestino médio de A. mellifera com 0.71 ±0.01 μm e 0.83 ±0.02 μm em nutridoras e 0.84 ±0.02 μm e 0.99 ±0.04 μm em campeiras, respectivamente. Em M. quadrifasciata a espessura foi de 0.66 ±0.02 μm na região anterior e 0,90 ±0.02 μm na região posterior de nutridoras, 0.68 ±0.01 μm na região anterior e 0.96 ±0.04 μm na região posterior de campeiras. Em operárias de A. mellifera, moléculas de dextran com peso molecular de 40 KDa atravessam a matriz peritrófica, enquanto aquelas ≥ 70 KDa não atravessam a matriz. Em M. quadrifasciata o limite de passagem ficou entre as moléculas de 4 e 40 KDa, sendo as de peso molecular 4 KDa, as únicas evidenciadas no espaço ectoperitrófico. Abelhas alimentadas com anticorpo anti-peritrofina 55 apresentaram aumento na permeabilidade das moléculas de dextran em ambas as espécies. A matriz peritrófica possui diferenças morfométricas quanto as regiões do intestino médio mas não apresenta diferença na porosidade ao longo do intestino, tendo porém, alteração na permeabilidade da matriz quando em presença do anticorpo anti-peritrofina 55. / The peritrophic matrix is formed by non-cell layers of chitin fibrils associated with glycoproteins and proteoglycans lining the food in the arthropods midgut, serving as a barrier between the food and the midgut epithelium. The permeability of the peritrophic matrix depends on the presence of pores, which is one of its functional features. The objective of this work was to determine the permeability of the matrix peritrophic and changes over midgut in the presence of antibody anti-peritrophin 55 in Melipona quadrifasciata (Apidae: Meliponini) and Apis mellifera (Apidae: Apini). The peritrophic matrix has average variable thickness between the anterior and posterior regions of the midgut of A. mellifera with 0.71 ± 0.01 μm and 0.83 ± 0.02 μm in nurturing and 0.84 ± 0.02 μm and 0.99 ± 0.04 μm in foragers, respectively. In M. quadrifasciata the thickness was 0.66 ± 0:02 μm in the anterior and 0.90 ± 0:02 μm in the posterior region of nurturing, 0.68 ± 0.01 μm in the anterior and 0.96 ± 0:04 μm in the posterior region of foragers. In workers of A. mellifera, dextran molecules with molecular mass of 40 KDa cross the peritrophic matrix, while those ≥ 70 KDa do not pass through the matrix. In M. quadrifasciata, the passage limit was among molecules of 4 and 40 KDa, being the 4 KDa, the only evident in ectoperitrophic space. Bees fed with the antibody anti- peritrophin 55 show an increase in the permeability of dextran molecules in both species. The peritrophic matrix has morphological differences in the midgut regions but shows no difference in porosity throughout the intestine, and however, show changes in permeability of the matrix in presence of antibody anti-peritrophin 55.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-20T15:50:22Z No. of bitstreams: 1 texto completo.pdf: 1478810 bytes, checksum: be9e97e7629b09268a428ed4728be5c8 (MD5) / Made available in DSpace on 2016-12-20T15:50:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1478810 bytes, checksum: be9e97e7629b09268a428ed4728be5c8 (MD5) Previous issue date: 2016-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A leishmaniose visceral (LV) é uma doença causada por protozoários parasitas intracelulares da espécie Leishmania infantum chagasi nas Américas. Esses parasitos possuem forte tropismo por órgãos viscerais, especialmente baço, fígado e medula óssea, onde se proliferam e provocam lesões podendo ser letal caso não sejam tratadas. O parasito possui moléculas consideradas fatores de virulência que vem sendo intensamente investigadas. Entre elas destacamos as proteínas ligante de heparina (PLH), glicoproteínas relacionadas em diversos trabalhos na literatura com o processo de adesão e internalização do parasito à célula hospedeira. Nesse trabalho utilizamos a PLH de L. infantum chagasi (PLHLc) em experimentos de imunização de camundongos BALB/c para avaliar a proteção conferida a esses animais após desafio com as formas promastigotas do parasito. Foram avaliadas a carga de parasitos no baço e no fígado, produção de citocinas (IFN- , TNF, IL-2, IL-17, IL-6, IL-10 e IL-4) e de óxido nítrico (NO) após imunização com PLHLc isolada ou associada ao Adjuvante Incompleto de Freund (AIF) e Adjuvante Saponina (SAP), quatro semana após o desafio. Os camundongos foram imunizados por via intraperitoneal ou subcutânea, sendo submetidos a duas doses de reforço utilizando o mesmo protocolo da primeira imunização. Quinze dias após a última dose os camundongos foram infectados com 1 x 10 7 promastigotas de L. infantum chagasi em fase final logarítmica de crescimento. Quatro semanas após a infecção os animais foram eutanasiados e o baço e o fígado foram coletados para ensaio da quantificação da carga parasitária e avaliação da produção de citocinas e NO por citometria de fluxo e pelo método de Griess, respectivamente. Nossos resultados mostraram que o grupo imunizado apenas com PLHLc não apresentou redução da carga parasitária e os esplenócitos estimulados in vitro com antígeno particulado de L. infantum chagasi (AgLc) produziram citocinas IL-6 e IL-17, ambas de padrão de resposta imune Th17 e a citocina IL-10. O grupo imunizado com PLHLc + AIF apresentou redução da carga de parasitos apenas no baço e produziram citocinas IFN- e IL-2 (Th1), IL-6 e IL-17 (Th17) e IL-10. O grupo imunizado com PLHLc + SAP apresentou redução da carga parasitária no baço e no fígado. Os esplenócitos desse grupo estimulados in vitro com AgLc produziram maiores médias de citocinas de padrão de resposta imune Th1 e Th17 e a citocina reguladora IL-10 comparado com os grupos anteriores. Em relação a produção de NO por esplenócitos estimulados in vitro com AgLc níveis basais foram detectados pelo método de Griess em todos os grupos avaliados. Os resultados alcançados nos mostra que a PLHLc associada ao adjuvante Saponina representa uma forte candidata a vacina contra LV e nos direciona para uma nova etapa de investigação, a proteção conferida pela PLHLc recombinante. / Visceral leishmaniasis (VL) is a disease caused by the protozoan intracellular parasite Leishmania infantum chagasi specie in the Americas. These parasites exhibit a strong tendency to invade visceral organs, especially the spleen, liver and bone marrow, where they proliferate and cause injuries and can be lethal if not treated. The parasite exhibit molecules considered as virulence factors that have been intensively searched. Among then we highlight the heparin-binding proteins (HBP), glycoproteins related in several studies in the literature with the process of adhesion and internalization of the parasite to the host cell. In this work, we used L. infantum chagasi HBP (HBPLc) in immunization experiments using BALB/c mice to evaluate the protection given to the animals after challenge with promastigote forms of the parasite. Were evaluate the parasite load in the spleen and liver, cytokine production (IFN- , TNF, IL-2, IL-17, IL-6, IL-10 e IL-4) and nitric oxide (NO) after immunization with isolated HBPLc or associated with Incomplete Freund’s Adjvant (IFA) and Saponin Adjuvant (SAP), four weeks after challenge. The mice were immunized by intraperitoneal or subcutaneous route and submitted two times to booster doses using the same protocol of the first immunization. Fifteen days after the last dose the mice were infected with 1 x 10 7 L. infantum chagasi promastigotes end logarithmic phase of growth. Four weeks after infection the animals were euthanized and spleen and liver were collected for assay quantification of the parasite load and evaluation of the cytokine and NO production by flow cytometry and by Griess method, respectively. Our results showed that the group immunized with only HBPLc showed no reduction in parasite load and spleen cells stimulated in vitro with crude extract of L. infantum chagasi (AgLc) produced cytokines IL-6 and IL-17, both standard immune response Th17 and IL-10 cytokine. The group immunized with HBPLc + IFA decreased the parasite load only in the spleen and produced cytokines IFN- and IL-2 (Th1), IL-6 and IL-17 (Th17) and IL-10 cytokine. The group immunized with HBPLc + SAP decreased the parasite load in the spleen and liver. The spleen cells this group in vitro stimulated with AgLc generated pattern cytokines of Th1 and Th17 and regulatory cytokine IL-10 compared to previous groups. About NO production by spleen cells stimulated in vitro with AgLc baseline levels were detected by Griess method in all groups evaluated. The results achieved shows that the HBPLc associated with Saponin adjuvant is a strong candidate against VL vaccine and directs us to a new stage of research, the protection given by recombinant HBPLc.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-23T12:26:14Z No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) / Made available in DSpace on 2016-12-23T12:26:14Z (GMT). No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) Previous issue date: 2016-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os insetos respondem à infecção através da montagem de reações imunes do tipo celular e humoral. Os reguladores primários dessas respostas são células chamadas hemócitos, os quais medeiam importantes respostas celulares, incluindo a fagocitose, encapsulamento, nodulação, e também segregam fatores humorais, tais como opsoninas, fatores de melanização e peptídeos antimicrobianos. Os hemócitos circulam ao longo da hemocele (cavidade corporal do inseto) pelo fluxo rápido da hemolinfa (sangue), além disso, partes desses hemócitos também existem como células sésseis que estão associados aos tecidos. As larvas de Galleria mellonella são uma alternativa viável para os modelos tradicionais dos mamíferos para estudar a eficácia de drogas antimicrobianas e a patogênese de microrganismos in vivo. No entanto, apesar da sua importância como um modelo de infecção, aspectos biológicos sobre as células do sistema imunológico, tais como a densidade e dinâmica dos hemócitos das larvas são mal compreendidos. No presente trabalho, investigamos a resposta imune celular dos hemócitos circulantes das larvas de G. mellonella contra diferentes cepas da bactéria Gram-negativa Actinobacillus pleuropneumoniae: baixa virulência (780), alta virulência (1022), e cepas de referência do sorotipo 8 (R8). Os hemócitos foram classificados com base no seu tamanho, morfologia, coloração e seus papeis na resposta imune, incluindo cinco tipos: prohemócitos, plasmatócitos, granulócitos, esferulócitos, e oenocitóides. Contagem total de hemócitos, contagem diferencial de hemócitos, atividade dos fagolisossomos, resposta autofágica, viabilidade celular, e a ativação da caspase-3 (como indicador de apoptose) foram determinados em hemócitos circulantes provenientes de larvas desafiadas e controle. Demostramos pela primeira vez no modelo de G. mellonella que os plasmatócitos e granulócitos ativam suas respostas autofágicas através da formação dos autofagossomos após o contato com A. pleuropneumoniae. Além disso, nossos dados demonstram que a imunidade celular do presente modelo de infecção muda dependendo do grau de virulência das cepas bacterianas. / Insects respond to infection by mounting cellular and humoral immune reactions. The primary regulators of these immune responses are cells called hemocytes, which mediate important cellular immune responses including phagocytosis, encapsulation, nodulation and also secrete immune factors such as opsonins, melanization factors and antimicrobial peptides. Hemocytes circulate through the hemocoel (body cavity) by the swift flow of hemolymph (blood), and part of these hemocytes population are sessile and are attached to tissues. Larvae of Galleria mellonella is a widely used factitious host as a viable alternative to traditional mammalian models to study the efficacy of antimicrobial drugs and the microbial pathogenesis in vivo. However, despite their importance as an infection model, biological aspects about the immune cells, such as density and hemocyte dynamic of larvae are poorly understood. In the present study, we investigated the cellular immune response of hemocytes from G. mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low virulent (780), high virulent (1022), and the serotype 8 reference strain (R8). Five types of larval hemocytes, prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes, were distinguished according to size, morphology, detection by molecular probes, dye-staining properties, and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability, and caspase-3 activation were determined in circulating hemocytes of naïve and infected larvae. Granulocytes and plasmatocytes were the major hemocyte types involved in the cellular defense against A. pleuropneumoniae; these hemocytes activated phagolysosome activities associated with an autophagic response against the bacteria. Moreover, our results showed that apoptosis in circulating hemocytes after exposure to virulent bacterial strains was related to an excessive autophagic cell death response induced by stress and subsequent caspase-3 activation.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-05-30T17:13:22Z No. of bitstreams: 1 DIS_BELISA_AVILA_RODRIGUES_PARCIAL.pdf: 420085 bytes, checksum: 77f9b7ddcd1250899386df6d319c1934 (MD5) / Made available in DSpace on 2017-05-30T17:13:22Z (GMT). No. of bitstreams: 1 DIS_BELISA_AVILA_RODRIGUES_PARCIAL.pdf: 420085 bytes, checksum: 77f9b7ddcd1250899386df6d319c1934 (MD5) Previous issue date: 2016-07-29 / Acinetobacter calcoaceticus-baumannii (ACB) are opportunistic pathogens responsible for respiratory tract infections, wound infections, and bacteremia in intensive care units patients. These microorganisms became a problem in health care units due to their ability to acquire and accumulate resistance determinants. They are resistant to important antibiotics commonly used to treat infections caused by them. Thus, this study aimed to determine the antimicrobial susceptibility and detect resistance determinants in clinical ACB isolates. For this, 200 clinical ACB isolates, resistant to carbapenems, were collected from a hospital in Porto Alegre, Brazil. To determine susceptibility to antimicrobial agents, 16 antimicrobials were used in the disk diffusion technique and the minimum inhibitory concentration (MIC) for polymyxin B and meropenem was performed by broth microdilution. Additionally, MIC for polymyxin B in 17 isolates was determined using different methods in comparison with broth microdilution, adopted as reference method. The presence of blaOXA-23, blaOXA-51, blaIMP and blaNDM, as well as class 1 and 2 integrons, were detected by PCR. A total of 82.5% were extremely resistant (XDR) and 17.5% were multidrug resistant (MDR). Forty-six non-susceptibility patterns were found among the isolates, with polymyxins, tetracyclines, and aminoglycosides being the classes with higher rate of susceptibility. When different methods to determining MIC for polymyxin B were evaluated, very major errors and major errors were found in E-test and major errors in agar dilution, as compared with the reference method. The broth microdilution with polysorbate 80 showed a reduction of two or more dilutions in most isolates (82.3%). The blaOXA-23 and blaOXA-51 genes were found in 100% of the isolates, while 49% harbored blaIMP. The blaNDM gene was not found in any isolate. Class 1 and class 2 integrons were found in 68% and 88% of the isolates, respectively. The high percentage of XDR isolates, as well as the detection of important resistance determinants and the high rate of integrons found, reinforce the concern regarding ACB microorganisms and their spread in health care units. / Acinetobacter calcoaceticus-baumannii (ACB) s?o pat?genos oportunistas respons?veis por infec??es do trato respirat?rio, infec??es de feridas e bacteremia em pacientes internados em unidades de terapia intensiva. Esses microrganismos tornaram-se um problema nas unidades de assist?ncia ? sa?de devido ? sua capacidade de adquirir e acumular determinantes de resist?ncia a antimicrobianos, sendo respons?veis por altas taxas de resist?ncia aos principais antimicrobianos utilizados terapeuticamente. Desta forma, esse estudo teve por objetivo determinar a suscetibilidade a antimicrobianos, assim como detectar determinantes de resist?ncia em isolados cl?nicos de ACB. Para isso, foram utilizados 200 isolados cl?nicos, resistentes aos carbapen?micos, pertencentes ao complexo ACB, coletados de um hospital em Porto Alegre, Brasil, no per?odo de janeiro de 2012 a maio de 2015. Para determinar a suscetibilidade a antimicrobianos, 16 f?rmacos foram utilizados na t?cnica de disco-difus?o, assim como foi determinada a concentra??o inibit?ria m?nima (CIM) para polimixina B e meropenem por meio da t?cnica de microdilui??o em caldo. Adicionalmente, a CIM para polimixina B foi determinada em 17 isolados, empregando diferentes m?todos em compara??o com a microdilui??o em caldo, adotada como refer?ncia. Os genes blaOXA-23, blaOXA-51, blaIMP e blaNDM, bem como os integrons de classe 1 e 2 foram detectados por PCR. Um total de 82,5% dos isolados foram extremely resistant (XDR) e 17,5% foram multidrug resistant (MDR). Quarenta e seis padr?es de n?o-suscetibilidade foram encontrados entre os isolados, sendo que polimixinas, tetraciclinas e aminoglicos?deos foram as classes com maior taxa de suscetibilidade. Na determina??o de CIM para polimixina B com diferentes m?todos, very major errors e major errors foram encontrados em E-test, e major errors em dilui??o em ?gar, quando comparados com o m?todo de refer?ncia. A microdilui??o em caldo suplementado com polissorbato 80 mostrou redu??o de duas ou mais dilui??es na maioria dos isolados (82,3%). Os genes blaOXA-23 e blaOXA-51 foram encontrados em 100% dos isolados, enquanto 49% apresentaram blaIMP. O gene blaNDM n?o foi encontrado nos isolados analisados. Integrons de classe 1 foram encontrados em 68% dos isolados e integrons de classe 2 em 88%. O elevado percentual de isolados XDR, assim como a detec??o de importantes determinantes de resist?ncia e a alta taxa de integrons encontrada, refor?am a preocupa??o com microrganismos do complexo ACB e sua dissemina??o nas unidades de assist?ncia ? sa?de.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-01T14:56:39Z No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) / Made available in DSpace on 2017-06-01T14:56:39Z (GMT). No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) Previous issue date: 2016-03-29 / Solanum lycopersicum L., tomato, is an herbaceous plant belonging to the Solanaceae family. Its fruits are consumed worldwide, reaching the world production of 160 million tons per year. In Brazil, it is the second vegetable in economic importance. However, the tomato is attacked by numerous diseases that lead to loss of production and /or poor quality of the fruit, such as the hollow stem, the disease caused by Pectobacterium spp. Due to the large number of diseases caused by plant pathogens, the tomato is a culture in which a significant amount of agrochemicals is used. Therefore, this species is among the vegetables with the greatest amount of residual pesticides. Under these circumstances, it is essential to develop sustainable plant defense techniques in order to reduce the use of agrochemicals. Then, changes in plant metabolism related to defense must be understood so that new strategies and new products can be developed. Disease control using soil microorganisms has been considered as an alternative, since the rhizobacteria, in addition to promoting plant growth, may induce resistance as the result of activation of the natural plant defenses. These, calls plant growth promoting rhizobacteria (PGPR), has been explored for their biofertilizers, biopesticides and phyto-stimulating abilities. The aims of this study were to characterize biochemically the Streptomyces spp. isolates, to determine the antagonism against Pectobacterium carotovorum subsp. brasiliensis (Pcb), to determine the ability of Streptomyces spp. on promoting growth of tomato plants and to evaluate the modulation of the defense-related metabolism of tomato plants when treated with Streptomyces spp. The possible influence of Streptomyces spp. on reducing soft rot disease in tomato plants was also evaluated. Biochemical characterization was evaluated through the ability of Streptomyces spp. on producing siderophores, solubilizing phosphate, and activity of amylase and lipase, as well as volatile organic compounds (VOC) production. Antagonism of Streptomyces spp. against Pcb was determined by dual-culture method and I-plate for VOC effect analysis. Plant growth promotion was evaluated through VOC emission and by direct interaction with Streptomyces spp. isolates (PM1, PM3, PM4, PM5, PM6 e PM9). Enzymes related to plant defense were colorimetric analyzed in plants treated with isolates of Streptomyces spp. Evaluation of soft rot disease was performed on plants treated with Streptomyces spp. and challenged with Pcb through the area under the disease progression curve (AUDPC) and plant mortality. Isolates of Streptomyces spp. displayed characteristics of PGPR and 32 volatile compounds were identified from the different isolates. PM3 was the isolate showing efficient antagonism against Pcb. Most of the isolates promoted increase of root and shoot length of tomato plants by VOC although PM5 was efficient on promoting growth by direct interaction with Streptomyces spp. Treatment with Streptomyces spp. modulated the activity of defense-related enzymes and decrease incidence of soft rot disease. / O tomateiro (Solanum lycopersicum L.) ? uma planta herb?cea pertencente ? fam?lia Solanaceae. Seus frutos s?o consumidos mundialmente, chegando ? produ??o mundial de 160 milh?es de toneladas por ano. No Brasil, ? a segunda hortali?a em import?ncia econ?mica. Contudo, o tomateiro ? alvo de in?meras doen?as que levam ? perda de produ??o e/ou m? qualidade dos frutos, como por exemplo, a doen?a Talo oco causada por Pectobacterium spp. Devido ao elevado n?mero de doen?as causadas por fitopat?genos, o tomateiro ? uma cultura onde se utiliza uma quantidade expressiva de agroqu?micos, estando entre as hortali?as que apresentam maior quantidade de agrot?xicos residuais. Por isso, torna-se imprescind?vel o desenvolvimento de t?cnicas sustent?veis de defesa para o vegetal, a fim de reduzir o uso destes compostos. Para este fim, ? fundamental compreender as altera??es no metabolismo vegetal relacionado ? defesa, para que novas estrat?gias e novos produtos agr?colas possam ser desenvolvidos. O controle de doen?as utilizando microrganismos de solo tem sido considerado uma alternativa, uma vez que as rizobact?rias, al?m de promoverem o crescimento vegetal, podem induzir ? resist?ncia como consequ?ncia da ativa??o da defesa vegetal. Estas, chamadas rizobact?rias promotoras de crescimento vegetal (PGPR), v?m sendo exploradas quanto ? capacidade biofertilizante, fito-estimuladora e biopesticida. Os objetivos deste estudo foram caracterizar seis isolados de Streptomyces spp. como PGPR, determinar o antagonismo contra Pectobacterium carotovorum subsp. brasiliensis (Pcb), determinar a capacidade de isolados de Streptomyces spp. na promo??o do crescimento de plantas de tomate e avaliar a modula??o do metabolismo relacionado ? defesa das plantas de tomate quando tratadas com Streptomyces spp. A poss?vel influ?ncia de Streptomyces spp. na redu??o da doen?a Talo oco em plantas de tomate tamb?m foi avaliada. A caracteriza??o bioqu?mica de isolados de Streptomyces spp. foi realizada por meio da capacidade de produzir sider?foros, solubilizar fosfato, e da atividade de amilase e lipase, bem como a produ??o de compostos org?nicos vol?teis. O antagonismo de Streptomyces spp. contra Pcb foi determinado pelo m?todo de dupla cultura e placa com barreira para an?lise do efeito de compostos org?nicos vol?teis (VOC). A promo??o do crescimento das plantas foi avaliada por meio de emiss?o de VOC e pela intera??o direta com os isolados de Streptomyces spp. (PM1, PM3, PM4, PM5, PM6 e PM9). Enzimas relacionadas ? resposta de defesa foram analisadas colorimetricamente em plantas tratadas com isolados de Streptomyces spp. A avalia??o da doen?a Talo oco foi realizada em plantas tratadas com Streptomyces spp. e desafiadas com Pcb atrav?s da ?rea sob a curva de progresso da doen?a e da mortalidade das plantas em 24 dias. Os isolados de Streptomyces spp. mostraram caracter?sticas de PGPR e 32 compostos vol?teis foram identificados como produtos dos diferentes isolados. PM3 foi o isolado mais eficiente quanto ao antagonismo contra Pcb. A maioria dos isolados promoveu o aumento do comprimento de raiz e da parte a?rea do tomateiro por VOC, embora PM5 tenha sido tamb?m eficiente na promo??o do crescimento atrav?s da intera??o direta com Streptomyces spp. O tratamento com Streptomyces spp. modulou a atividade de enzimas relacionadas ? defesa e diminuiu a incid?ncia da doen?a Talo oco.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-23T14:56:57Z No. of bitstreams: 1 DIS_FERNANDA_TEIXEIRA_SUBTIL_COMPLETO.pdf: 1495988 bytes, checksum: 70f1cb1dccabfa7c302effb51db76e03 (MD5) / Made available in DSpace on 2017-06-23T14:56:57Z (GMT). No. of bitstreams: 1 DIS_FERNANDA_TEIXEIRA_SUBTIL_COMPLETO.pdf: 1495988 bytes, checksum: 70f1cb1dccabfa7c302effb51db76e03 (MD5) Previous issue date: 2017-03-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The high incidence of tuberculosis is a great concern worldwide. Different strategies are being developed by the World Health Organization to fight tuberculosis. Amongst the three pillars that are part of the End TB Strategy, we can highlight the intensive research and innovation pillar. In this extent, the development of new drugs for tuberculosis treatment is a field that is gaining importance. The 2(quinolin-4-yloxy) acetamides are molecules that showed promising bactericidal effects in Mycobacterium tuberculosis, which motivated us to continue studying to improve their mycobactericidal activity and also perform their chemical and biological characterization. In order to continue the 2(quinolin-4-yloxy) acetamides derivatives development, the target identification of these molecules is a keystone and is also the focus of this study. Initially, it was hypothesized that DNA gyrase was their molecular target due to the great chemical similarities between the 2(quinolin-4-yloxy) acetamides and the fluoroquinolones. Despite our results that the 2(quinolin-4-yloxy) acetamides have diminished effects in ofloxacin resistant clinical isolates, the results obtained with the gyrA point mutant and with DNA gyrase protein revealed that this enzyme is not the molecular target of these compounds. The new target identification strategy involved the selection of spontaneous mutants for our lead compound 12L, characterization of this mutant strain against other 2(quinolin-4-yloxy) acetamides derivates and whole genome sequencing. Whole genome sequencing data allowed the identification of a single mutation (T313A) in the QcrB protein, which is the B subunit of cytochrome bc1 complex. This mutation was confirmed by Sanger sequencing and molecular docking results confirmed the importance of this residue for proteindrug interaction. The cytochrome bc1 complex is involved in the electron transport of the bacilli?s respiratory chain, and therefore it appears to be an interesting target, especially to treat the latent form of the disease. We hope that this work contributes to the 2(quinolin-4-yloxy) acetamides development for tuberculosis treatment. / A alta incid?ncia da tuberculose, em ?mbito mundial, ? de grande preocupa??o. Para combater esta doen?a, diferentes estrat?gias v?m sendo desenvolvidas pela Organiza??o Mundial da Sa?de (OMS). Dentre os pilares que comp?em a End TB strategy, podemos destacar a pesquisa intensiva e a inova??o. Neste ?mbito, o desenvolvimento de novos f?rmacos para tuberculose vem ganhando destaque. As 2(quinolin-4-il?xi) acetamidas s?o mol?culas que demonstraram resultados bactericidas promissores frente ao Mycobacterium tuberculosis, o que nos motivou a realizar novos estudos para melhorar a atividade e realizar a caracteriza??o qu?mica e biol?gica destas mol?culas. A fim de continuar o desenvolvimento da s?rie quinol?nica, a identifica??o do seu alvo molecular foi o foco deste trabalho. Inicialmente, levantou-se a hip?tese de que a DNA girase seria o alvo molecular, uma vez que as 2(quinolin- 5-il?xi) acetamidas possuem grande similaridade estrutural com as fluoroquinolonas. Apesar das 2(quinolin-4-il?xi) acetamidas terem apresentado menor atividade frente a isolados cl?nicos resistentes a ofloxacino, os resultados de atividade obtidos frente a uma mutante pontual de gyrA e frente ? prote?na indicam que a DNA girase n?o ? o alvo destas mol?culas. A nova estrat?gia para identifica??o de alvo envolveu a sele??o de mutantes espont?neos para o composto l?der 12L, caracteriza??o desta cepa frente aos demais compostos da s?rie e sequencimento total do genoma. Este permitiu a identifica??o de uma ?nica muta??o (T313A) localizada na prote?na QcrB, que ? a subunidade B do complexo citocromo bc1. Esta muta??o foi confirmada por sequenciamento de Sanger e o docking molecular aferiu a import?ncia deste res?duo na intera??o prote?na-composto. O complexo citocromo bc1 est? envolvido no transporte de el?trons na cadeia respirat?ria do bacilo, e por isso ? um alvo molecular interessante, principalmente para combater a forma latente da doen?a. Esperamos que este trabalho contribua no processo de desenvolvimento das 2(quinolin-4-il?xi) acetamidas para o tratamento da tuberculose.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:23:57Z No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:24:07Z (GMT) No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) / Made available in DSpace on 2017-06-30T13:24:16Z (GMT). No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) Previous issue date: 2017-02-13 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Metals are some of the more toxic substances in the environment. Nickel is one of them and this heavy metal is naturally present in the earth?s crust. However, excessive levels of nickel lead to environmental contamination and can cause serious and irreversible health problems. The aim of this study was to evaluate the toxicological effects of nickel exposure on cognition and behavior, in larvae and adult zebrafish (Danio rerio). Larvae and adult zebrafish were exposed to four different concentrations (0.025, 2, 5, and 15 mg/L NiCl2) or water (control group) in two treatments: acute and subchronic. Larvae were exposed to NiCl2 for 2 hours (acute treatment: 5-day-old larvae treated for 2 hours) or 11 days (subchronic treatment: 11-day-old larvae treated since fertilization) and adults were exposed for 12 hours (acute treatment) or 96 hours (subchronic treatment). In both treatments, for larvae and adults, exposed animals had a significant concentration-dependent increase in nickel levels compared to control group. For larvae, the survival rate was similar in both treated groups compared to control. However, a significant delay of hatching, a decreased heartbeat rate and morphological alterations (decrease of body length and ocular area at 5 and 8 days post-fertilization, dpf) were observed in subchronically-treated animals. Aversive and exploratory behavior showed no significant differences among doses in acute treatment. In contrast, larvae analyzed at 5 dpf in subchronic treatment displayed differences in exploratory behavior, showing decrease in distance traveled and mean speed, at 0.025 mg/L, whereas there was an increase at same parameters in higher doses (5 and 15 mg/L). Over the 11 days of treatment, the locomotor behavior decreased significantly, at 15 mg/L, at 8 and 11 dpf. Furthermore, subchronic-treated larvae showed impaired aversive long-term memory in the inhibitory avoidance task in high doses analyzed (2, 5 and 15 mg/L). For adults, acute treatment did not alter the locomotor activity. Besides, animals submitted to the concentration of 15.0 mg/L, in subchronic treatment, showed anxiogenic effects. The social behavior was not altered by treatments. However, the exposure to NiCl2 caused a decrease in aggressive behavior (subchronic treatment) and impaired memory (acute and subchronic treatments) in all doses compared to controls. In order to evaluate if nickel exposure produced alterations in the hematological system, we analyzed different blood cells in adult animals. The results showed that treated animals submitted to the concentration of 2.0 mg/L, in acute and subchronic treatment, presented an increase of monocytes. Furthermore, to verify if the behavioral alterations observed in treated animals were related to mechanisms of neuronal death, analyzes of apoptotic death in larvae and specific markers of neuronal death in larvae and adults were performed. The results of apoptotic death demonstrated a significant increase in cell death in acutely treated larvae at a concentration of 15 mg/L. In the subchronic treatment, at 5 dpf, there was an increase in apoptotic death at concentration of 5 mg/L, at 8 dpf, in concentrations of 5 and 15 mg/L and at 11 dpf, in concentrations of 2 and 5 mg/L. In the analysis of specific markers of neuronal death, the results suggest that NiCl2 concentrations tested in larvae and adults did not alter the protein levels of cell death markers. These results suggest that prolonged exposure to nickel in early life stages of zebrafish development leads to morphological and physiological alterations and cognition and locomotor deficits, whereas it may cause anxiogenic effects, impaired memory and decrease aggressive behavior in adult stage. These morphological and behavioral alterations may be associated to neurotoxic effects damage caused by this metal. / Metais s?o algumas das subst?ncias mais t?xicas de grande preocupa??o ambiental. O n?quel ? um destes agentes, sendo que este metal pesado ? naturalmente presente na crosta terrestre. Entretanto, n?veis excessivos de n?quel levam ? contamina??o ambiental e podem causar s?rios e irrevers?veis problemas de sa?de. O presente estudo visa avaliar os efeitos t?xicos da exposi??o aguda e subcr?nica ao n?quel, em peixe-zebra (Danio rerio), no est?gio larval e adulto. Os animais foram expostos a quatro diferentes concentra??es (0,025, 2, 5 e 15 mg/L NiCl2) ou ?gua (grupo controle) e submetidos a dois tratamentos: agudo e subcr?nico. As larvas foram expostas por 2 horas (tratamento agudo: larvas de 5 dias p?s-fertiliza??o - dpf - tratadas por 2 horas) ou 11 dias (tratamento subcr?nico: larvas tratadas do momento da fertiliza??o at? 11 dpf). Os animais adultos foram expostos por 12 horas (tratamento agudo) ou 96 horas (tratamento subcr?nico). Nos dois tratamentos, larvas e adultos expostos apresentaram um aumento dependente da concentra??o nos n?veis de NiCl2, comparados com animais do grupo controle. A taxa de sobreviv?ncia das larvas e embri?es n?o foi alterada pelos tratamentos. Entretanto, atraso na eclos?o dos ovos, diminui??o dos batimentos card?acos e altera??es morfol?gicas (diminui??o do comprimento corporal e ?rea ocular aos 5 e 8 dpf) foram observados em animais tratados subcronicamente. O comportamento aversivo e explorat?rio n?o mostrou diferen?as significativas entre as concentra??es de NiCl2, no tratamento agudo. Diferentemente, larvas subcronicamente tratadas, analisadas aos 5 dpf, apresentaram diferen?as no comportamento explorat?rio, exibindo diminui??o na dist?ncia percorrida e na velocidade m?dia, na concentra??o de 0,025 mg/L, enquanto houve um aumento dos mesmos par?metros em altas concentra??es (5 e 15 mg/L). Ao longo dos 11 dias de tratamento, o comportamento explorat?rio diminuiu significativamente, na concentra??o de 15 mg/L, aos 8 e 11 dpf. Al?m disso, larvas submetidas ao tratamento subcr?nico com altas concentra??es de NiCl2 (2, 5 e 15 mg/L) apresentaram comprometimento no comportamento aversivo. Em animais adultos, o tratamento agudo n?o promoveu altera??es na atividade locomotora. Por outro lado, animais expostos ? concentra??o de 15 mg/L no tratamento subcr?nico demonstraram efeito ansiog?nico. A intera??o social n?o foi alterada pelos tratamentos. Entretanto, a exposi??o ao NiCl2 levou a uma diminui??o do comportamento agressivo (tratamento subcr?nico) e comprometimento de mem?ria (tratamento agudo e subcr?nico), em todas as concentra??es testadas, quando comparados ao grupo controle. No sentido de avaliar se a exposi??o ao n?quel produziu altera??es no sistema hematol?gico, avaliamos diferentes c?lulas leucocit?rias em animais adultos. Os resultados encontrados mostraram um aumento significativo de mon?citos em animais expostos de forma aguda e subcr?nica na concentra??o de 2 mg/L comparado aos controles. Al?m disso, para verificar se as altera??es comportamentais observadas em animais tratados estavam relacionadas com mecanismos de morte celular e neuronal, foram realizadas an?lises de morte apopt?tica em larvas e de marcadores espec?ficos de morte neuronal em larvas e adultos. Os resultados de morte apopt?tica, demonstraram um aumento significativo de morte celular em larvas tratadas de forma aguda na concentra??o de 15 mg/L. No tratamento subcr?nico, aos 5 dpf, houve um aumento de morte apopt?tica na concentra??o de 5 mg/L, em 8 dpf nas concentra??es de 5 e 15 mg/L e em 11 dpf nas concentra??es de 2 e 5 mg/L. Na an?lise de marcadores espec?ficos de morte neuronal, os resultados sugerem que as concentra??es de NiCl2 testadas em larvas e adultos n?o alteraram os n?veis de prote?na dos marcadores de morte celular. Estes resultados sugerem que a exposi??o prolongada ao n?quel, em est?gios iniciais do desenvolvimento do peixe-zebra, leva a altera??es morfol?gicas, fisiol?gicas e d?ficits na cogni??o e locomo??o, enquanto que, em animais adultos, foi observado um efeito ansiog?nico, comprometimento de mem?ria e diminui??o do comportamento agressivo. Estas altera??es morfol?gicas e comportamentais podem estar associadas a efeitos neurot?xicos causados por este metal.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T17:30:00Z No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T17:30:08Z (GMT) No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) / Made available in DSpace on 2017-06-30T17:30:17Z (GMT). No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) Previous issue date: 2017-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The plant growth promoting rhizobacteria (PGPR) can increase agricultural productivity by promoting growth through production of plant hormones, facilitating the uptake of nutrients and chemicals on the soil, as well as inhibiting plant stress factors. Streptomyces spp. (Stm) are bacteria with great biotechnological potential, because in addition to its growth promotion and plant defense induction, they are also known as great producers of secondary metabolites, including antibiotics and phenazines. Soybean [Glycine max (L) Merrill] is one of the main legume crop grown around the world and Brazil is the second largest producer. Its production is affected by many diseases, among which bacterial pustule caused by the pathogen Xanthomonas axonopodis pv. glycines (Xag). The objective of this project was to evaluate isolates of Streptomyces spp. obtained from the rhizosphere of Fabaceae plants regarding characteristics of PGPR, as well as the modulation capacity of soybean defenses in response to the phytopathogen Xag. Eleven isolates of Streptomyces spp. were screened for PGPR traits by siderophores production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA) and phenazines. For a taxonomic identification and growth evaluation of soybean plants, three isolates were selected for their biochemical characteristics. The growth promoting assay was performed in greenhouse using bacterized seeds with the selected isolate and sterile distilled water was use for the control. Length, fresh and dry weight from shoot and root at 15, 30 and 45 days of cultivation were the evaluated parameters. For evaluation of the induction capacity of defense mechanisms of soybean plants, the isolate that obtained the best performance in the growth promotion test was selected. Seeds of soybeans, from sensitive cultivars and resistant to Xag, were bacterized with the selected Stm isolate and grown under greenhouse conditions. The plants were challenged with Xag 15 days after emergence. The treatments consisted of (a) plants treated with sterile distilled water (absolute control); (b) plants bacterized with Stm CLV45 (Stm45); (c) water-treated and Xag challenged plants; and (d) plants bacterized with StmCLV45 and challenged with Xag (Stm45+Xag). The enzymatic responses related to the defense pathways were evaluated biochemically, by analyzing the activity of phenylalanine ammonia lyase (PAL) and by the production of phenolic compounds at times 0, 24, 48, 72 and 144 hours post infection (hpi) of Xag. The expression of the genes related to the defense in Xag challenged soybean plants was determined by the relative expression of the genes PAL, JAZ, ERF5 and PR1 by qPCR at times 0, 12, 24 and 48 hpi. The results of the biochemical analysis indicated the isolates CLV42, CLV44 and CLV46 as the major producers of siderophores and CLV41, CLV45 and CLV46 isolates with higher ACC deaminase activity. All isolates were able to produce IAA, highlighting the isolate CLV45, which produced 398.53 ?g AIA g-1 cell. Phenazine pyocyanin (PYO) was also detected in all isolates, but the same did not occur for the 1-carboxylic acid phenazine (PCA), only produced by CLV41, CLV43 and CLV45. The isolates CLV42, CLV44 and CLV45 were selected for their PGPR characteristics for the growth promotion trial of greenhouse soybean plants and taxonomically characterized as species of the genus Streptomyces. None of the isolates evaluated in the trial caused a growth deficit in soybean plants. The CLV45 isolate significantly promoted the growth of soybean shoots in 36.63%, corroborated by the highest dry mass, 17.97%, in relation to the control group, being selected for soybean defense pathways induction. Expression of PAL gene was moderately enhanced in susceptible Stm45+Xag plants at 12 hpi, followed by increase of PAL enzyme activity from 48 to 144 hpi, although corresponding accumulation of phenolic compounds was not recorded. In the resistant cultivar, the highlighted expression of PAL in Stm45+Xag plants resulted in high activity of this enzyme. Enhanced expression of ERF5 and decrease on JAZ gene at 12 hpi in Stm45+Xag plants from both cultivars suggested that ET and JA play a concert role on induced systemic defense by Streptomyces sp. CLV45 against Xag in soybean. / As rizobact?rias promotoras de crescimento de plantas (PGPR) podem aumentar a produtividade agr?cola, atuando atrav?s da promo??o de crescimento vegetal por meio de fitorm?nios reguladores de crescimento, facilitando a capta??o de nutrientes e de compostos qu?micos no solo, bem como inibindo fatores de estresse vegetal. Bact?rias do g?nero Streptomyces spp. (Stm) apresentam grande potencial biotecnol?gico, pois al?m de promoverem o crescimento e a indu??o de defesa vegetal, tamb?m s?o conhecidas pela grande produ??o de metab?litos secund?rios, incluindo antibi?ticos e fenazinas. A soja [Glycine max (L.) Merrill] ? uma das principais leguminosas cultivadas no mundo, sendo o Brasil o segundo maior produtor. Sua produ??o ? afetada por in?meras doen?as, como a P?stula bacteriana causada pelo fitopat?geno Xanthomonas axonopodis pv. glycines (Xag). O objetivo deste trabalho foi avaliar 11 isolados de Streptomyces spp. oriundos da rizosfera de plantas de Fabaceae quanto ?s caracter?sticas de PGPR, bem como, ? capacidade de modula??o das vias de defesa de plantas de soja em resposta ? fitobact?ria patog?nica Xag. Os isolados foram avaliados quanto ?s caracter?sticas de PGPR pela produ??o de sider?foros, de ?cido indolac?tico (AIA), da enzima 1-aminociclopropano-1-?cido carbox?lico (ACC) desaminase e de fenazinas. Para a identifica??o taxon?mica e a avalia??o da promo??o de crescimento de plantas de soja foram selecionados tr?s isolados com caracter?sticas de PGPR. O ensaio de promo??o de crescimento ocorreu em casa de vegeta??o por meio da microbioliza??o das sementes pelos isolados selecionados e o controle com ?gua destilada est?ril. Os par?metros avaliados foram: comprimento, massa fresca e seca, de parte a?rea e raiz, aos 15, 30 e 45 dias de cultivo. Para a avalia??o da capacidade de indu??o dos mecanismos de defesa de plantas de soja, foi selecionado o isolado que obteve o melhor desempenho no ensaio de promo??o de crescimento. Sementes de soja, de cultivar sens?vel e resistente ? Xag, foram microbiolizadas com o isolado de Stm selecionado e cultivadas em casa de vegeta??o. As plantas obtidas foram desafiadas com Xag, 15 dias ap?s a sua emerg?ncia. Os tratamentos consistiram de (a) sementes tratadas com ?gua destilada est?ril (controle absoluto); (b) sementes microbiolizadas com StmCLV45 (Stm45); (c) sementes tratadas com ?gua destilada est?ril e plantas desafiadas com Xag (Xag); e (d) sementes microbiolizadas StmCLV45 e plantas desafiadas com Xag (Stm45+Xag). As respostas enzim?ticas relacionadas ?s vias de defesa foram avaliadas bioquimicamente, pela an?lise da atividade da fenilalanina am?nia liase (PAL) e pela produ??o dos compostos fen?licos, nos tempos 0, 24, 48, 72 e 144 horas p?s inocula??o (hpi) da Xag. A express?o dos genes relacionados ? defesa das plantas de soja desafiadas com Xag foi determinada pela express?o relativa de JAZ, ERF5, PAL e PR1 por qPCR, nos tempos 0, 12, 24 e 48 hpi. Os resultados da an?lise bioqu?mica indicaram os isolados CLV42, CLV44 e CLV46 como maiores produtores de sider?foros e os isolados CLV41, CLV45 e CLV46 com maior atividade de ACC desaminase. Todos os isolados foram capazes de produzir AIA, com destaque para o isolado CLV45, que produziu 398,53 ?g AIA g-1 de c?lulas. A fenazina piocianina (PYO) tamb?m foi detectada em todos os isolados, entretanto o mesmo n?o ocorreu para a fenazina 1-?cido carbox?lico (PCA), somente produzida por CLV41, CLV43 e CLV45. Os isolados CLV42, CLV44 e CLV45 foram selecionados por suas caracter?sticas de PGPR para o ensaio de promo??o do crescimento de plantas de soja em casa de vegeta??o e caracterizados taxonomicamente como esp?cies do g?nero Streptomyces. Nenhum dos isolados avaliados no ensaio causou d?ficit de crescimento em plantas de soja. O isolado CLV45 promoveu significativamente o crescimento de parte a?rea de plantas soja, em 36,63%, corroborado pela maior massa seca, 17,97%, em rela??o ao grupo controle, sendo selecionado para avalia??o nas vias de defesa da soja. A express?o do gene PAL foi moderadamente aumentada em plantas suscet?veis Stm45+Xag em 12 hpi, seguido por aumento da atividade da enzima PAL de 48 a 144 hpi, embora o ac?mulo correspondente de compostos fen?licos n?o tenha sido registrado. Na cultivar resistente, a express?o de PAL em plantas Stm45+Xag resultou em alta atividade desta enzima. A express?o aumentada de ERF5 e a diminui??o de express?o do gene JAZ em 12 hpi em plantas Stm45+Xag de ambas as cultivares sugeriram que etileno e ?cido jasm?nico desempenharam fun??o na defesa sist?mica induzida por Streptomyces sp. CLV45 contra Xag em plantas de soja.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:09:29Z No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:09:36Z (GMT) No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) / Made available in DSpace on 2017-06-30T18:09:44Z (GMT). No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) Previous issue date: 2017-03-06 / Neurotoxic agents, such as pesticides, organic solvents and metals, are capable to alter normal CNS activity. Manganese (Mn) is a metal found in the environment, which when in excess, accumulates in the CNS making it a potential hazard to health. In addition to Mn, other neurotoxic agents can cause changes, leading to deficits in neurotransmission systems. The growth of crop pests has exponentially increased the use of agrochemicals to maintain plantations. The fungicide tebuconazole is a triazole used in crops, such as barley, wheat, peanuts and orchard fruits and related to behavioral and oxidative changes. The insecticide dichlorvos, belongs to organophosphate family, acts through the acetylcholinesterase (AChE) inhibition, leading to hyperstimulation of cholinergic receptors. Cholinergic system is characterized by the action of acetylcholine (ACh) on muscarinic and nicotinic receptors. The level of this molecule is regulated by acetylcholinesterase (AChE), which catalyzes degradation of ACh into choline and acetate. Purinergic system is characterized by the action of ATP and adenosine on purinoreceptor P2 and P1, respectively. The levels of these molecules are regulated by ectonucleotidases, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by action of adenosine deaminase (ADA). Dopamine (DA), synthesized from L-DOPA, is the neurotransmitter of the dopaminergic system and it acts through its D1-D5 receptors, controlling the start and execution of movement. This study aims to evaluate the effects of exposure to manganese and pesticides, such as dichlorvos and tebuconazole, on behavioral parameters in larval and adult zebrafish, as well as its effects on purinergic, cholinergic and dopaminergic signaling. Exposure to MnCl2 in the early stages of development (1 hpf - 5 dpf) was able to reduce the distance traveled, absolute turn angle and mobile time of the larvae at 5, 7 and 10 dpf. In addition, it caused an increase in TH levels at 5 and 7 dpf, followed by a reduction at 10 dpf. There was also an increase in markers of cell death. In adult animals, exposure during 96 hours caused a reduction in distance traveled, number of line crossings and absolute turn angle, and an inhibition on memory acquisition through the inhibitory avoidance task. In addition, adult animals exposed to MnCl2 showed increased levels of markers of cell death, TH and DOPAC (3,4-dihydroxyphenylacetic acid) in the brain. The 96-hour exposure to this metal was also able to inhibit the NTPDases and ecto-ADA activities and to reduce ADA2.1-mRNA transcripts, besides for increasing ADP levels and reducing the amount of AMP, ADO and INO in the incubation medium. Tebuconazole was able to increase the ocular distance of larvae at 5 dpf and reduce the exploratory behavior of these animals, and still decrease the locomotor activity of 96 hours exposed adult zebrafish. Moreover, this fungicide decreased AChE activity in both 5 dpf larvae and brains of adult animals. Exposure to dichlorvos (1 hpf - 7 dpf) reduced body length, heartbeat rate and larval surface at 7 dpf as well as affected the escape capacity of larvae at 7 and 14 dpf. Further, it reduced exploratory behavior at 7 and 14 dpf, maintaining this pattern when animals became young adults at 30 dpf, and adults at 70 dpf. However, the analysis performed at 120 dpf showed a recovery of behavioral activity equal to the control. In addition, the analysis of exposure to dichlorvos in the early stages of development at 120 dpf on the enzymes of the purinergic system, showed that this insecticide is able to elevate the ecto-5'-nucleotidase activity and reduce the ecto-ADA activity. The results showed that neurotoxic agents, such as Mn, dichlorvos and tebuconazole, play a role in the larval and adult behavior of zebrafish, and affect different neurotransmission systems. Such behavioral changes may be associated with the deficits observed in neurochemical and molecular mechanisms investigated. / Agentes neurot?xicos, tais como pesticidas, solventes org?nicos e metais, s?o capazes de alterar a atividade normal do Sistema Nervoso Central (SNC). O mangan?s (Mn) ? um metal encontrado no ambiente que, quando em excesso, acumula-se no SNC, tornando-se um perigo potencial para a sa?de. Al?m do Mn, outros agentes neurot?xicos podem causar altera??es, levando a d?ficits em sistemas de neurotransmiss?o. O crescimento de pragas de lavoura tem aumentado exponencialmente o uso de agrot?xicos para manuten??o de planta??es. O fungicida tebuconazol ? um triazol utilizado em culturas como a cevada, trigo, amendoim e frutas do pomar e relacionado com altera??es comportamentais e oxidativas. O inseticida dicl?rvos, pertence a fam?lia dos organofosforados atuando atrav?s da inibi??o da acetilcolinesterase (AChE), que leva ? hiperestimula??o dos receptores colin?rgicos. O sistema colin?rgico ? caracterizado pela a??o da acetilcolina (ACh) nos receptores muscar?nicos e nicot?nicos. O n?vel dessa mol?cula ? regulado pela acetilcolinesterase (AChE), que catalisa a degrada??o da ACh em colina e acetato. O sistema purin?rgico ? caracterizado pela a??o do ATP e adenosina (ADO) nos purinoreceptores P2 e P1, respectivamente. Os n?veis dessas mol?culas s?o regulados pela a??o das ectonucleotidases que catalisam a hidr?lise do ATP a adenosina (ADO). A ADO pode ser desaminada a inosina (INO) pela a??o da adenosina desaminase (ADA). A dopamina (DA), sintetizada a partir da L-DOPA, ? o neurotransmissor do sistema dopamin?rgico e age atrav?s dos seus receptores D1-D5, controlando o in?cio e execu??o do movimento. Portanto, este estudo visa avaliar os efeitos da exposi??o ao mangan?s e agrot?xicos, tais como dicl?rvos e tebuconazol, sobre par?metros comportamentais em peixe-zebra nos est?gios larval e adulto, bem como os seus efeitos sobre a sinaliza??o purin?rgica, colin?rgica e dopamin?rgica. A exposi??o ao MnCl2 nos est?gios iniciais de desenvolvimento (1 hpf - 5 dpf) foi capaz de reduzir a dist?ncia percorrida, o ?ngulo de giro absoluto e o tempo de mobilidade das larvas aos 5, 7 e 10 dpf. Al?m disso, causou um aumento nos n?veis de TH aos 5 e 7 dpf, seguido por uma redu??o em 10 dpf. Tamb?m foi observado um aumento dos marcadores de morte celular. Em animais adultos, a exposi??o ao longo de 96 horas causou redu??o na dist?ncia percorrida, n?mero de cruzamentos e ?ngulo de giro absoluto, e ainda uma redu??o na aquisi??o de mem?ria atrav?s da tarefa de esquiva inibit?ria. Al?m disso, os animais adultos expostos a MnCl2 apresentaram aumento nos n?veis de marcadores de morte celular, al?m de TH e DOPAC (?cido 3,4-di-hidroxifenilac?tico) no enc?falo. A exposi??o por 96 horas a este metal tamb?m foi capaz de inibir a atividade das NTPDases e da ecto-ADA e reduzir os transcritos de RNAm do gene ADA2.1, al?m de aumentar os n?veis de ADP e reduzir a quantidade de AMP, ADO e INO no meio. O tebuconazol foi capaz de aumentar a dist?ncia ocular de larvas aos 5 dpf e reduzir a capacidade explorat?ria desses animais, e ainda diminuir a atividade locomotora de peixe-zebra adultos expostos por 96 horas. Al?m disso, este fungicida diminuiu a atividade da AChE tanto em larvas de 5 dpf, quanto em enc?falo de animais adultos. Al?m do mais, o tebuconacol aumentou a express?o relativa de mRNA da AChE em larvas, no entanto, n?o alterou em adultos. A exposi??o ao dicl?rvos, nas fases iniciais do desenvolvimento (1 hpf - 7 dpf), reduziu o tamanho corporal, a taxa de batimentos card?acos e a superf?cie dos olhos de larvas de 7 dpf, bem como afetou a capacidade de escape de larvas aos 7 e 14 dpf. Al?m disso, reduziu a capacidade explorat?ria aos 7 e 14 dpf, padr?o mantido quando os animais tornaram-se adultos jovens aos 30 dpf, e adultos aos 70 dpf. No entanto, as an?lises realizadas aos 120 dpf mostraram recupera??o da atividade comportamental similar ao grupo controle. A an?lise aos 120 dpf da exposi??o ao dicl?rvos nas fases iniciais do desenvolvimento sobre as enzimas do sistema purin?rgico mostrou que este inseticida ? capaz de elevar a atividade da ecto-5?-nucleotidase e reduzir a atividade da ecto-ADA. Os resultados mostram que os agentes neurot?xicos, tais como Mn, dicl?rvos e tebuconazol t?m atua??o significativa sobre o comportamento do peixe-zebra em est?gio larval e adulto, al?m de afetar diferentes sistemas de neurotransmiss?o. Tais altera??es comportamentais podem estar associadas aos d?ficits observados nos mecanismos neuroqu?micos e moleculares investigados.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:19:34Z No. of bitstreams: 1 DIS_JESSICA_CAVALHEIRO_SOARES_PARCIAL.pdf: 613600 bytes, checksum: 2e577069eeb48be6afd6a79bac3f8992 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:19:41Z (GMT) No. of bitstreams: 1 DIS_JESSICA_CAVALHEIRO_SOARES_PARCIAL.pdf: 613600 bytes, checksum: 2e577069eeb48be6afd6a79bac3f8992 (MD5) / Made available in DSpace on 2017-06-30T18:19:49Z (GMT). No. of bitstreams: 1 DIS_JESSICA_CAVALHEIRO_SOARES_PARCIAL.pdf: 613600 bytes, checksum: 2e577069eeb48be6afd6a79bac3f8992 (MD5) Previous issue date: 2017-02-21 / Recent studies have shown several applications of graphene oxide (GO) in neurology in the imaging area as a contrast agent for diagnostic purposes, for drug delivery of anticarcinogenic drugs, proteins and peptides and in the field of tissue engineering for applications in the regeneration of nervous lesions. Advances in GO applications research in neurology require addressing its potential toxic mechanisms of action. Zebrafish (Danio rerio) has been widely used in neurotoxicological trials due to the homology of biochemical processes and neural structures with vertebrates. To investigate the potential neurotoxic effects of GO exposure in different concentrations at development period, we evaluated in vivo parameters for initial toxicological screening. We found that GO did not induce changes in survival, hatching and spontaneous movement. However, an increase in heart rate at 48 hpf was found and a reduction in body length without altering the ocular area at 5 dpf. Additionally, molecular gene expression analyses of nervous system-related proteins were performed, showing synapsin IIa expression is increased and dopamine transporter (dat) gene expression is reduced, suggesting a potential compensatory mechanism. The evaluation of the locomotion behavior of animals exposed at GO showed an increase in the absolute turn angle. Based on these initial parameters, we performed biochemical analyzes to determination of the enzyme acetylcholinesterase (AChE) activity and dopamine levels. The exposure at GO did not change AChE activity and decreased dopamine levels. Additionally, the gene expression of B cell lymphoma 2 (bcl2) gene and caspase 3 (casp3) gene were evaluated for the group of larvae exposed to GO, demonstrating an increase of bcl2 and unchanged casp3 expression, suggesting no apoptosis involvement. The tissue and cellular structure of zebrafish larvae brain exposed at GO was evaluated by transmission electron microscopy (TEM) and cell dead by autophagosome formation with loss of cellular architecture was observed most likely due to exposure to the nanomaterial. Further studies are necessary to the complete understanding of the neurological changes observed in zebrafish by exposure to GO and the mechanisms involved in this process, are necessary. / Estudos recentes t?m mostrado diversas aplica??es do ?xido de grafeno (GO) como agente de contraste para fins de diagn?stico, para o endere?amento de drogas (do ingl?s drug delivery) de f?rmacos anticarcinog?nicos, prote?nas e pept?deos e na ?rea de engenharia tecidual para aplica??es na regenera??o de les?es nervosas. O avan?o em pesquisas relacionadas a aplica??es do GO em neurologia exige a investiga??o dos mecanismos de a??o potencialmente t?xicos desencadeados pela exposi??o ao nanomaterial. O zebrafish (Danio rerio) tem sido amplamente utilizado em ensaios neurotoxicol?gicos devido ? homologia de processos bioqu?micos e estruturas neurais com outros vertebrados, vis?veis desde o in?cio de seu desenvolvimento externo. Para investigar os efeitos neurot?xicos da exposi??o a diferentes concentra??es do GO no per?odo de desenvolvimento, foram avaliados diversos par?metros in vivo para avalia??o toxicol?gica preliminar. Foi verificado que o GO n?o induziu altera??es na sobreviv?ncia, eclos?o e movimentos espont?neos. Contudo, o aumento dos batimentos card?acos em 48 hpf foi constatado e redu??o do tamanho do eixo axial, sem altera??o na ?rea ocular em 5 dpf. Adicionalmente, an?lises moleculares da express?o de genes espec?ficos para prote?nas do sistema nervoso foram realizadas mostrando que o GO aumenta a express?o de sinapsina IIa e reduz a express?o do transportador de dopamina (dat), sugerindo um prov?vel mecanismo compensat?rio. A avalia??o da locomo??o dos animais expostos ao GO mostra o aumento do ?ngulo absoluto de virada. Com base nestes par?metros iniciais, foram realizadas an?lises bioqu?micas para a determina??o da atividade da enzima acetilcolinesterase (AChE) e dosagem dos n?veis de dopamina. A exposi??o ao GO n?o alterou a atividade da AChE e reduziu os n?veis de dopamina. Adicionalmente, a express?o do gene de linfoma de c?lulas B2 (do ingl?s B cell lymphoma 2 (bcl2) ) e do gene que codifica para caspase 3 (casp3) foram avaliadas para o grupo de larvas expostas ao GO onde foi mostrado aumento da express?o de bcl2, enquanto que a express?o de casp3 permaneceu inalterada, sugerindo que o GO parece n?o induzir mecanismo de apoptose. A estrutura tecidual e celular do c?rebro de larvas de zebrafish expostas ao GO foi avaliada por microscopia eletr?nica de transmiss?o (TEM) onde morte celular por necrose com perda da arquitetura celular foi constatada. Estudos adicionais para o completo entendimento das altera??es neurol?gicas observadas pela exposi??o ao GO e dos mecanismos envolvidos nesse processo s?o necess?rios.