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.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:29:48Z No. of bitstreams: 1 TES_LEONARDO_PEDRAZZA_PARCIAL.pdf: 9312449 bytes, checksum: e82cef97eb17ae6eefdb30bc5fe133ed (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:29:54Z (GMT) No. of bitstreams: 1 TES_LEONARDO_PEDRAZZA_PARCIAL.pdf: 9312449 bytes, checksum: e82cef97eb17ae6eefdb30bc5fe133ed (MD5) / Made available in DSpace on 2017-06-30T18:30:01Z (GMT). No. of bitstreams: 1 TES_LEONARDO_PEDRAZZA_PARCIAL.pdf: 9312449 bytes, checksum: e82cef97eb17ae6eefdb30bc5fe133ed (MD5) Previous issue date: 2017-03-24 / Mesenchymal stem cells (MSC) were first identified by Friedenstein and Petrakova (1966), who isolated these progenitor cells from rat bone marrow and found that these cells are able to differentiate into connective tissue lineage including bone, adipose tissue, cartilage and muscle. MSCs have emerged in recent years as therapeutic tools based on four important features: differentiation potential, capacity to modulate immune responses, pro-angiogenic and repair promoting capacities, and low immunogenicity, the latter feature may allow allogeneic treatments. Based on their immunomodulatory properties and paracrine effects through trophic factors with anti-fibrotic, anti-apoptotic or pro-angiogenic properties, MSCs are considered a promising instrument for cell therapy, in particular for inflammatory diseases. MSCs regulate the function of a broad range of immune cells, and are activated by inflammatory mediators released from activated immune cells. The mechanisms involved in the immunoregulatory activity of MSCs are still under investigation. Therefore, MSCs become a potential treatment alternative for sepsis and for acute lung infection, which may lead to the interruption of the sequence in the pathogenesis and cause mortality reduction of both pathologies. The principal objective of this study was to evaluate the therapeutic and immunomodulatory effect of MSCs in the treatment of sepsis and acute lung injury and search for their possible mechanisms of action. Our results demonstrated for the first time that the reduction of inflammation in sepsis caused by treatment with MSCs is directly involved in the inhibition of the pathway of mitogen-activated proteins (MAPKs) and that MSCs were unable to modulate the expression of toll-like receptors. During acute lung injury (ALI), the immunomodulation caused by the treatment and the decrease of the oxidative stress that consequently led to a decrease in the formation of extracellular neutrophil network (NETs), leading to an increase in the survival of animals with LPA. The promising results obtained in these studies are encouraging and suggest that MSCs might be a therapeutic option to treat sepsis and acute lung infection in patients in the future. / As c?lulas-tronco mesenquimais (MSC) foram identificadas primeiramente por Friedenstein e Petrakova (1966), que isolaram estas c?lulas progenitoras a partir da medula ?ssea de rato e observaram serem capazes de se diferenciarem em linhagem de tecido conectivo, incluindo osso, tecido adiposo, cartilagem e m?sculo. As MSCs surgiram nos ?ltimos anos como ferramentas terap?uticas baseadas em quatro caracter?sticas importantes: potencial de diferencia??o, capacidade para modular a resposta imune, capacidades pr?-angiog?nicas promovendo regenera??o tecidual, e baixa imunogenicidade, sendo que esta ?ltima caracter?stica pode permitir tratamentos alog?nicos. Com base nas suas propriedades imunomoduladoras e efeitos par?crinos atrav?s de fatores tr?ficos com propriedades anti-fibr?ticas, anti-apopt?ticas ou pr?-angiog?nicas, as MSCs s?o consideradas um instrumento promissor para a terapia celular, em particular para doen?as inflamat?rias. As MSCs regulam as fun??es de uma ampla gama de c?lulas imunes, e s?o ativadas por mediadores inflamat?rios liberados de c?lulas imunes ativadas. Os mecanismos envolvidos na atividade imunorreguladora de MSCs est?o ainda sob investiga??o. Desta forma, as c?lulas-tronco mesequimais se tornam uma potencial alternativa de tratamento para a sepse e para infec??o pulmonar aguda, podendo levar a interrup??o do curso da patog?nese, e provocar a redu??o da mortalidade de ambas patologias. O principal objetivo deste trabalho foi avaliar o efeito terap?utico e imunomodulador de c?lulas-tronco mesenquimais no tratamento da sepse e da les?o pulmonar aguda e buscar os seus poss?veis mecanismos de a??o. Nossos resultados demonstraram pela primeira vez, que a redu??o da inflama??o na sepse provocada pelo tratamento com c?lulas-tronco mesenquimais est? diretamente envolvido a inibi??o da via das prote?nas ativadas por mit?genos (MAPKs) e que as MSCs foram incapazes de modular a express?o de receptores do tipo toll. Durante a les?o pulmonar aguda (LPA) ficou evidente a imunomodula??o provocada pelo tratamento e a diminui??o do estresse oxidativo que consequentemente ocasionou a uma diminui??o da forma??o das redes extracelulares de neutr?filos (NETs), levando a um aumento na sobrevida dos animais com LPA. Os resultados promissores obtidos neste estudo s?o encorajadores e sugerem que as MSCs podem ser uma op??o terap?utica para tratar a sepse e a les?o pulmonar aguda em pacientes no futuro.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:31:20Z No. of bitstreams: 1 TES_ANDRE_ZORATTO_GASTALDO_COMPLETO.pdf: 1361129 bytes, checksum: dbc7c8c4f5ab004118129ecf678044b8 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:31:27Z (GMT) No. of bitstreams: 1 TES_ANDRE_ZORATTO_GASTALDO_COMPLETO.pdf: 1361129 bytes, checksum: dbc7c8c4f5ab004118129ecf678044b8 (MD5) / Made available in DSpace on 2017-06-30T18:31:34Z (GMT). No. of bitstreams: 1 TES_ANDRE_ZORATTO_GASTALDO_COMPLETO.pdf: 1361129 bytes, checksum: dbc7c8c4f5ab004118129ecf678044b8 (MD5) Previous issue date: 2017-03-21 / The evolution in human identification through the study of genetic markers STR (Short Tandem Repeats) induced and propitiated the growth also of animal genetic identification. However, contrary to what occurs in the human domain, animal molecular identification still lacks validation of analysis systems and population studies to be considered reliable to the degree required. Only recently the International Society for Animal Genetics (ISAG) has recommended STR markers suitable for individual discrimination in domestic animal species. Accurate animal genetic identification is sought to provide entities, associations, breeders and owners in general with assurances that their animals are in fact derived from reliable breeders. Only reliable and authentic genealogical records will ensure, for example, efficiency in genetic breeding processes. Even though it is a promising area, there are currently only two options in the market for commercial kits for animal genotyping. But its use has been deferred by specialized laboratories in the field, which end up preparing their own identification panels, using the recommended genetic markers to meet their local demands more specifically. However, if the frequencies of the alleles present in these genetic markers in the study population are not known and the real power of discrimination that they provide is not estimated, the analyzes performed and the results obtained may generate misleading or imprecise interpretations. In the present study, three panels of STR genetic markers were used with all loci recommended by ISAG, capable of identifying equine, bovine and canine individuals. Allelic frequencies and other parameters such as heterozygosity, polymorphism information content, power of exclusion and power of discrimination were obtained from significant samples in breeds present in Rio Grande do Sul (Southern Brazil), Uruguay and Paraguay, to be used in the practice of animal genetics for identification purposes. In addition, comparative population studies within and among breeds were also carried out, in order to evaluate the genetic diversity of the animal populations studied. / O avan?o da identifica??o molecular humana pelo estudo dos marcadores gen?ticos STR (Short Tandem Repeats) induziu e propiciou o crescimento tamb?m da identifica??o gen?tica animal. Contudo, ao contr?rio da ?rea humana, a identifica??o molecular animal ainda carece de valida??es dos sistemas de an?lise e de estudos populacionais para que seja considerada fidedigna no grau que se necessita. Apenas recentemente a ISAG (International Society for Animal Genetics) recomendou marcadores STR adequados para discrimina??o individual em esp?cies de animais dom?sticos. A identifica??o gen?tica animal exata e precisa ? buscada para fornecer, a entidades, associa??es, criadores e propriet?rios em geral, garantias de que seus animais s?o, de fato, oriundos de reprodutores confi?veis. Apenas registros geneal?gicos fidedignos e aut?nticos garantir?o, por exemplo, efici?ncia nos processos de melhoramento gen?tico. Mesmo sendo uma ?rea promissora, neste momento h? somente duas op??es no mercado de kits comerciais para genotipagem animal. Mas seu uso tem sido preterido por laborat?rios do ramo, os quais acabam preparando seus pr?prios pain?is (in house) de identifica??o, utilizando os marcadores gen?ticos recomendados para atender suas demandas locais de forma customizada. Contudo, se n?o forem conhecidas as frequ?ncias dos alelos presentes nestes marcadores gen?ticos na popula??o em estudo e n?o for estimado o real poder de discrimina??o que estes fornecem, as an?lises realizadas e os resultados obtidos poder?o gerar interpreta??es equivocadas ou imprecisas. No presente trabalho, foram utilizados tr?s pain?is de marcadores gen?ticos STR com todos os loci recomendados pela ISAG, capazes de identificar indiv?duos equinos, bovinos e caninos. Frequ?ncias al?licas e outros par?metros como heterozigosidade, conte?do de informa??o de polimorfismo, poder de exclus?o e de discrimina??o foram obtidos a partir de amostras significativas em ra?as presentes no Rio Grande do Sul/Brasil, no Uruguai e no Paraguai, para que sejam usados na pr?tica da gen?tica animal com fins de identifica??o. Al?m disso, estudos populacionais comparativos dentro e entre ra?as tamb?m foram realizados, com o intuito de avaliar a diversidade gen?tica das popula??es animais estudadas.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-25T18:53:17Z No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-26T14:20:44Z (GMT) No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) / Made available in DSpace on 2017-07-26T14:27:43Z (GMT). No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) Previous issue date: 2017-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The redox-active chemical properties of trace element copper make it an essential cofactor for several cellular mechanisms, as for ROS production. Polyphenols have been used in the pro-oxidant mechanism of action of interaction with endogenous Cu (II) ions for the production of reactive oxygen species in excess as a treatment for malignancies, leading to apoptosis. This work studied Cu (II) ions in the complex formation mechanism with Resveratrol in MCF-7 cells model. We analyzed the selectivity of Resveratrol in relation to the cupric metal ion using HPLC (High-Performance Liquid Chromatography) and the formation of the compound Resveratrol-Copper through UV-VIS (Ultra-Violet Visible Spectrophotometry). We analyzed the cellular morphology and location of the metal ion by MET-EDS (Electronic Transmission microscopy with Dispersive X-ray Spectroscopy). Cell death by apoptosis and quantification of ROS in said cell line with copper enrichment and treated with Resveratrol was made by flow cytometry. The results show the selectivity of the polyphenol compound by copper ion as well as the formation of the complex Resveratrol-Copper in extracellular conditions, however even with verification of endogenous copper accumulation in physiological conditions in vitro, the formation of that complex did not occur because there was no production of ROS and therefore, no cell death. In short, our research reveals that for in vitro conditions for the MCF-7 line, there's no Resveratrol-Copper complex formation as observed in sub-lethal quantities of chemically enriched cells with CuSO4 and treated with Resveratrol. / As propriedades qu?micas redox-ativas do oligoelemento cobre o tornam cofator essencial para diversos mecanismos celulares como o de produ??o de ROS. Polifen?is v?m sendo utilizados no mecanismo de a??o pr?-oxidante de intera??o com ?ons Cu (II) end?geno para a produ??o dessas esp?cies oxig?nio reativas em excesso como tratamento de malignidades, levando a apoptose. O presente trabalho estudou os ?ons Cu (II) no mecanismo de forma??o do complexo com Resveratrol em modelo de c?lulas MCF-7. Analisamos a seletividade do Resveratrol frente ao ?on met?lico c?prico utilizando CLAE (Cromatografia L?quida de Alta Efici?ncia) e verificou-se a forma??o do complexo Resveratrol-Cobre por meio de UV-VIS (Espectrofotometria de Ultra-Violeta Vis?vel). Analisou-se a morfologia celular e localiza??o do ?on met?lico por MET-EDS (Microscopia de Transmiss?o Eletr?nica com Espectroscopia Dispersiva de Raios-X). Morte celular por apoptose e quantifica??o de ROS na linhagem enriquecida com cobre e tratadas com Resveratrol foi feita por Citometria de Fluxo. Os resultados mostram a seletividade do composto polifen?lico pelo ?on cobre bem como a forma??o do complexo Resveratrol-Cobre em condi??es extracelulares, por?m mesmo com a verifica??o de ac?mulo de cobre end?geno, em condi??es fisiol?gicas in vitro n?o foi constatada a forma??o do referido, pois n?o houve forma??o de ROS e morte celular conseguinte. Em suma, Nossa pesquisa revela que em condi??es in vitro para a linhagem MCF-7, n?o h? forma??o de complexo Resveratrol-Cobre como observado quimicamente em quantidades sub-letais de c?lulas enriquecidas com CuSO4 e tratadas com Resveratrol.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-18T12:45:18Z No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-28T18:46:03Z (GMT) No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Made available in DSpace on 2017-07-28T18:54:15Z (GMT). No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) Previous issue date: 2017-03-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Liver fibrosis is the wound healing response to repeated injury of the liver. This process begins with the damage of the parenchymal cells and subsequent inflammation, characterized by the rupture of the hepatic architecture associated to the increase of the expression of the components of the extracellular matrix. The development of hepatic fibrosis is based on the activation of hepatic stellate cells (HSC) that undergo phenotypic changes and are characterized by loss of vitamin A deposition and increased cell proliferation, triggering hepatic microcirculatory dysfunction and fibrogenesis in patients with chronic liver disease (CLD). Liraglutide is a GLP-1 agonist (glucagon-like peptide 1) well established as an antidiabetic drug, but also has anti-inflammatory properties, in addition to the effectiveness for NAFLD (non-alcoholic fatty liver disease). Therefore, the aim of this study was to evaluate the effects of liraglutide on the HSC phenotype and liver microvascular function using diverse pre-clinical models of CLD. The results obtained demonstrate that Liraglutide de-activated human and rat HSC phenotype through a GLP1-Rindependent mechanism. Liraglutide did not affect the HSC viability but decreased cell proliferation. CLD-rats receiving liraglutide exhibited significantly lower portal pressure (-20%) with a consequent reduction in intrahepatic vascular resistance. There was also a marked improvements in hepatic vascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue. In conclusion, this study demonstrates for the first time that liraglutide improves hepatic sinusoidal endothelium in clinically relevant experimental models of cirrhosis, which leads to improvement in fibrosis and portal hypertension, and therefore is valid in the treatment of advanced chronic liver disease. / A fibrose hep?tica ? a resposta cicatricial do f?gado ? les?es repetidas. Este processo inicia com o dano das c?lulas parenquimatosas e consecutiva inflama??o, caracterizado pelo rompimento da arquitetura hep?tica associada ao aumento da express?o dos componentes da matriz extracelular. O desenvolvimento da fibrose hep?tica ? baseado na ativa??o das c?lulas hep?ticas estreladas (HSC) que sofrem mudan?as fenot?picas e se caracterizam pela perda do dep?sito de vitamina A e aumento da prolifera??o celular, desencadeando disfun??o microcirculat?ria hep?tica e fibrog?nese nos pacientes com doen?a hep?tica cr?nica (CLD). A liraglutida ? um an?lago do GLP-1 (glucagon-like peptide 1) bem estabelecido como f?rmaco antidiab?tico, mas que tamb?m possui propriedades antinflamat?rias, al?m da efetividade para NAFLD (doen?a hep?tica gordurosa n?o alco?lica). Por essa raz?o, o objetivo deste estudo foi avaliar os efeitos da liraglutida sobre o fen?tipo das HSC e a fun??o microvascular hep?tica utilizando diversos modelos pr?-cl?nicos de CLD. Os resultados obtidos demonstram que a liraglutida desativou o fen?tipo das HSC humanas e de ratos atrav?s de um mecanismo independente do receptor GLP1. A liraglutida n?o afetou a viabilidade das HSC mas diminuiu a prolifera??o celular. Os ratos com CLD que receberam liraglutida apresentaram press?o portal significativamente menor (-20%) com consequente redu??o da resist?ncia vascular intra-hep?tica. Houve tamb?m uma acentuada melhoria na fun??o vascular hep?tica, fibrose, fen?tipo das HSC e fen?tipo endotelial sinusoidal. Os efeitos anti-fibr?ticos da liraglutida tamb?m foram confirmados em tecido hep?tico humano. Como conclus?o, este estudo demonstra pela primeira vez que a liraglutida melhora o endotelio sinusoidal hep?tico em modelos experimentais clinicamente relevantes de cirrose, o que leva a melhora no quadro fibr?tico e na hipertens?o portal e, portanto, pode ser v?lido no tratamento da doen?a hep?tica cr?nica avan?ada.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-10-11T12:59:22Z No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-10-11T20:09:27Z (GMT) No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Made available in DSpace on 2017-10-11T20:19:08Z (GMT). No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) Previous issue date: 2017-03-10 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Glioblastoma multiform (GBM) is considered the most aggressive tumors of central nervous system (CNS), and the most lethal among primary tumors presenting low survival prognosis, resistance to radiotherapy and chemotherapy. P2Y12 is considered a chemoreceptor for adenosine diphosphate (ADP). Its expression is documented in some cancer types, such as the C6 lineage of rat glioma, renal carcinoma and colon carcinoma. However, its role in the development of tumour progression and the resistance mechanism in chemotherapy are not well elucidated. Currently, it is well established that normal platelet function is an important factor for the progression of tumors, with thrombocytopenic rats demonstrating a significant reduction of metastases. Clopidogrel bisulfate is a drug, which belongs to the class of antiplatelet agents, being a P2Y12 receptor antagonist whose endogenous ligand is ADP. The aim of the present study was to determine the effects of tumor cells exposed to the treatment with clopidogrel. The C6 rat cell line was sensitive to the drug tested (150 ?M) in the viability and cell counts (69.63 ? 8.70) and (53.05 ? 20.06). Both cell lines showed a significant reduction in the number of colonies after 10 days of treatment with clopidogrel. The U251-MG strain demonstrated a significant increase in the G1 phase and a significant reduction in the S phase in the cell cycle (20.32 ? 3.05) and (19.45 ? 2.35) in the concentration of 500 ?M. Other results demonstrate important antiproliferative activity in both tumor lines, suggesting an important participation of the P2Y12 receptor in this process. / O glioblastoma multiforma (GBM) ? considerado o mais agressivo dos tumores do sistema nervoso central (SNC) e o mais letal entre os tumores prim?rios apresentando baixo progn?stico de sobrevida, resist?ncia ? radioterapia e a quimioterapia. O P2Y12 ? considerado um quimiorreceptor para a adenosina difosfato (ADP). Sua express?o ? documentada em algumas linhagens de c?ncer, como a linhagem C6 de glioma de rato, carcinoma renal e carcinoma de c?lon. Contudo, seu papel no desenvolvimento da sinaliza??o e resposta ? quimioterapia n?o est? bem elucidado. Atualmente, est? bem estabelecido que o funcionamento normal das plaquetas ? um importante fator para a progress?o de tumores, sendo que ratos com trombocitopenia demonstraram uma redu??o significativa de met?stases. O bissulfato de clopidogrel ? um f?rmaco pertencente ? classe dos antiagregantes plaquet?rios, sendo um antagonista do receptor P2Y12, cujo ligante end?geno ? o ADP. O presente trabalho teve como objetivo averiguar quais os efeitos ocasionados ?s c?lulas de gliomas quando expostas ao clopidogrel. Para isto, foram utilizadas duas linhagens de gliomas, U251-MG de glioma humano e C6 de ratos, e o tratamento com clopidogrel (150, 300, e 500 ?M), foi avaliado nos par?metros de viabilidade celular, contagem de c?lulas, RT-PCR e citometria de fluxo. A linhagem de rato C6 mostrou-se sens?vel ao f?rmaco testado (150 ?M) com redu??o da viabilidade e contagem celular (69.63?8,70) e (53,05 ? 20.06), respectivamente. Ambas as linhagens tapresentaram uma redu??o significativa no n?mero de col?nias ap?s 10 dias tratamento com clopidogrel. A linhagem U251-MG demonstrou um aumento significativo na fase G1 e uma redu??o significativa na fase S e do ciclo celular (20,32 ? 3.05) e (19.45? 2.35 ) na concentra??o de 500 ?M, por?m nos ensaios de viabilidade e contagem n?o houve diferen?a significativa. Nossos resultados demonstram atividade antiproliferativa importante em ambas as linhagens tumorais, sugerindo uma participa??o importante do receptor P2Y12 neste processo.