Made available in DSpace on 2015-04-14T14:51:22Z (GMT). No. of bitstreams: 1 444695.pdf: 624430 bytes, checksum: e4547549fd33f8db2dfcdef98c9ca071 (MD5) Previous issue date: 2012-08-31 / The two parasites in the genus Angionstrongylus that cause disease in humans are Angiostrongylus costarisensis and Angiostrongylus cantonensis. They have different target organs: A. costarisensis is located in the mesentery and causes eosinophilic ileocolitis, whereas A. cantonensis is a neurotropic parasite responsible for eosinophilic meningoencephalitis. Based on several indications that the sexual maturation is associated with increased pathogenesis in angiostrongylid worms, proteomic analysis was performed on A. cantonensis protein samples, to describe differential protein expression between 21 and 42 days post infection. Triplicated bidimensional electrophoresis was submitted to analysis and 11 proteins were found to be exclusively expressed after sexual maturation. Acetate kinase was the only protein that could be identified after mass spectrometry (LC-MS/MS). Since acetate is an important end-product of the energy metabolism among many parasites but not among their mammalian hosts, acetate formation is an attractive target for the development of new anti-parasitic drugs. Furthermore, studying the adaptations in parasite metabolisms can result in an increased understanding of the host-parasite interaction. This data open opportunities for control interventions and new strategies for molecular diagnosis. / Os principais parasitos do g?nero Angionstrongylus que causam doen?a nos seres humanos s?o Angiostrongylus costarisensis e Angiostrongylus cantonensis. Eles t?m distintos ?rg?os alvos: A. costarisensis est? localizado no mesent?rio e causa ileocolite eosinof?lica, enquanto que A. cantonensis ? um parasita neurotr?pico respons?vel pela meningoencefalite eosinof?lica. Com base em v?rias evid?ncias de que a matura??o sexual est? associada ao aumento da patog?nese em vermes deste g?nero, uma an?lise prote?mica foi realizada em vermes de A. cantonensis para descrever a express?o diferencial de prote?nas entre 21 e 42 dias ap?s a infec??o. Eletroforese bidimensional em triplicata foi submetida ? an?lise e 11 prote?nas foram encontradas exclusivamente expressas ap?s a matura??o sexual. A acetato quinase foi a ?nica prote?na identificada por espectrometria de massa (LC-MS/MS). O acetato ? um importante produto final do metabolismo energ?tico de muitos parasitos, mas n?o de seus hospedeiros mam?feros. Sendo assim, ? um alvo atrativo para o desenvolvimento de novas drogas anti-parasit?rias. Al?m do mais, o estudo das adapta??es no metabolismo do parasito pode resultar em uma maior compreens?o da rela??o parasito-hospedeiro. Este trabalho abre oportunidades para interven??es de controle e novas estrat?gias de diagn?stico molecular.

Made available in DSpace on 2015-04-14T14:51:22Z (GMT). No. of bitstreams: 1 444924.pdf: 1000917 bytes, checksum: f4d5f486dd1a714bf1f2ecdcc7d68781 (MD5) Previous issue date: 2012-08-30 / I offer this thesis, which serves the purposes of the Programa de P?s-Gradu??o em Biologia Celular e Molecular, PUCRS and Coordena??o de Aperfei?oamento de Pessoal de N?vel Superiror (CAPES), in order to corroborate the routine practice in forensic genetics. Fired cartridge cases are commonly present in crime scenes with firearms and sometimes they are the only evidence available for the elucidation of a fact, which makes them investigation key players. To load a firearm the individual has to touch the ammunition, resulting in deposition of epithelial cells on the surface of the cartridges. However, in forensic genetic laboratories routine, DNA tests are not required for these signs as often as they are found. The reason for the underuse of these materials is the low concentration and high rate DNA degradation wich occurs due to overheating of the cartridge that can reach 1800oC. Besides that, the inhibition of PCR, few reports of success in obtaining genetic profiles from fired cases and the scarce verifying feasibility studies of these samples are factors that discourage this practice. In order to corroborate the genetic research routine, we have established a partnership between the sectors of Instituto Geral de Per?cias do Rio Grande do Sul (IGP/RS) Forensic Genetics and Ballistics and Laborat?rio de Gen?tica Humana e Molecular da PUCRS (LGHMPUCRS). The present study developed a controlled research to obtaining and analysis of nuclear DNA from fired cartridge cases. The study was based on standard protocols and/or indicated by the Forensic Science Department of Virginia. This study demonstrated that is possible to use fired, or not, cartridge cases as source of DNA to identify individuals who involved with its handle. However, considering the limited efficiency, the restricted effectiveness and the cost-benefit, means that the DNA analysis strategy from cells left in cartridges/cases is not priority in forensics lab routine. But, in many situations it could be the only option to investigators and, at this moment, our results and protocols herein will have main importance. This study concluded that following protocols here presented it is possible to produce data for human identification. The use of these protocols and their results will be definitive when used as an additional part of a police investigation and/or as evidence in criminal proceedings / Apresento esta disserta??o, a qual atende aos prop?sitos do Programa de P?s- Gradua??o em Biologia Celular e Molecular da PUCRS e da Coordena??o de Aperfei?oamento de Pessoal de N?vel Superiror (CAPES), com o prop?sito de corroborar com a rotina de pr?ticas em gen?tica forense. Estojos resultantes da deflagra??o de cartuchos s?o vest?gios comumente presentes em cenas de crime com armas de fogo e, por vezes, ?nicos ind?cios de que se disp?e para a elucida??o de um fato, o que os torna pe?as chave em uma investiga??o. Ao municiar uma arma de fogo ? necess?rio que o indiv?duo toque a muni??o, resultando na deposi??o de c?lulas epiteliais na superf?cie dos cartuchos. Entretanto, na rotina de laborat?rios de gen?tica forense, testes de DNA n?o s?o requeridos para estes ind?cios com tanta frequ?ncia quanto s?o encontrados. A justificativa para a subutiliza??o destes materiais ? a baixa concentra??o e a alta taxa de degrada??o do DNA, que ocorre devido ao superaquecimento do cartucho que pode chegar a 1800oC, al?m disso, a inibi??o da PCR, os poucos relatos de sucesso na obten??o de perfil gen?tico a partir de estojos deflagrados e os ex?guos estudos que verificam a viabilidade de amostras como estas, s?o fatores que desestimulam esta pr?tica. Com o intuito de corroborar com a rotina de investiga??o gen?tica, estabeleceu-se uma parceria entre os Setores de Gen?tica Forense e de Bal?stica Forense do Instituto-Geral de Per?cias do Rio Grande do Sul (IGP/RS) e o Laborat?rio de Gen?tica Humana e Molecular da Faculdade de Bioci?ncias da Pontif?cia Universidade Cat?lica do Rio Grande do Sul (LGHMPUCRS). O presente estudo desenvolveu uma pesquisa controlada para obten??o e an?lise de DNA nuclear oriundos de estojos de cartuchos deflagrados. O estudo de foi realizado adaptando-se protocolos padr?o e/ou indicados pelo Departamento de Ci?ncias Forenses da Virg?nia. Este estudo demonstrou que ? poss?vel utilizar estojos deflagrados ou n?o, como fonte de DNA com a finalidade de identificar os envolvidos com o seu manuseio. Contudo, considerando o rendimento limitado, a efic?cia restrita e, sobretudo, o custo-benef?cio, entende-se que a estrat?gia de an?lise de DNA oriundo de c?lulas deixadas em cartuchos/estojos n?o seja a priorit?ria na rotina do Laborat?rio Forense. Mas, em muitas situa??es, essa pode ser a ?nica op??o dos investigadores e, nesse momento, nossos resultados e protocolos aqui apresentados ter?o import?ncia fundamental. Com este estudo conclu?mos que seguindo os protocolos aqui apresentados ? poss?vel produzir dados para fins de identifica??o humana. O uso destes protocolos, e de seus resultados, poder? ser definitivo quando usados como elemento adicional de um inqu?rito policial e/ou como prova em um processo penal

Made available in DSpace on 2015-04-14T14:51:22Z (GMT). No. of bitstreams: 1 445049.pdf: 985835 bytes, checksum: 6999f9525f1cf085ddde3a6a0fb67e0b (MD5) Previous issue date: 2012-10-31 / The presence of Bacillus spp. in milk is an important problem for the dairy industry due to their capability of sporulation and the possibility of spore resistance to heat treatment by ultra high temperature (UHT). Bacillus sporothermodurans survive to the UHT system, germinating and growing in stored milk and, if not correctly identified and quantified, can exceed the criterion established for mesophilic aerobic, besides altering the quality of dairy products when in high concentrations. On the other hand, contamination of milk by Bacillus cereus is not only an important cause of deterioration, but is also associated with the occurrence of diarrhea and emetic syndromes. Traditionally, these microorganisms are identified and quantified in food using conventional microbiological techniques, but the Polymerase Chain Reaction (PCR) based methods have been widely used for the same purpose. However, PCR cannot distinguish between viable and dead cells, which can be overcame with the use of DNA intercalating, such as propidium monoazide (PMA). PMA binds to DNA derived from cells with damaged membranes, preventing their amplification by PCR, allowing, thus, the selective detection of viable cells. Therefore, this thesis aimed to characterize the thermal resistance of B. sporothermodurans and to develop methods of detection and quantificatification of viable cells of B. sporothermodurans and B. cereus in milk samples by qPCR associated with PMA. Isothermal and non-isothermal treatments allowed the determination of the profile of heat resistance of B. sporothermodurans spores to heat UHT process, predicting that to 121?C was found a D value between 2 a 4 min. The selective detection and quantification of B. sporothermodurans and B. cereus by PMA-qPCR were developed targeting 16S rRNA gene and hemolysin gene, respectively.The treatment with PMA from pure culture and artificially contaminated UHT milk were standardized by end-point PCR for the detection of viable cells of these microorganisms. The inhibition of amplification of DNA from dead cells was obtained at a concentration of 30μg/mL PMA. The standardization of qPCR assays were performed using hydrolysis probes (TaqMan? system) specific to each target gene. The quantification limit from UHT milk artificially contaminated was 2.5 x 102 CFU/mL for B. sporothermodurans and 7.5 x 102 CFU/mL for B. cereus. The assays were applied to 135 samples of UHT milk of different commercial brands, comparing with the conventional method of cultivation for each microorganism. B. sporothermodurans and B. cereus were respectively detected in 14 (10.4%) and 44 (32.6%) of the samples by molecular methods developed, and in 11 (8.1%) and 15 (11.1%) by conventional culturing methods. The PMA-qPCR methods developed in this study were specific and sensitive for the detection and quantification of viable B. sporothermodurans and B. cereus cells, being applicable for the evaluation of milk samples, reducing the time for the analysis of this product. Furthermore, the results showed that B. cereus can be found in UHT milk / A presen?a de Bacillus spp. em leite representa um importante problema para a ind?stria de latic?nios devido ? sua capacidade de esporula??o e ? possibilidade de resist?ncia do esporo ao tratamento t?rmico por ultra alta temperatura (UAT). O Bacillus sporothermodurans sobrevive ao sistema UHT, germinando e se multiplicando no leite estocado e, caso n?o seja corretamente quantificado e identificado, pode ultrapassar o limite estabelecido pela legisla??o para microrganismos mes?filos aer?bios, al?m de alterar a qualidade dos produtos l?cteos quando em altas concentra??es. Por outro lado, a contamina??o de leite por Bacillus cereus constitui n?o somente uma importante causa de deteriora??o, mas tamb?m est? associada com a ocorr?ncia das s?ndromes em?tica e diarreica. Tradicionalmente, estes microrganismos s?o identificados e quantificados em alimentos atrav?s de t?cnicas cl?ssicas de cultivo, mas m?todos baseados na Rea??o em Cadeia pela Polimerase (PCR) tamb?m t?m sido amplamente utilizados. Entretanto, a PCR n?o distingue c?lulas mortas de c?lulas vi?veis, o que pode ser contornado com o emprego de intercalantes de DNA, como o prop?dio monoazida (PMA). O PMA se liga ao DNA derivado de c?lulas com membranas rompidas, impedindo suas amplifica??es na PCR, permitindo, assim, a detec??o seletiva de c?lulas vi?veis. Portanto, a presente tese teve por objetivo caracterizar a resist?ncia t?rmica de B. sporothermodurans, bem como desenvolver m?todos de detec??o e quantifica??o de c?lulas vi?veis de B. sporothermodurans e de B. cereus em amostras de leite atrav?s de PCR associada ao PMA. Tratamentos isot?rmicos e n?o isot?rmicos permitiram a determina??o do perfil de resist?ncia t?rmica de esporos de B. sporothermodurans ao processo UHT, predizendo que a 121?C foi encontrado um valor D entre 2 a 4 min.A detec??o e quantifica??o seletivas de B. sporothermodurans e de B. cereus atrav?s de PMA-qPCR foram desenvolvidas utilizando o gene RNAr 16S e o gene da hemolisina como alvos, respectivamente. O tratamento com PMA a partir de cultura pura e leite UHT artificialmente contaminado foi padronizado atrav?s da PCR convencional para a detec??o de c?lulas vi?veis destes microrganismos. A inibi??o da amplifica??o de DNA de c?lulas mortas foi obtida na concentra??o de 30μg/mL de PMA. A padroniza??o dos ensaios de qPCR foram realizados utilizando sondas de hidr?lise (sistema TaqMan?) espec?ficas para cada gene alvo. O limite de quantifica??o a partir de leite UHT artificialmente contaminado foi de 2,2 x 102 UFC/mL para B. sporothermodurans e de 7,5 x 102 UFC/mL para B. cereus. As t?cnicas foram aplicadas a 135 amostras de leite UHT de diferentes marcas comerciais, comparando com a metodologia cl?ssica de cultivo para cada microrganismo. B. sporothermodurans e B. cereus foram, respectivamente, detectados em 14 (10,4%) e 44 (32,6%) das amostras analisadas pelos m?todos moleculares desenvolvidos, e em 11 (8,1%) e 15 (11,1%) pelos m?todos convencionais de cultivo. Os m?todos de PMA-qPCR desenvolvidos neste estudo foram espec?ficos e sens?veis para a detec??o e quantifica??o de c?lulas vi?veis de B. sporothermodurans e de B. cereus, mostrando-se aplic?veis para serem utilizados na avalia??o de amostras de leite, reduzindo o tempo de an?lise deste produto. Al?m disso, os resultados demonstraram que B. cereus pode ser encontrado em leite tratado pelo sistema de UHT

Made available in DSpace on 2015-04-14T14:51:23Z (GMT). No. of bitstreams: 1 447380.pdf: 675913 bytes, checksum: bfb87e6d734478e3c7bb6275c3132761 (MD5) Previous issue date: 2013-02-26 / Liver fibrosis is the wound healing response to repeated injury of the liver. It is characterized by disruption of the liver architecture associated with increased expression of extracellular matrix components. Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. In normal liver, HSC are quiescent and its main function is to store vitamin A. During liver injury, these cells undergo activation, become myofibroblasts and acquire fibrogenic properties. Activation of PPARγ (peroxisome proliferator-activated receptor gamma) and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. Aware that the process of hepatic fibrosis involves inflammatory mediators, various anti-inflammatory substances have been studied in an attempt to revert fibrosis. The purpose of this study was to investigate the in vitro effects of fructose-1,6-bisphospahte (FBP) on HSC phenotype. The results demonstrated that FBP induced quiescent phenotype in HSC via PPARγ activation. Significant decrease in type I collagen mRNA expression was observed in the first 24h of treatment. These events preceded the reduction of TGF-β1 (transforming growth factor-beta) and total collagen secretion. Thus, FBP promoted downregulation of HSC activation by its antifibrotic and anti-inflammatory actions. These findings demonstrate that FBP may have potential as a novel therapeutic agent for the treatment of liver fibrosis. / A fibrose hep?tica ? a resposta cicatricial do f?gado a les?es continuadas, caracterizada pelo rompimento da arquitetura hep?tica associada ao aumento da express?o dos componentes da matriz extracelular. As c?lulas estreladas hep?ticas (HSC) desempenham um papel fundamental no processo de fibrog?nese. No f?gado normal, as HSC encontram-se em sua forma quiescente de dep?sito de vitamina A. Durante a les?o hep?tica, essas c?lulas passam por uma ativa??o fenot?pica, tornam-se miofibroblastos e adquirem propriedades fibrog?nicas. O processo de fibrose hep?tica envolve v?rios mediadores inflamat?rios e, portanto, subst?ncias anti-inflamat?rias tem sido empregadas na tentativa de reverter a fibrose e bloquear a ativa??o e diferencia??o das HSC. A ativa??o de PPARγ (receptor ativado por proliferador de peroxissomo Gama) e a inibi??o de mol?culas fibrog?nicas s?o poss?veis estrat?gias para estes fins. O objetivo deste estudo foi investigar os efeitos in vitro da frutose-1,6-bisfosfato (FBP) sobre o fen?tipo das HSC. Os resultados demonstraram que a FBP ? capaz de induzir o fen?tipo quiescente das HSC via ativa??o de PPARγ. Foi observado nas primeiras 24h de tratamento uma diminui??o significativa da express?o de mRNA de col?geno tipo I. Posteriormente, houve uma redu??o do col?geno total e de TGF-β1 (fator de transforma??o do crescimento beta). Assim, a FBP diminui o estado de ativa??o das HSC por suas a??es antifibr?ticas e anti-inflamat?rias. Estas descobertas demonstram que a FBP pode ser um potencial novo agente terap?utico para o tratamento de fibrose hep?tica.

Made available in DSpace on 2015-04-14T14:51:23Z (GMT). No. of bitstreams: 1 447376.pdf: 1656350 bytes, checksum: c0a2b1db197a97d3100dfa1ffa101f1f (MD5) Previous issue date: 2012-12-26 / Benzodiazepines, such as diazepam and midazolam, are a widely used class of drugs for anxiety treatment, with anxiolytic, hypnotic, and anticonvulsant properties. The use of zebrafish (Danio rerio) as a model for evaluating pharmacological mechanisms has gained importance due to their rapid development and high sensitivity to drugs. Studies have shown that behavioral parameters were altered in zebrafish after benzodiazepine treatment. Many neurotransmitter systems have been identified in this species, including purinergic and cholinergic system. 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, especially nucleoside triphosphate diphosphohydrolase (NTPDases) and ecto-5'-nucleotidase, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by action of adenosine deaminase (ADA). ATP is coreleased with other neurotransmitters, including acetylcholine, and has been demonstrated that adenosine can control the release of acetylcholine. 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. Since there are few reports relating these enzyme activities and the action mechanism of benzodiazepines, the aim of this study was evaluated the in vitro and ex vivo effects of classical benzodiazepines, such as diazepam and midazolam, on NTPDase, ecto-5'nucleotidase, ADA, and AChE activities in zebrafish brain and gene expression pattern in treatments that induced changes in enzyme activity in the ex vivo experiments. In order to elucidate whether diazepam or midazolam has direct effects on these enzymes, we performed in vitro experiments. Diazepam, at 500 μM, promoted a decrease on ATP hydrolysis (66%), whereas this drug, at 10-500 μM, reduced ADP hydrolysis (40-54%, respectively). Midazolam also decreased ATP (16-71% for 10-500 μM, respectively) and ADP hydrolysis (48-73% for 250-500 μM, respectively), and ecto-ADA activity (26-27.5% for 10-500 μM, respectively). Diazepam and midazolam did not induce significant changes on ecto-5?-nucleotidase activity at the concentrations tested. Concerning to AChE activity, 500 μM diazepam promoted a decrease on ACh hydrolysis (19%), whereas midazolam, at 50-500 μM, reduced AChE activity (18-79%, respectively). For ex vivo experiments, diazepam or midazolam exposures did not alter NTPDase activities in zebrafish brain membranes. AMP hydrolysis was decreased in animals treated with of 0.5 and 1mg/L midazolam (31.5% and 36.1%, respectively) when compared to the control group. However, diazepam was unable to alter ecto-5 -nucleotidase. Both drugs significantly decreased the ecto-ADA activity, whereas diazepam and midazolam reduced the adenosine hydrolysis at a concentration of 1.25 mg/L (30.85%) and 1 mg/L (32.8%), respectively. Diazepam did not alter cytosolic-ADA activity; however, the exposure to 0.1 mg/L midazolam induced a significant increase in cytosolic-ADA (39.9%) when compared with the control group. The gene expression pattern demonstrated that the CD73 transcript levels were increased (41.7%) after treatment with 0.5 mg/L midazolam. Moreover, the changes caused by diazepam and midazolam in the ADA activity are not related to the transcriptional control. Concerning the cholinerg signaling, diazepam decreased ACh hydrolysis at 1.25 mg/L (30.7%) when compared to the control group. Similarly, the exposure to 0.5 mg/L midazolam also changed the enzymatic activity of AChE promoting an increase in the ACh hydrolysis (36.7%). It is possible to suggest that these drugs can induce a direct effect on the enzyme activities, since we observed a decreased on nucleotide and nucleoside hydrolysis after in vitro exposure. In addition, the alteration on AMP hydrolysis, ADA and AChE activities suggest a modulation of extracellular adenosine and ACh levels induced by benzodiazepine exposure. / F?rmacos benzodiazep?nicos, como diazepam e midazolam, s?o muito usados na pr?tica cl?nica para o tratamento da ansiedade, possuindo propriedades ansiol?ticas, hipn?ticas e anticonvulsivantes. O uso do zebrafish (Danio rerio) como modelo para avaliar mecanismos farmacol?gicos tem ganhado grande import?ncia devido ao r?pido desenvolvimento e alta sensibilidade a drogas que essa esp?cie possui. Estudos t?m demonstrado que par?metros comportamentais mostraram-se alterados em zebrafish ap?s tratamento com benzodiazep?nicos. Muitos sistemas de neurotransmiss?o foram identificados nessa esp?cie, incluindo os sistemas purin?rgico e colin?rgico. 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, especialmente as nucleos?deo trifosfato difosfoidrolases (NTPDases) e a ecto-5 -nucleotidase, que catalisam a hidr?lise do ATP a adenosina. A adenosina pode ser desaminada a inosina pela a??o da adenosina desaminase (ADA). O ATP ? coliberado com outros neurotransmissores, entre eles a acetilcolina, e tem sido demonstrado que a adenosina pode controlar a libera??o de acetilcolina. 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. Uma vez que existem poucos relatos relacionando esses sistemas enzim?ticos e a a??o de f?rmacos benzodiazep?nicos, o objetivo deste estudo foi avaliar o efeito in vitro e ex vivo do tratamento com f?rmacos benzodiazep?nicos, tais como diazepam e midazolam, sobre a atividade das NTPDases, ecto-5'-nucleotidase, ADA and AChE no enc?falo de zebrafish e o padr?o de express?o g?nica nos tratamentos que induziram altera??es na atividade enzim?tica nos experimentos ex vivo. A fim de elucidar se o diazepam e o midazolam t?m efeitos diretos nessas enzimas, experimentos in vitro foram realizados. Na concentra??o de 500 μM, o diazepam diminuiu a hidr?lise de ATP (66%) e, nas concentra??es de 10-500 μM, este f?rmaco reduziu a hidr?lise de ADP (40-54%, respectivamente). O midazolam tamb?m diminuiu a hidr?lise do ATP (16-71% para 10-500 μM, respectivamente), ADP (48-73% para 250-500 μM, respectivamente) e a atividade da ecto-ADA (26-27,5% para 10-500 μM, respectivamente). Diazepam e midazolam n?o induziram altera??es significativas sobre a atividade da ecto-5?-nucleotidase nas concentra??es testadas. Com rela??o ? atividade da AChE, o diazepam, 500 μM, promoveu uma diminui??o na hidr?lise de ACh (19%) e o midazolam, nas concentra??es de 50-500 μM, reduziu a atividade da AChE (18-79%, respectivamente). Nos experimentos ex vivo, as exposi??es ao diazepam e midazolam n?o alteraram a atividade enzim?tica das NTPDases em membranas cerebrais de zebrafish. A hidr?lise do AMP diminuiu em animais tratados com 0.5 mg/L e 1 mg/L de midazolam (31.5% e 36.1%, respectivamente) quando comparados com o grupo controle. Entretanto, o diazepam foi incapaz de alterar a atividade da ecto-5 -nucleotidase. Ambos os f?rmacos diminu?ram significativamente a atividade da ecto-ADA, sendo que o diazepam e o midazolam reduziram a hidr?lise da adenosina na concentra??o de 1.25 mg/L (30.85%) e 1 mg/L (32.8%), respectivamente. O diazepam n?o alterou a atividade da ADA citos?lica, no entanto a exposi??o a 0.1 mg/L de midazolam induziu um significativo aumento na atividade dessa enzima (39.9%) quando comparado ao grupo controle. O padr?o de express?o g?nica demonstrou que os n?veis de transcritos do CD73 apresentaram-se reduzidos (41,7%) ap?s o tratamento com 0.5 mg/L de midazolam. Com rela??o a sinaliza??o colin?rgica, diazepam diminuiu a hidr?lise da ACh na concentra??o de 1.25 mg/L (30.7%) quando comparado ao grupo controle. Similarmente, a exposi??o ? concentra??o de 0.5 mg/L de midazolam tamb?m alterou a atividade enzim?tica da AChE, promovendo um aumento na hidr?lise da ACh (36.7%). ? poss?vel sugerir que essas drogas podem induzir um efeito direto na atividade enzim?tica, uma vez que foi observada uma diminui??o na hidr?lise de nucleot?deos e nucleos?deos ap?s a exposi??o in vitro. Al?m disso, as altera??es na hidr?lise do AMP e atividade da ADA e da AChE sugerem uma modula??o dos n?veis extracelulares de adenosina e acetilcolina induzidos pela exposi??o aos f?rmacos benzodiazep?nicos.

Made available in DSpace on 2015-04-14T14:51:23Z (GMT). No. of bitstreams: 1 447525.pdf: 8523375 bytes, checksum: f1de0a2db10e3435b1a38c1f14c98be9 (MD5) Previous issue date: 2012-11-28 / Periodontitis has a bacterial etiology associated with the presence of a susceptible host. Immunogenetics factors have been studied in an attempt to explain the more aggressive disease, to establish diagnosis and to determine a more reliable prognosis. The present study had as objectives to evaluate the HLA-G polymorphisms (14 bp insertion/deletion and C/G +3142) and the cytokines profile (Th1 and Th2) in patients with chronic periodontitis, aggressive periodontitis and healthy controls. In relation to the 14 bp polymorphism, in chronic periodontitis patients, it was observed a significant increase in homozygous frequency for the deletion allele, when compared to controls. This same group presented a higher frequency of this allele, which was marginally not significant. Furthermore, no significant difference was observed between aggressive periodontitis patients and controls in relationship to the polymorphisms of 14 bp and C/G +3142.When haplotypes were estimated, an increased frequency of the deletion/G and decreased of the insertion/G was observed in chronic periodontitis patients compared to controls, but with no statistical difference. When evaluating serum cytokines concentration (IL-2, IL-4, IL-5, IL-10, TNF-α and IFN-γ), although no statistical difference could be seen between groups, a tendency to lower levels of IL-5 and IL-10 in aggressive periodontitis group was observed. Our results suggest that having HLA-G homozygosis for the deletion allele, yields three more times chance to present chronic periodontitis (OR = 3.07, 95% CI: 1.24-7.87), inferring a susceptibility role of this polymorphism in the pathogenesis of this condition. Yet considering the cytokine profiles, the aggressive periodontitis patients presented a tendency towards the Th2 profile, suggesting a contribution to the development of this exacerbated manifestation of the disease / A periodontite apresenta etiologia bacteriana associada ? presen?a de um hospedeiro suscet?vel. Fatores imunogen?ticos t?m sido estudados para tentar explicar as formas mais agressivas da doen?a, estabelecer um diagn?stico precoce e definir um progn?stico mais confi?vel. O presente estudo teve como objetivos avaliar os polimorfismos do gene HLA-G (inser??o e dele??o de 14 pb e C/G +3142) e o perfil de citocinas (Th1 e Th2) em pacientes com periodontites cr?nica, periodontite agressiva e controles saud?veis. Em rela??o ao polimorfismo de 14 pb foi observado, nos pacientes com periodontite cr?nica, um aumento significante na frequ?ncia de homozigotos para o alelo de dele??o, quando comparados aos controles. Este mesmo grupo apresentou a maior frequ?ncia deste alelo, o que foi marginalmente n?o significante. Al?m disso, nenhuma diferen?a significativa foi observada entre os pacientes com periodontite agressiva e os controles em rela??o aos polimorfismos de 14 pb e C/G +3142.Quando os hapl?tipos foram estimados, uma frequ?ncia aumentada do dele??o/G e diminu?da do inser??o/G foi observada nos pacientes com periodontite cr?nica comparados aos controles, mas sem diferen?a estat?stica. Com rela??o ? concentra??o s?rica de citocinas (IL-2, IL-4, IL-5, IL-10, TNF-α e IFN-γ), n?o foi verificada diferen?a significativa entre os grupos estudados, embora os achados revelaram uma tend?ncia a menores n?veis de IL-5 e IL-10 no grupo com periodontite agressiva. Nossos resultados sugerem em rela??o ao HLA-G, que os pacientes homozigotos para o alelo de dele??o, t?m 3 vezes mais chance de apresentar periodontite cr?nica (OR = 3.07, 95% CI: 1.24-7.87), inferindo um papel de suscetibilidade deste polimorfismo na patog?nese desta condi??o. J? os pacientes com periodontite agressiva, quando avaliados em rela??o ao perfil de citocinas, apresentaram uma tend?ncia direcionada ao perfil Th2, sugerindo uma contribui??o para o desenvolvimento da manifesta??o exacerbada da doen?a

Made available in DSpace on 2015-04-14T14:51:25Z (GMT). No. of bitstreams: 1 448546.pdf: 1722481 bytes, checksum: a40adfe8bfc8e436fd504a30736f22aa (MD5) Previous issue date: 2013-02-08 / Temperament can be regarded as the basis of mood, behavior and personality. It has a strong biological basis, manifested early in the development of the individual, guiding the formation of habits and is relatively stable over time. Although it is known that there are environmental influences, the relationship of temperament with biological markers and social environment is not well defined. Evidence suggests that the temperament and personality traits predict psychiatric disorders and that most of them are recurrent and chronic. Thus, this study aims to investigate the biological and social basis of behavior and in animal models of traits to assess the relationship of temperament with hyperucemia and with self-report of having been bullied in humans. To study the neurobiological basis of this trait, we selected mice with high and low exploration of a central object in an open field. Out of one hundred mice tested, the ten mice with higher (HE) and lower exploratory (LE) activity were evaluated with gene expression (Genechip Mouse Gene 1.0 ST Array Affymetrix) in the striatum and frontal cortex. The results showed 118 and 86 differentially expressed genes (DEGs) in the striatum and frontal cortex, respectively. Through analysis of DEGs biological processes were significantly more enriched in nervous system were development and function and cell-to-cell signaling, particularly in the striatum. These results suggest the involvement of translational and post-translational processes as well as striatal synaptic elements in the trait differences of exploratory behavior. Human studies were conducted with the data collected in a large web-survey on psychological and psychiatric measures (BRAINSTEP). In the study of biological basis of behavior we analyzed temperament in 7.155 males (5.1% hyperuricemic) and 25.225 women (1.8% hyperuricemic). Hyperuricemic subjects scored higher in anger and lower in inhibition and control, but hyperuricemic women also showed a higher emotional sensitivity and a lower degree of volition and coping. Subjects with hyperuricemia present more more externalizing and unstable emotional traits and affective temperaments In the study of the social bases of temperament assessed bullying during childhood and adolescence, through a question on time of exposure to bullying (none, <1 year, 1-3 years and> 3 years). Emotional traits and affective temperaments were evaluated with The Affective and Emotional Composite Temperament Scale (AFECTS). About half of the sample reported exposure to bullying and 10% reported being victimized by peers for longer than 3 years. Longer exposure to bullying was associated with lower Volition, Coping and Control. Bullying victimization was also associated with a much lower proportion of euthymic and hyperthymic types in both genders, which was compensated by an increase mainly in the proportion of depressive, cyclothymic and volatile types. Being bullied was associated with a broad and profound impact on emotional and cognitive domains in all dimensions of emotional traits, and with internalized and unstable affective temperaments. These results, taken together, show the importance of social factors and serum markers, as well as genetic markers of temperament. / O temperamento pode ser considerado como a base do humor, do comportamento e da personalidade, tem uma base biol?gica forte, manifesta-se cedo no desenvolvimento do indiv?duo, norteia a forma??o dos h?bitos sendo relativamente est?vel no decorrer do tempo. Apesar de se saber que h? influ?ncias do meio, n?o est? bem definida a rela??o entre temperamento e o meio social. Evid?ncias sugerem que o temperamento e os tra?os de personalidade predisp?em aos transtornos psiqui?tricos e que a maioria deles ? recorrente e cr?nico. Dessa forma, o presente estudo tem como objetivo investigar as bases biol?gicas e sociais do temperamento em modelos animais de temperamento e avaliar a rela??o do temperamento com hiperuricemia (eleva??o dos n?veis s?ricos de ?cido ?rico) e com o autorrelato de ter sido v?tima de bullying em humanos. Na avalia??o das bases neurobiol?gicas do temperamento foram usados modelos animais, no qual foram selecionados camundongos com alta e baixa explora??o em um teste campo aberto. Foram testados cem camundongos, e posteriormente selecionados os dez camundongos mais exploradores (HE) e os dez menos exploradores (LE), cujo mRNA de c?rtex frontal e estriado foi coletado para posteriormente ser avaliado atrav?s de chips para avalia??o da express?o g?nica (Genechip Mouse Gene 1.0 ST Array - Affymetrix). Os resultados mostraram 86 e 118 genes expressos diferencialmente (DEGS) no estriado e no c?rtex frontal, respectivamente. Atrav?s da an?lise dos DEGs os processos biol?gicos mais significativamente enriquecidos foram o do desenvolvimento do sistema nervoso e da fun??o e sinaliza??o celular, especialmente no estriado, numa compara??o entre animas HE com LE. Estes resultados sugerem o envolvimento de processos de transla??o e p?s-tradu??o, assim como os elementos sin?pticos do estriado nas diferen?as de caracter?sticas de comportamento explorat?rio. Nos estudos em humanos, os dados foram coletados em um grande levantamento via Web atrav?s do Brazilian Internet Study on Temperament and Psychopathology (BRAINSTEP). No estudo de bases biol?gicas do temperamento em humanos, analisamos o temperamento em 7.155 homens (5,1% hiperuric?micos) e 22.225 mulheres (1,8% hiperuric?micas). Indiv?duos hiperuric?micos apresentaram escores mais elevados em raiva e inferiores na inibi??o e controle, j? as mulheres hiperuric?micas tamb?m mostram uma maior sensibilidade emocional e um menor grau de vontade e de enfrentamento. Os resultados demonstraram que indiv?duos com hiperuricemia t?m mais tra?os emocionais e temperamentos afetivos externalizados e inst?veis. No estudo sobre as bases sociais do temperamento avaliamos o bullying durante a inf?ncia e adolesc?ncia, atrav?s de uma pergunta sobre tempo de exposi??o ao bullying (nenhum, <1 ano, 1 a 3 anos e > 3 anos). Tra?os emocionais e temperamentos afetivos foram avaliados com a Escala de Temperamento Afetivo e Emocional (AFECTS). Cerca de metade da amostra relatou exposi??o ao bullying e 10% relataram ter sido v?timas por mais de 3 anos. Vitimas de bullying tamb?m apresentam uma propor??o muito menor de temperamentos eut?micos e hipert?micos em ambos os sexos, o que foi compensado por um aumento, principalmente, na propor??o de tra?os depressivos, ciclot?micos e vol?til. Sofrer bullying foi associado com um impacto amplo e profundo sobre os dom?nios cognitivos e emocionais em todas as dimens?es de tra?os emocionais, e com temperamento afetivo internalizado e inst?vel. Esses resultados, em conjunto, mostram a import?ncia de poss?veis marcadores s?ricos (?cido ?rico) e fatores gen?ticos e sociais sobre os tra?os de temperamento.

Made available in DSpace on 2015-04-14T14:51:25Z (GMT). No. of bitstreams: 1 448604.pdf: 1148296 bytes, checksum: ca8ff4b1f0199484ac8c3e8b70d970b8 (MD5) Previous issue date: 2013-03-05 / According to data from the World Health Organization (WHO), stress affects more than 90% of the world's population and is considered a global epidemy. Stress has become an integral part of human life and organisms are constantly subjected to stressful stimuli that change various physiological processes, representing a risk factor for many diseases, such as cancer, cardiovascular, metabolic, infectious and gastrointestinal deseases and depression. The zebrafish (Danio rerio) has many similarities with mammalian in terms of overall organization and the functioning of their stress regulatory systems. The zebrafish hypothalamic-pituitary-interrenal (HPI), analogous to the mammalian hypothalamic-pituitary-adrenal (HPA), has been found in zebrafish and the activation of this pathway is essential for the maintenance of vital functions in response to stressful events. Despite extensive knowledge regarding stress responses in mammals, data on the relationship between chronic unpredictable stress and its effects on purinergic signaling is limited. Purinergic signaling is mediated by ATP and adenosine receptor activation through P2 and P1, respectively. The catabolism of extracellular ATP to adenosine is promoted by the ectonucleotidases, such as Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5 '-nucleotidases (E-5?NT). Adenosine is deaminated by adenosine deaminase (ADA) producing inosine. Thus, these enzymes control the levels of extracellular nucleotides and nucleosides, exerting an influence on purinergic signaling. Considering some models of stress can affect the signalling systems, the objective of this study was to verify whether the unpredictable chronic stress changes ectonucleotidases and adenosine deaminase activities in zebrafish brain. In addition, we analyzed the metabolism of ATP as well as the expression of genes related to ADA, ada1, ada2.1, ada2.2, adaL, adaasi and the effects of agonists and antagonists of adenosine receptors in an unpredictable chronic stress model in zebrafish. Our results demonstrated that there are no changes on ATP (P = 0.1499), ADP (P = 0.3615) and AMP (P = 0.8987) hydrolysis in zebrafish brain membranes submitted to unpredictable chronic stress. In contrast, ecto-ADA activity significantly decreased (26.8%; 8,164 ? 0.78 NH3 min &#8722; 1 mg&#8722; 1 of protein; p 0.05; n = 5 <) in brain membranes of animals exposed to the unpredictable chronic stress when compared to the control group (11.15 ? 2.16 NH3 min &#8722; 1 mg &#8722; 1 of protein; p 0.05; n = 5). However, the activity of cytosolic ADA was not changed after the unpredictable chronic stress. Quantitative analysis by RT-qPCR did not show significant changes after chronic unpredictable stress exposure on gene expression in the genes ada1, ada2.1, ada2.2, adaL, adaasi. ATP metabolism in brain membrane preparations of zebrafish in the control group and stressed was measured by high-performance liquid chromatography (HPLC) and showed a significant increase of adenosine in stressed animals. Considering adenosine A1 receptor agonists have an anxyolitic activity and the A2A receptor antagonists can exert neuroprotective effects, we investigated the effect of administration of an agonist of A1 receptor, N&#8310;-ciclopentiladenosine (CPA), and an antagonist of A2A receptors, (ZM241385) on behavioral parameters of animals submitted to unpredictable chronic stress to check a possible relationship between the adenosinergic system and the stress. As a result we showed that these compounds have not been able to reverse the anxiogenic effect caused by unpredictable chronic stress. Since adenosine has neuromodulatory and anxiolytic effects, changes in its levels could play a role in counteracting the stress, which could be related to a compensatory mechanism in order to restore the homeostasis. / De acordo com dados da Organiza??o Mundial de Sa?de (OMS), atualmente o estresse afeta mais de 90% da popula??o mundial, sendo considerado uma epidemia global. O estresse tornou-se parte integrante da vida humana e os organismos s?o constantemente submetidos a est?mulos estressantes que alteram v?rios processos fisiol?gicos, sendo um fator de risco para diversas patologias, como c?ncer, doen?as cardiovasculares, metab?licas, infecciosas, gastrointestinais e depress?o. O peixe-zebra (Danio rerio) tem muitas semelhan?as com mam?feros em termos de organiza??o geral e o funcionamento dos respectivos sistemas reguladores do estresse. O eixo hipot?lamo-pituit?ria-interrenal (HPI), um sistema an?logo ao eixo hipot?lamo-pituit?ria-adrenal (HPA), foi descrito em zebrafish e a ativa??o desta via ? essencial para a manuten??o das fun??es vitais como resposta a eventos estressantes. Apesar do amplo conhecimento das respostas do estresse em mam?feros, os dados sobre a rela??o entre o estresse cr?nico imprevis?vel e seus efeitos na sinaliza??o purin?rgica s?o limitados. A sinaliza??o purin?rgica ? mediada por ATP e adenosina por meio da ativa??o dos receptores P2 e P1, respectivamente. O catabolismo de ATP extracelular at? adenosina ? promovido pelas ectonucleotidases, como as Ecto-nucleos?deo trifosfato difosfoidrolases (E-NTPDases) e Ecto-5 - nucleotidases (E-5?NT). A adenosina produzida pode ser desaminada pela adenosina desaminase (ADA), produzindo inosina. Assim, estas enzimas controlam os n?veis de nucleos?deos e nucleot?deos extracelulares, exercendo uma influ?ncia na sinaliza??o purin?rgica. Considerando que alguns modelos de estresse podem afetar os sistemas de sinaliza??o, o objetivo deste estudo foi verificar se o estresse cr?nico imprevis?vel altera as atividades ectonucleotid?sicas e adenosina desaminase em c?rebro de zebrafish. Al?m disso, analisamos o metabolismo do ATP, bem como a express?o de genes relacionados ? ADA, ada1, ada2.1, ada2.2, adaL, adaasi e os efeitos de agonistas e antagonistas de receptores de adenosina em zebrafish submetidos a um modelo de estresse cr?nico imprevis?vel. Nossos resultados demonstraram que n?o h? altera??es na hidr?lise de ATP (P = 0.1499), ADP (P = 0.3615) e AMP (P = 0.8987) em membranas cerebrais de zebrafish submetidos ao estresse cr?nico imprevis?vel. Em contraste, a atividade da ecto- ADA diminuiu significativamente (26,8%; 8.164 ? 0,78 NH3 min&#8722;1 mg&#8722;1 de prote?na; p < 0,05; n = 5) em membranas de c?rebro de animais expostos ao estresse cr?nico imprevis?vel quando comparado ao grupo controle (11.15 ? 2,16 NH3 min&#8722;1 mg&#8722;1 de prote?na; p < 0,05; n = 5). No entanto, a atividade da ADA citos?lica n?o foi alterada. A an?lise quantitativa por RT-qPCR n?o apresentou altera??es significativas ap?s a exposi??o do estresse cr?nico imprevis?vel na express?o g?nica nos genes ada1, ada2.1, ada2.2, adaL, adaasi. O metabolismo do ATP em prepara??es de membrana de c?rebro de zebrafish no grupo controle e estressado foi medido por cromatografia l?quida de alta efici?ncia (HPLC) e mostrou um aumento significativo de adenosina nos animais estressados. Considerando que os agonistas de receptores A1 t?m uma atividade ansiol?tica e os antagonistas dos receptores A2A podem exercer efeitos neuroprotetores, investigamos o efeito da administra??o de um agonista do receptor A1, N&#8310;-ciclopentiladenosina (CPA), e de um antagonista de receptores A2A, (ZM241385) em par?metros comportamentais de animais submetidos ao estresse cr?nico imprevis?vel, para verificar uma poss?vel rela??o entre o sistema adenosin?rgico e o estresse. Como resultado, observamos que esses compostos n?o foram capazes de reverter o efeito ansiog?nico causado pelo estresse cr?nico imprevis?vel. Uma vez que a adenosina tem efeitos neuromodulat?rios, as altera??es nos n?veis de adenosina podem desempenhar um papel nos mecanismos relacionados ao estresse, representando um mecanismo de compensa??o a fim de restaurar a homeostase.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-05-14T11:12:13Z No. of bitstreams: 1 468517 - Texto Completo.pdf: 6892081 bytes, checksum: c5aa659f71c5093d831d4810c3ab482c (MD5) / Made available in DSpace on 2015-05-14T11:12:13Z (GMT). No. of bitstreams: 1 468517 - Texto Completo.pdf: 6892081 bytes, checksum: c5aa659f71c5093d831d4810c3ab482c (MD5) Previous issue date: 2014-08-27 / The understanding of gene function in the externally visible characteristcs (EVC) expression has several uses in human population evolution studies or in forensic investigations. To this last, some effort has been done to discover an efficient and easy model for prediction of skin and eye color in humans. The obvious advantage of the prediction of such EVCs through the use of DNA is to be incorporated as routine in forensic labs and to be applied to police investigations. In our study we combined the genotyping of eight SNPs in pigment-related genes (rs4778138 - OCA2; rs12913832 - HERC2; rs16891982 - SLC45A2; rs8045560 - MC1R; rs1426654 - SLC24A5; rs2733832 - TYRP1; rs1042602 - TYR; rs916977 - HERC2) with different analytical approaches. Considering this SNP panel we evaluated allele frequencies from HAPMAP and ALFRED data obtained from subjects with High Melanin Content (HMC; from African populations), and Low Melanin Content (LMC; from European populations) and defined the alleles H (to predict HMC subjects) and alleles L (to predict LMC subjects). The cumulative distribution of alleles H and alleles L in two phenotypically different color groups of 134 South Brazilian subjects showed that 82% of HMC subjects (N = 61) had eight or more allele H and 100% of LMC subjects (N = 73) had less than eight allele H, with accuracy value of 96.3%. We performed other analyses using AUC (Area Under the Receiver Operating Characteristic Curve), PGL (Calculation of Pathway Genetic Load), and GP (Genetic Probability) approaches. The AUC was 0.99 in predicting both HMC and LMC phenotypes; PGL showed the eight SNPs panel had 93% of concordance between genotype and HMC or LMC phenotypes; and GP approach showed 91% of concordance between prediction and HMC or LMC phenotypes. Our high-throughput genotyping technology combined with different analytical approaches reached very high accuracy to predict the extreme phenotypes of human pigmentation. We believe this forensic DNA phenotyping (FDP) technique would be particularly useful in cases in which the genetic profiles of crime scenes were not found in the DNA data banks or to help classify degraded cadavers skeletons, or biological clues of dismissed people. / A compreens?o da fun??o e express?o dos genes envolvidos nos tra?os externamente vis?veis (EVC; do ingl?s externally visible characteristics) t?m sido amplamente utilizada em v?rios estudos de evolu??o humana e investiga??es forenses. Para este ?ltimo prop?sito, v?rios esfor?os t?m sido feitos para descobrir um modelo eficiente e f?cil para a predi??o da cor da pele e dos olhos em seres humanos. A vantagem ?bvia da predi??o de tais EVCs, atrav?s da utiliza??o do DNA, ? sua incorpora??o na rotina em laborat?rios forenses, sendo assim aplicada em investiga??es policiais. Em nosso estudo, relacionamos o gen?tipo de oito SNPs em genes relacionados com a pigmenta??o (rs4778138 - OCA2; rs12913832 - HERC2; rs16891982 - SLC45A2; rs8045560 - MC1R; rs1426654 - SLC24A5; rs2733832 - TYRP1; rs1042602 - TYR; rs916977 - HERC2) com diferentes abordagens anal?ticas. Este painel de SNPs considerou as frequ?ncias al?licas obtidas de dados do HapMap e Alfred a partir de indiv?duos com Alto Conte?do de Melanina (HMC; do ingl?s High Melanin Content; a partir de popula??es africanas), e Baixo Conte?do de Melanina (LMC; do ingl?s Low Melanin Content; a partir de popula??es europeias) e definiu os alelos H (para predizer os HMC) e alelos L (para predizer os LMC). A distribui??o cumulativa dos alelos H e L nos dois grupos com caracter?sticas de pigmenta??o fenotipicamente distintas, dos 134 indiv?duos da nossa popula??o, mostrou que 82% dos indiv?duos HMC (N = 61) tinham oito ou mais alelos H e 100% dos indiv?duos de LMC (N = 73) tinham menos de oito alelo H, com o valor de precis?o de 96,3%. Outras abordagens como AUC (do ingl?s; Area Under the Receiver Operating Characteristic Curve), c?lculo de PGL (do ingl?s; Pathway Genetic Load) e GP (do ingl?s; Genetic Probability) foram realizadas. A an?lise AUC foi de 0,99 tanto para a predi??o fenot?pica dos HMC quanto LMC; as an?lises PGL, para o painel com 8 SNPs, teve 93% de concord?ncia gen?tipo-fen?tipo nos HMC ou LMC; e a abordagem GP mostrou 91% de concord?ncia para predi??o dos fen?tipos HMC e LMC. Nossa tecnologia de genotipagem de alto rendimento, combinada com diferentes abordagens anal?ticas, chegou a uma precis?o muito alta para predizer os fen?tipos extremos de pigmenta??o humana. Acreditamos que esta t?cnica de fenotipagem forense pelo DNA (FDP; do ingl?s forensic DNA phenotyping), seria particularmente ?til nos casos em que os perfis gen?ticos de locais de crime n?o fossem encontrados no bancos de dados de DNA ou para ajudar a classificar cad?veres degradados, esqueletos, ou vest?gios biol?gicos de pessoas desaparecidas.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-05-15T12:04:15Z No. of bitstreams: 1 468567 - Texto Completo.pdf: 3711593 bytes, checksum: 32dee061e07f3464cbf1a35564001f4c (MD5) / Made available in DSpace on 2015-05-15T12:04:15Z (GMT). No. of bitstreams: 1 468567 - Texto Completo.pdf: 3711593 bytes, checksum: 32dee061e07f3464cbf1a35564001f4c (MD5) Previous issue date: 2014-08-22 / Eucalyptus is an economically important woody species, especially as a raw material in many industrial sectors. Brazil ranks the second worldwide position in acreage, totalizing approximately three million hectares. Eucalyptus species are very susceptible to pathogens such as Botrytis cinerea (gray mold), which leads to mortality of cuttings in rooting phase. Biological control of plant diseases using soil microorganisms has been considered an alternative to reduce the use of pesticides and pathogen attack. Plant growth promoting rhizobacteria can act directly on plant development for production of phytohormones or indirectly as antagonists to pathogens, as well as promote changes in secondary metabolism, and hence inducing of systemic resistance. In this study, the direct roleof Streptomyces isolates in plant development was evaluated through the production of auxin and rhizogenic potential in Eucalyptus grandis and E. globulus plants, as well as indirectly, by modulation of the secondary metabolism, and induction of sistemic resistence in plantselicited with Streptomyces sp. and challenged with the pathogenic fungus B. cinerea. Metabolic responses were evaluated throughactivity of plant defense enzymes (PPO and POX) and induced secondary compounds (total phenolics and quercetinic-flavonoids fraction). The incidence and progression of gray mold disease on plants elicited Streptomyces sp. PM9, and coculture of these microorganisms (Streptomyces and B. cinerea) were also evaluated. Streptomyces sp. PM5 and PM9 isolates produced more auxin than the other isolates tested. Streptomyces sp. PM9 showed the highest rhizogenic potential on Eucalyptus sp. and modulated secondary metabolism of these plants. Antagonism of this isolated over B. cinerea was evidenced. Plants elicited with Streptomyces sp. PM9 and challenged with B. cinerea showed changes in PPO and POX enzymes and levels of phenolic compounds at different time points of analysis, which may be related to initial defense response. Phenolic compounds chlorogenic acid and gallic acid were, on average, the most abundant, while caffeic acid, benzoic acidand catechin were induced at specific time points. A delay in the onset of disease was significant in plants of E. grandis elicited with Streptomyces. The induction of resistance, disease delay and antagonism against B. cinereaindicate the capacity of Streptomyces sp. PM9 as an inducer of plant systemic resistance, and poses this microorganism as a potential candidate for biological control programs in nurseries of Eucalyptus. Interaction of rhizobacteria with eucalyptus plant, as well as the modulation of defense mechanisms may contribute to the establishment of new biocontrol strategies applied to forestry. / O eucalipto ? uma esp?cie lenhosa economicamente importante, destacando-se como mat?riaprima em diversos setores industriais. O Brasil ocupa a segunda posi??o mundial em ?rea plantada, totalizando aproximadamente tr?s milh?es de hectares. As esp?cies de eucalipto s?o muito suscet?veis a pat?genos como Botrytis cinerea (mofo-cinzento), o qual leva ? mortalidade de estacas em fase de enraizamento. O controle biol?gico de doen?as em plantas utilizando microrganismos do solo tem sido considerado uma alternativa para reduzir o uso de agroqu?micos e o ataque de pat?genos. Rizobact?rias promotoras de crescimento vegetal podem agir diretamente no desenvolvimento das plantaspela produ??o de fitorm?niosou indiretamente, como antagonistas a fungos patog?nicos, al?m de causar altera??es no metabolismo secund?rio, com consequente indu??o de resist?ncia sist?mica. Neste trabalho, avaliou-se a a??o direta no desenvolvimento vegetal de isolados rizobacterianos do g?nero Streptomyces atrav?s da produ??o de auxinas e potencial rizog?nico de Eucalyptus grandis e E. globulus, bem como oefeito modulador no metabolismo secund?rioe a indu??o de resist?ncia sist?mica em plantas eliciadas com Streptomyces sp. e desafiadas com o fungo patog?nico B. cinerea. As respostas metab?licas foram avaliadas atrav?s das atividades de enzimas realacionadas ? defesa vegetal (PPO e POX) e dos compostos secund?rios induzidos (compostos fen?licos totais e fra??o flavon?ides quercet?nicos). A incid?ncia e progress?o da doen?a mofo cinzento em plantas eliciadas com Streptomyces sp. PM9, e cocultivo destes microrganismos (Streptomyces e B.cinerea) tamb?m foram avaliados. Os isolados de Streptomycessp.PM5 e PM9 apresentaram maior produ??o de auxina que os demais isolados testados. Streptomyces sp.PM9 apresentou o maior potencial rizog?nico em plantas de Eucalyptus sp. emodulou o metabolismo secund?rio destas plantas. Oantagonismo deste isolado sobre B. cinerea foi evidenciado.As plantas eliciadas com Streptomyces sp. PM9 e desafiadas com B. cinereaapresentaram altera??es nas enzimas PPO e POXe nos n?veis de compostos fen?licos totais em diferentes tempos de an?lise, as quais foram relacionadas ? resposta inicial de defesa.Os compostos fen?licos ?cido g?lico e clorog?nico foram, em m?dia, os mais abundantes, embora os ?cidos cafeico e benzoico e a catequina tenham sido induzidos em momentos espec?ficos. O retardo no estabelecimento da doen?a foi significativo em plantas de E. grandis eliciadas com Streptomyces. Os resultados de indu??o de resist?ncia, retardo da doen?a eantagonismocontra B.cinerea, demonstram a capacidade de a??o de Streptomycessp. PM9 como indutor de resist?ncia sist?mica vegetal, colocando este microrganismo como potencial candidato aos programas de controle biol?gico em viveiros de mudas de Eucalyptus. A intera??o da planta de eucalipto com a rizobact?ria, bem como a modula??o dos mecanismos de defesa podem contribuir para o estabelecimento de novas estrat?giasde biocontrole aplicado ? silvicultura.