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.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-01-16T11:14:58Z No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-01-26T12:32:00Z (GMT) No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) / Made available in DSpace on 2018-01-26T12:40:54Z (GMT). No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) Previous issue date: 2017-11-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Using the classical hybridization approach series of 1H-benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones were synthesized and evaluated as inhibitors of Mycobacterium tuberculosis growth. Chemical modifications and structure-activity relationship studies yielding potent antitubercular agents with minimum inhibitory concentration values in submicromolar range. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to HepG2, HaCat, and Vero cells. In addition, some 3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or morphological alteration in zebrafish (Danio rerio) models. Therefore, these data denote that this class of molecules may furnish candidates for future development of novel anti-TB drug alternatives. / Usando a abordagem cl?ssica de hibrida??o molecular, s?ries de 1H-benzo[d]imidaz?is e 3,4-diidroquinazolin-4-onas foram sintetizadas e ensaiadas como inibidores de crescimento de Mycobacterium tuberculosis. Modifica??es qu?micas e estudos de rela??o estrutura-atividade nos conduziram a potentes agentes antituberculose com valores submicromolares de concentra??o inibit?ria m?nima. Os compostos sintetizados tamb?m foram ativos contra cepas resistentes ? f?rmacos e demonstraram desprovida citotoxicidade aparente em c?lulas HepG2, HaCat e Vero. Al?m disso, algumas 3,4-diidroquinazolin-4-onas apresentaram baixo risco de toxicidade card?aca, e nenhum sinal de neurotoxicidade ou altera??o morfol?gica em modelo de peixe-zebra (Danio rerio). Sendo assim, os resultados indicam que essa classe de mol?cular pode fornecer condidatos para o desenvolvimento futuro de novos f?rmacos contra a tuberculose.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-02-20T13:36:43Z No. of bitstreams: 1 MARIA_EDUARDA_AZAMBUJA_AMARAL_DIS.pdf: 3946234 bytes, checksum: 4e7a733b9d2337c3a887b3d9bbb56a93 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-23T18:05:39Z (GMT) No. of bitstreams: 1 MARIA_EDUARDA_AZAMBUJA_AMARAL_DIS.pdf: 3946234 bytes, checksum: 4e7a733b9d2337c3a887b3d9bbb56a93 (MD5) / Made available in DSpace on 2018-02-23T18:29:05Z (GMT). No. of bitstreams: 1 MARIA_EDUARDA_AZAMBUJA_AMARAL_DIS.pdf: 3946234 bytes, checksum: 4e7a733b9d2337c3a887b3d9bbb56a93 (MD5) Previous issue date: 2018-01-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Background Breast cancer is highly prevalent among women worldwide. It is classified into three main subtypes: the estrogen receptor positive (ER+), the human epidermal growth factor receptor 2 positive (HER2+), and the triple negative breast cancer (TNBC). This latter type is the most prevalent in clinics. Each type requires a specific treatment, being TNBC an actual challenge for treating. Drug repurposing is an easy and cheaper methodology to identify new therapies for cancer treatment. This study evaluated the effects of aspirin and metformin, isolated or in combination, in breast cancer cells of different subtypes. Methods The breast cancer cell lines MCF-7, MDAMB- 231, and SK-BR-3 were treated with aspirin and/or metformin (0.01 mM - 10 mM); functional in vitro assays were performed. The interactions with the estrogen receptors (ER) were evaluated in silico. Results Metformin altered the morphology, and reduced the viability and migration of the ER+ cell line MCF-7. A synergistic effect for the combination of metformin and aspirin was observed in the TNBC cell subtype MDAMB- 231, according to the evaluation of viability and colony formation. No significant effects were observed for either drugs in the HER2+ cell subtype SK-BR-3. The effects of metformin and aspirin partly rely on cyclooxigenase-2 (COX-2) upregulation, without any alteration of lipoxin production. In silico, metformin and aspirin bound the ERa receptor with the same energy. Conclusion We provide novel evidence on the mechanisms of action of aspirin and metformin in breast cancer cells, showing favorable outcomes for these drugs in the ER+ and TNBC subtypes. / O c?ncer de mama ? uma doen?a altamente prevalente entre mulheres nos cinco continentes, sendo uma das principais causas de morte associadas ao c?ncer. Esse tumor ? classificado em tr?s subtipos principais: c?ncer de mama estrog?nio positivo (ER+), c?ncer de mama HER2 positivo (HER2+) e, c?ncer de mama triplo negativo (TNBC). A necessidade da identifica??o do subtipo tumoral ? de extrema import?ncia para escolha do tratamento. Atualmente, o subtipo triplo negativo ? o que gera maiores desafios cl?nicos, pois o tratamento ainda ? pouco espec?fico, n?o sendo eficiente em muitos casos. O reposicionamento de drogas baseia-se na determina??o de indica??es diferentes daquelas para as quais os f?rmacos foram previamente aprovados. Representa um m?todo f?cil e r?pido para a identifica??o de novos tratamentos, se comparado com o desenvolvimento tradicional de novos f?rmacos. O objetivo deste estudo foi avaliar os efeitos da aspirina e da metformina, dois medicamentos j? aprovados internacionalmente, isolados ou em combina??o, em linhagens de c?ncer de mama de tr?s diferentes subtipos. As linhagens celulares utilizadas foram MCF-7 (ER+), MDA-MB-231 (triplo negativa) e SK-BR-3 (HER2+), as quais foram submetidas ao tratamento com metformina e/ou aspirina (0.01 mM - 10 mM). Ap?s o tratamento, foram realizados ensaios funcionais in vitro. Al?m disso, an?lises in silico foram empregadas para identificar a intera??o entre o receptor de estrog?nio (ER) e os f?rmacos. Na linhagem MCF-7, a metformina induziu altera??es morfol?gicas, redu??o da viabilidade e da migra??o celular. Foi observado um efeito sinerg?stico entre a combina??o de metformina e aspirina na linhagem MDA-MB-231, em rela??o ? viabilidade celular e ? capacidade de forma??o col?nias. Entretanto, na linhagem SK-BR-3, n?o foram obtidos efeitos significativos. Acredita-se que os efeitos da metformina e da aspirina podem estar relacionados ? indu??o da express?o de COX-2, independente da produ??o de lipoxinas. Dados in silico demonstraram que a aspirina e a metformina possuem a capacidade de se ligar, com a mesma energia, ao ERa. O presente estudo traz novas evid?ncias sobre os mecanismos de a??o da aspirina e da metformina em c?lulas de c?ncer de mama, com efeitos antitumorais predominantes nos subtipos ER+ e triplo negativo.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-06T17:32:47Z No. of bitstreams: 1 VANESSA_KAPPEL_DASILVA_TES.pdf: 5900313 bytes, checksum: 98206f797d5710ed04b0cd1d6529092e (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-04-16T19:58:40Z (GMT) No. of bitstreams: 1 VANESSA_KAPPEL_DASILVA_TES.pdf: 5900313 bytes, checksum: 98206f797d5710ed04b0cd1d6529092e (MD5) / Made available in DSpace on 2018-04-16T20:04:27Z (GMT). No. of bitstreams: 1 VANESSA_KAPPEL_DASILVA_TES.pdf: 5900313 bytes, checksum: 98206f797d5710ed04b0cd1d6529092e (MD5) Previous issue date: 2018-03-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Brain iron accumulation has been observed both in normal aging and in many neurodegenerative diseases. In previous studies, we have described that brain iron overload results in persistent memory deficits, accompanied by oxidative stress. The high metabolic rate of the nervous system makes mitochondria essential for nerve cells. These organelles control iron homeostasis in its interior and the management of reactive oxygen species. When deregulation in these activities occurs, mitochondrial functioning is compromised, resulting in failures in the energy supply mainly for the synapses. Inadequate functioning of neural circuits may culminate in the activation of cell death pathways, a feature strongly associated with neurodegenerative diseases. In the present study we analyzed the effects of neonatal iron overload on complex I deletions in the mitochondrial DNA ; on methylation and hydroxymethylation of mitochondrial DNA; on mitochondrial proteins involved on iron homeostasis, on the enzymatic activity of Succinate Dehydrogenase and Creatine Kinase, enzymes involved in the cells energy supply; and on proteins involved in apoptotic pathways, such as Caspase 8, Caspase 9, Caspase 3, Cytochrome c, APAF1, and PARP in the hippocampus of adult rats. In addition, we investigated the effects of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa, in reversing iron-induced effects on all parameters analyzed. Male rats received vehicle or iron carbonyl (30 mg / kg) from the 12th to the 14th postnatal day and were treated with vehicle or CBD (10mg / kg) for 14 days in adulthood. Iron treatment induced increased deletions of mitochondrial DNA and expression of proteins involved in apoptosis, while induced reductions of methylation and hydroxymethylation, enzymatic activity and mitochondrial ferritin, an iron storage protein. CBD reversed iron-induced effects, recovering hydroxymethylation levels, mitochondrial ferritin, Succinate dehydrogenase activity, apoptotic proteins Caspase 3, Caspase 9, PARP and APAF1 at levels comparable to controls. These results suggest that iron can affect mechanisms of mitochondrial functioning and trigger cell death pathways by apoptosis. The reversal of some of these effects by CBD indicates its neuroprotective potential. / O ac?mulo de ferro no c?rebro tem sido observado tanto no envelhecimento normal quanto em muitas doen?as neurodegenerativas. Nossos estudos anteriores mostraram que a sobrecarga de ferro cerebral resulta em d?ficits de mem?ria persistentes, acompanhados por estresse oxidativo. A elevada taxa metab?lica do sistema nervoso torna as mitoc?ndrias essenciais para c?lulas nervosas. Essas organelas t?m como fun??o o controle da homeostasia do ferro em seu interior e o gerenciamento das esp?cies reativas de oxig?nio. Uma vez que ocorre desregula??o nessas atividades, o funcionamento das mitoc?ndrias fica comprometido, resultando em falhas no aporte energ?tico principalmente para as sinapses. O funcionamento inadequado de circuitos neurais pode culminar na ativa??o de vias de morte celular, uma caracter?stica bastante associada ?s doen?as neurodegenerativas. No presente trabalho analisamos os efeitos da sobrecarga de ferro neonatal sobre as dele??es no complexo I do DNA mitocondrial; sobre os mecanismos de metila??o e hidroximetila??o do DNA mitocondrial; sobre prote?nas envolvidas no metabolismo de ferro mitocondrial (Ferritina mitocondrial e Mitoferrina 2), sobre a atividade enzim?tica da Succinato desidrogenase e Creatina quinase, envolvidas no aporte energ?tico para as c?lulas; e sobre prote?nas envolvidas nas vias apopt?ticas, como a Caspase 8, Caspase 9, Caspase 3, Citocromo c, APAF1 e PARP em hipocampos de ratos adultos. Al?m disso, investigamos os efeitos do canabidiol (CBD), principal componente n?o psicotr?pico da Cannabis sativa, na revers?o dos efeitos induzidos pelo ferro sobre todos os par?metros analisados. Ratos machos receberam ve?culo ou ferro carbonila (30 mg/kg) do 12? ao 14? dia p?s-natal e na idade adulta foram tratados com ve?culo ou CBD (10 mg/kg) durante 14 dias. O tratamento com ferro induziu o aumento das dele??es do DNA mitocondrial e das prote?nas envolvidas na via intr?nseca da apoptose, enquanto induziu a redu??o de metila??o e hidroximetila??o no DNA mitocondrial, bem como da atividade enzim?tica e da ferritina mitocondrial, prote?na de armazenamento de ferro. O CBD reverteu os efeitos induzidos pelo ferro, recuperando os n?veis de hidroximetila??o, de ferritina mitocondrial, da atividade da Succinato dehidrogenase, e das prote?nas apopt?ticas Caspase 3, Caspase 9, PARP e APAF1 a n?veis compar?veis com o controle. Os resultados sugerem que o ferro pode afetar mecanismos de funcionamento mitocondrial e desencadear vias de morte celular por apoptose. A revers?o de alguns desses efeitos pelo CBD indica o seu potencial neuroprotetor.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-04T12:33:24Z No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-04-19T16:26:33Z (GMT) No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) / Made available in DSpace on 2018-04-19T16:43:13Z (GMT). No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) Previous issue date: 2018-02-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Mosquitoes are responsible for the transmission of various pathogens. Over the past three years, the world has witnessed epidemics affecting all age groups, such as dengue fever, yellow fever, chikungunya and zika virus. To control and eliminate Aedes vectors responsible for the transmission of these diseases, it is necessary to use larvicides and insecticides such as pyriproxyfen. Pyriproxyfen is a larvicide used to control mosquitoes, mainly of the genus Aedes. This study evaluated the effects of pyriproxyfen on survival, morphology, behavior, and endocrine parameters at different stages of zebrafish development. We have demonstrated that pyriproxyfen can cause changes in the survival rate, behavior and morphology of exposed larvae, adults exposed at the larval stage, and adult offspring of exposed parents. Larvae exposed to pyriproxyfen for 1-96 hpf (hours post-fertilization) showed a decrease in survival rate, heart rate 3 dpf (days post-fertilization), and body length at 5 and 8 dpf. The distance traveled and the mean velocities of the exposed larvae were reduced in comparison to the vehicle group (1% DMSO). Therefore, pyriproxyfen changes the morphology and behavior of zebrafish in early stages of development and may affect the next generations. However, when the exposed larvae were maintained until the adult stage (6 months), we did not observe differences in locomotor parameters. Parental exposure to pyriproxyfen induced decrease in locomotion of zebrafish adult offspring as well as an anxiolytic-like behavior. We also tested pyriproxyfen exposure for 96 hours in adult zebrafish and the locomotion, anxiety, memory and endocrine parameters were analyzed as well as the gene expression of the glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF). These results showed that there was no significant difference in locomotion, anxiety and endocrine parameters. In addition, there was an impairment of habituation memory in adult zebrafish exposed to pyriproxyfen. Therefore, it is important to control the use of larvicides due to their toxic effects on non-target species. Our findings demonstrated the importance of studies related to the use of larvicides, since they are potential causes of morphological and behavioral alterations in aquatic species, such as zebrafish. / Mosquitos s?o respons?veis pela transmiss?o de v?rios agentes patog?nicos. Nos ?ltimos tr?s anos, o mundo testemunhou epidemias que afetam todas as faixas et?rias, tais como dengue, febre amarela, chikungunya e zika v?rus. Para controlar e eliminar vetores do g?nero Aedes, respons?vel pela transmiss?o destas doen?as, ? necess?rio usar larvicidas e inseticidas como o piriproxifeno. O piriproxifeno ? um larvicida usado para eliminar mosquitos, principalmente do g?nero Aedes. Este estudo avaliou os efeitos do piriproxifeno sobre a sobreviv?ncia, morfologia, comportamento e par?metros end?crinos em diferentes est?gios de desenvolvimento de peixe-zebra. N?s demonstramos que o piriproxifeno pode causar altera??es na taxa de sobreviv?ncia, comportamento e morfologia de larvas expostas, adultos expostos no est?gio larval e descendentes adultos de pais expostos. As larvas expostas ao piriproxifeno durante 1-96 hpf (horas p?s-fertiliza??o) mostraram uma diminui??o na taxa de sobreviv?ncia, frequ?ncia card?aca 3 dpf e comprimento corporal nos 5 e 8 dpf (dias p?s- fertiliza??o). A dist?ncia percorrida e a velocidade m?dia das larvas expostas foram reduzidas em rela??o ao grupo ve?culo (1% de DMSO). Portanto, o piriproxifeno altera a morfologia e o comportamento do peixe-zebra em est?gios iniciais de desenvolvimento e pode afetar as pr?ximas gera??es. No entanto, quando as larvas expostas foram mantidas at? o est?gio adulto (6 meses), n?o observamos diferen?as nos par?metros locomotores. A exposi??o parental ao piriproxifeno induziu uma diminui??o na locomo??o da prole adulta, bem como um comportamento do tipo ansiol?tico. N?s tamb?m avaliamos o efeito da exposi??o ao piriproxifeno em peixe-zebra na fase adulta durante 96 horas sobre a locomo??o, ansiedade, mem?ria e par?metros end?crinos, como express?o g?nica do receptor de glicocorticoide (GR) e fator de libera??o de corticotrofina (CRF). Os resultados demonstraram que n?o houve diferen?a significativa na locomo??o, ansiedade e par?metros end?crinos. Al?m disso, houve um preju?zo da mem?ria de habitua??o do peixe-zebra adulto exposto ? piriproxifeno. Portanto, ? importante controlar o uso de larvicidas devido aos seus efeitos t?xicos em esp?cies n?o-alvo. Nossos achados demonstram a import?ncia de estudos relacionados com a utiliza??o de larvicidas, uma vez que s?o potenciais causadores de altera??es morfol?gicas e comportamentais em esp?cies aqu?ticas, como o peixe-zebra.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-17T11:40:30Z No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-04T16:48:04Z (GMT) No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) / Made available in DSpace on 2018-05-04T16:56:22Z (GMT). No. of bitstreams: 1 KELLY_GOULART_LIMA_TES.pdf: 13016256 bytes, checksum: b2a78492cbc6e285501d0eed5538a8ee (MD5) Previous issue date: 2018-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Octyl gallate (OG) is an antioxidant used in the food, cosmetic and medications industries that has shown antitumor effect on cell lineage of leukemia, melanoma and B cell lymphoma, as well as on animal model of pulmonary metastasis. Hepatocellular carcinoma (HC) is the main primary liver cancer affecting the world's population. Although surgical resection, ablation and liver transplantation are curative options, few patients are eligible for these therapies. The application of these therapeutic options is indicated only in the early stages of the disease and unfortunately most patients are diagnosed at advanced stage. In addition, there are reports in the literature of resistance to the only drug approved for systemic treatment, sorafenibe. In this study, we investigated the effect of OG on cell proliferation and lipidic metabolism in hepatocellular carcinoma HepG2 cells. Moreover, we developed a protocol for the quantitative evaluation of lipid droplets. We report, for the first time, that treatment with OG for 24 h inhibited HepG2 cell growth by decreasing mitochondrial activity and mass, which led to the reduction of ATP levels. This reduction in the energy supply triggered a decrease in Ki67 protein expression, leading to cycle arrest in S phase. In addition, the use of two treatments with OG with interval 24 hours induced loss of mitochondrial functionality and apoptosis without inducing resistance. These results showed that OG targets the mitochondria and is a candidate for new research on therapies for CH. We also report, for the first time, the effect of OG on lipidic metabolism, since our results showed that OG was able to increase the amount of lipids, triglyceride levels and the area of lipid droplets without involving the mTOR/SREBP-1c signaling pathway or modification of PPAR-? and PPAR-? gene expression. As the ability of OG to inhibit mitochondrial activity and induce apoptosis is known, it is strongly suggested that reduction of mitochondrial fatty acid b-oxidation is involved in the OG mechanism in the accumulation of lipids. We also describe, for the first time, a protocol for the quantitative evaluation of lipid droplets using confocal laser scanning microscopy that compared to conventional fluorescence microscopy, provided great gain in the quality of the images. / O galato de octila (GO) ? um antioxidante utilizado na ind?stria de alimentos, cosm?ticos e de medicamentos que tem mostrado efeito antitumoral em linhagem celular de leucemia, melanoma e linfoma de c?lulas B, assim como em modelo animal de met?stase pulmonar. O Carcinoma hepatocelular (CH) ? o principal c?ncer prim?rio hep?tico que afeta a popula??o mundial. Embora a ressec??o cir?rgica, a abla??o e o transplante hep?tico sejam considerados terapias curativas, poucos pacientes s?o eleg?veis. A aplica??o dessas op??es terap?uticas ? indicada somente para est?gios iniciais da doen?a e infelizmente a maioria dos pacientes ? diagnosticado em est?gio avan?ado. Al?m disso, existem relatos na literatura de resist?ncia ? ?nica droga aprovada para tratamento sist?mico, o sorafenibe. Nesse estudo, n?s investigamos o efeito do GO sobre a prolifera??o celular e metabolismo lip?dico nas c?lulas de carcinoma hepatocelular HepG2. Al?m disso, desenvolvemos um protocolo para avalia??o quantitativa de gotas lip?dicas. N?s reportamos, pela primeira vez, que o tratamento com GO por 24 horas inibiu a prolifera??o das c?lulas HepG2 por reduzir a atividade e massa mitocondrial, que levou ? redu??o dos n?veis de ATP. Essa redu??o no fornecimento de energia desencadeou a diminui??o na express?o da prote?na Ki67, levando ? parada do ciclo celular em fase S. Al?m disso, o uso de dois tratamentos com GO com intervalo de 24 horas induziu perda da funcionalidade mitocondrial e apoptose, sem induzir resist?ncia. Esses resultados mostraram que o GO tem como alvo a mitoc?ndria, sendo um candidato para novas pesquisas sobre terapias para o CH. Reportamos tamb?m, pela primeira vez, o efeito do GO sobre o metabolismo lip?dico, visto que nossos resultados mostraram que o composto foi capaz de aumentar a quantidade de lip?deos, os n?veis de triglicer?deos e a ?rea de gotas lip?dicas, sem envolver a via de sinaliza??o mTOR/SREBP-1c ou a modifica??o da express?o dos genes PPAR-? e PPAR-?. Uma vez que a capacidade do GO em inibir a atividade mitocondrial e induzir a apoptose ? conhecida, ? fortemente sugerido que a redu??o da ?-oxida??o mitocondrial de ?cidos graxos esteja envolvida no mecanismo do GO no ac?mulo de lip?deos. Descrevemos tamb?m, pela primeira vez, um protocolo para avalia??o quantitativa de gotas lip?dicas usando microscopia confocal de varredura a laser que comparado ? microscopia de fluoresc?ncia convencional, proporcionou grande ganho na qualidade das imagens.