Made available in DSpace on 2018-08-01T21:35:02Z (GMT). No. of bitstreams: 1 tese_11116_Dissertação_Jéssica Aflávio dos Santos.pdf: 5393933 bytes, checksum: 008d621bda9822eefd27b026bf3f023d (MD5) Previous issue date: 2017-04-20 / Atualmente, tanto a prematuridade quanto nascimentos ocorridos no período pré-termo tardio e a termo inicial têm se destacado pela sua influência negativa na mortalidade e morbidade infantil. Vários fatores de risco têm sido associados à diminuição da idade gestacional de nascimento. Dentre os fatores de risco ambientais pode-se citar o cigarro, ingestão excessiva de álcool, etnia, peso e idade materna. Em relação aos fatores genéticos, polimorfismos genéticos envolvidos no estresse oxidativo e na metabolização de xenobióticos tem sido fortemente associados ao parto prematuro. Este é o caso do gene CYP1B1, que também desempenha um papel importante na síntese de estrogênio, um dos hormônios mais importantes na manutenção da gravidez. A ação da enzima CYP1B1 pode resultar na produção de compostos considerados reativos e carcinogênicos, devido à hidroxilação preferencial do 17β-estradiol na posição 4-hidroxi. Isto pode gerar estresse oxidativo, considerado um fator etiológico primário para o parto prematuro por poder causar um comprometimento na placenta. Os polimorfismos rs10012, rs1056827, rs1056836 foram selecionados para este trabalho estarem relacionados a variações na atividade desta enzima. Desta forma, o presente estudo tem como objetivo investigar a possível influência destes polimorfismos, assim como de dados clínicos e de hábitos de vida maternos na idade gestacional de nascimento. Para isto, foi realizado um delineamento de coorte prospectivo com gestantes do Município de Santo Antônio de Jesus, no Estado da Bahia, Brasil. As mães foram acompanhadas durante a gestação, parto e puerpério entre os anos de 2009 e 2016. As características e hábitos maternos foram obtidas por meio de questionários padronizados. Foi realizado a genotipagem a partir de amostras de sangue das gestantes com ensaios TaqMan® pela técnica de Reação em Cadeira da Polimerase (PCR) em Tempo Real. Gestantes que realizaram o parto no período a termo inicial possuíam idade materna e o Índice de Massa Corporal (IMC) pré-gestacional médios maiores em comparação com as gestantes que tiveram parto nos períodos prematuro e a termo. Os intervalos de IMC pré-gestacional, etnia, hábito tabagista e etilista não demonstraram associação significativa com a idade gestacional de nascimento. Foi detectada uma correlação significativa entre o SNP rs1056836 e a idade materna e o IMC pré-gestacional. Nenhuma das demais variáveis analisadas demonstraram associação significativa com nenhum dos outros polimorfismos. Não foi detectada uma associação direta entre nenhum dos polimorfismos analisados e a idade gestacional de nascimento. A partir dos resultados obtidos por este trabalho, não se pode descartar uma possível relação do gene CYP1B1 com a idade gestacional de nascimento. Trabalhos posteriores são necessários para analisar a possível associação destes polimorfismos com a idade gestacional de nascimento em outros grupos étnicos, além de realizar investigações mais abrangentes incluindo outros genes e polimorfismos, assim como fatores ambientais de risco para a prematuridade.

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Environ 3500 tonnes de pesticides sont étendues chaque année sur les terres agricoles du Québec. L’utilisation de plusieurs de ces substances a été interdite, car les ingrédients actifs qui les composaient avaient des effets toxiques sur les humains et/ou l’environnement. Malheureusement, certains qui sont toujours en vente ont aussi des effets secondaires non désirables. En effet, ces molécules exogènes ont le potentiel de moduler l’activation de protéines régulatrices comme le récepteur aux dioxines (AhR). AhR active, entre autres, l’expression de gènes faisant partie de la famille du cytochrome P450 (CYP1A1 et CYP1B1) qui sont impliqués dans la détoxification. Or, dans certains cas, ces enzymes mènent à la production de molécules mutagènes en augmentant la toxicité des ingrédients actifs en les métabolisant. Le projet global dans lequel s’insère ce projet de maîtrise vise à déterminer les effets génomiques des pesticides environnementaux sur des organismes vivants en milieu naturel dans les environs de la région de l’Estrie. Les espèces choisies comme modèles d’étude sont des insectivores, car les pesticides s’accumulent dans les lipides et que les insectes en sont une excellente source. Les consommateurs d’insectes sont donc d’excellents marqueurs du niveau de contamination de leur environnement par les pesticides. Les deux espèces sélectionnées sont l’hirondelle bicolore (Tachycineta bicolor) et la grande musaraigne (Blarina brevicauda). De façon plus spécifique, le projet de maîtrise se divise en deux principaux objectifs. Le premier objectif est de valider que les pesticides présents dans l’environnement sont en concentration suffisante pour modifier la régulation génétique d’animaux en milieu naturel. Cette validation se fera en comparant le taux d’expression de CYP1A1 et CYP1B1 (suite de leur activation par AhR) chez des bêtes ayant été exposées (ou non) à des pesticides. Comme le génome des deux modèles d’étude n’est pas encore séquencé, le clonage partiel des gènes à étudier (AhR et CYP1) a été entamé de même que la conception d’amorces qui permettra de quantifier le niveau d’expression de ces gènes par des réactions en chaîne par polymérase en temps réel (qPCR). À ce jour, une partie de la séquence d’AhR, de CYP1B1 et de trois gènes contrôles ont été séquencés pour l’hirondelle, ce qui a permis de concevoir des amorces pour la quantification de l’expression d’AhR et de deux contrôles. Pour la musaraigne, ce sont les gènes AhR, potentiellement CYP1A1 et trois contrôles qui ont été séquencés partiellement et ce sont les gènes AhR et les contrôles pour lesquels des amorces sont prêtes à être utilisées pour la quantification par qPCR. Le deuxième objectif est d’observer les effets génétiques des pesticides de manière globale sur des organismes vivants. Un génome de référence est donc nécessaire pour identifier les régions qui vont être régulées (directement ou indirectement) par les polluants d’origine agricole. Pour la grande musaraigne, c’est le génome de la musaraigne commune (Sorex araneus) qui sera utilisé, car ces deux espèces sont très proches phylogénétiquement. Par contre, pour l’hirondelle bicolore, le séquençage, l’assemblage et l’annotation de son génome seront essentiels parce que les espèces d’oiseaux actuellement séquencées sont trop éloignées pour permettre l’identification des régions qui seront régulées différemment en présence de pesticides. Le séquençage a été fait et l’assemblage a permis de couvrir 55% du génome de l’hirondelle bicolore. Cependant, il est très fractionné avec un N50 de 1339 et un peu plus de 600 000 contigs. Quelques étapes restent à accomplir pour optimiser l’assemblage tel que l’élimination de la contamination (estimée à 5%). L’annotation pourra être faite lorsque l’étape précédente sera finie. Compte tenu de l’ampleur du projet, ce qui a été effectué dans le cadre de la maîtrise contribuera grandement à la bonne continuation de celui-ci. Le projet global permettra de mieux comprendre l’impact des pesticides sur des organismes sauvages.

Submitted by André Chamelete null (andre_ecco@hotmail.com) on 2016-02-12T14:02:00Z No. of bitstreams: 1 Dissertação de Mestrado - FINAL.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-12T17:20:57Z (GMT) No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Made available in DSpace on 2016-02-12T17:20:57Z (GMT). No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) Previous issue date: 2016-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O BaP é um contaminante ambiental capaz de causar inflamação e desregulação de vias celulares. Pela ação da CYP1a1 e CYP1b1, é convertido a metabólitos mais reativos. A literatura mostra que o BaP aumenta a expressão de algumas citocinas próinflamatórias, como a IL-1, porém, são bem contraditórios os relatos sobre o efeito do BaP no cFOS, o qual apresenta papel importante na proliferação, na formação de tumores e, possivelmente, na inflamação. O objetivo deste estudo foi de elucidar a participação do receptor purinérgico P2X7 sobre a expressão dos genes IL-1 e cFOS, durante exposição ao BaP. Foi empregado as técnicas de qPCR para quantificação de expressão gênica, e testes de correlação e regressão entre IL-1 e cFOS. A exposição ao BaP induziu a expressão dos dois genes, além das enzimas do seu metabolismo. Quando bloqueado o receptor P2X7, além de uma menor indução das CYPs, os níveis de IL-1 e cFOS caíram abaixo dos níveis controle, sugerindo a participação do P2X7. Os testes de correlação e regressão mostraram uma relação forte direta entre IL-1 e cFOS, reforçando o papel do cFOS na inflamação. / BaP is an environmental contaminant capable to cause inflammation and impair cellular pathways. CYP1a1 and CYP1b1 convert it to more reactive metabolites. Studies show that BaP enhances some proinflammatory citokines expression, like IL-1, yet reports about BaP affecting cFOS, which plays important role in proliferation, tumor formation and inflammation, are controversial. This work aimed to elucidate whether P2X7 purinergic receptor plays a role in IL-1 and cFOS expression during BaP exposure. We applied qPCR techniques to quantify gene expression, correlation and regression assays. Our results showed that BaP raised both IL-1 and cFOS genes expression, besides CYPs ones. Morevoer, when blocking P2X7 receptor, IL-1 and cFOS expression dropped under normal levels, which suggest P2X7 participation, in addition to a smaller enzymes induction. Correlation and regression assays exhibited a strong straight relationship between IL-1 and cFOS expression, reinforcing the role of cFOS in inflammation.

No / The cytochrome P450 family of enzymes is involved in the Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, overexpression of one family member, cytochrome P450 1B1 (CYP1B1), has been associated with human epithelial tumors. As such, CYP1B1 was hypothesized to be a novel target for the development of anticancer therapies. We investigated expression of CYP1B1 protein in 61 human colorectal adenocarcinomas and compared this to that observed in 14 histologically normal human large bowel samples removed from patients undergoing surgery for large bowel tumors. Although we confirmed that CYP1B1 was expressed at high levels in human colorectal tumor epithelia, we also found that CYP1B1 was not absent from normal colonic epithelia but was expressed at low levels. The expression of CYP1B1 in colon tumors does not correlate with tumor stage or degree of lymph node invasion in this study. Furthermore, in addition to expression in colon epithelia, CYP1B1 is also observed in blood vessels within the colon. As with the epithelia, levels of CYP1B1 were higher in tumor vasculature than that of the normal colon. Although these observations greatly support the development of CYP1B1 targeted anticancer therapies, they also indicate the caution that should be observed when developing such drugs.

Orientador: Mônica Barbosa de Melo / Dissertação (mestrado - Universidade Estadual de Campinas, Intituo de Biologia / Made available in DSpace on 2018-08-20T18:09:19Z (GMT). No. of bitstreams: 1 Braghini_CarolinaAyumi_M.pdf: 2316733 bytes, checksum: 1031a8585bbf2541b2b39db621d9f45d (MD5) Previous issue date: 2012 / Resumo: O glaucoma compreende um grupo heterogêneo de neuropatias ópticas, caracterizadas pela escavação do disco óptico e perda progressiva do campo visual, representando uma das maiores causas mundiais de perda irreversível da visão. Em 1997, o gene Myocilin (MYOC) foi descoberto, e mutações neste gene foram envolvidas no desenvolvimento do glaucoma primário de ângulo aberto (GPAA) e do GPAA do tipo juvenil (GPAA-J). No Brasil, 35,7% e 3,85% dos pacientes com GPAA-J e GPAA, respectivamente, são portadores de mutações no gene MYOC. O gene citocromo P450, família 1, subfamília B, polipeptídeo 1 (CYP1B1), primeiramente associado ao glaucoma congênito primário, tem sido apontado como modulador do fenótipo do GPAA na presença de alterações no gene MYOC. Recentemente, observaram-se mutações no gene CYP1B1 associadas ao GPAA e GPAA-J, independentemente da presença de alterações estruturais no gene MYOC, em diferentes populações. Este projeto se propôs a avaliar mutações nos genes MYOC e CYP1B1, utilizando técnicas de PCR e sequenciamento direto, em 100 indivíduos pertencentes a famílias com GPAA ou GPAA-J e 43 pacientes não relacionados portadores de GPAA-J, bem como avaliar a possível modulação do gene CYP1B1 no fenótipo da doença. Uma nova mutação no gene MYOC, c.1187_1188insCCCAGA, foi identificada segregando com a doença em três gerações de uma família. De acordo com análises in silico, esta mutação pode alterar os contatos internos da proteína, além de comprometer um sítio de fosforilação de caseína quinase II. Nas demais três famílias foi detectada a mutação C433R, já descrita anteriormente. Como os membros das famílias apresentavam fenótipos bastante variados, foi realizada a análise da possível modulação do fenótipo da doença pelo gene CYP1B1. Contudo, as análises mostraram a não associação de variantes deste gene na modulação do fenótipo do glaucoma nestas famílias. Nos casos não relacionados de GPAA-J, foram observadas as mutações P370L, Q368X e C433R. Nenhuma mutação no gene CYP1B1 foi identificada nestes casos, mas somente polimorfismos: R48G, A119S, V243V, V432L, A443G, D449D e N453S. De acordo com este e outros estudos, é possível concluir que mutações no gene MYOC tem papel importante no desenvolvimento do GPAA e GPAA-J, sendo a mutação C433R a mais frequente na população brasileira. Além disso, o gene CYP1B1 parece ter menor contribuição no desenvolvimento destes tipos de glaucoma em nossa população / Abstract: Glaucoma comprises a group of heterogeneous optic neuropathies characterized by excavation of the optic disc and progressive loss of visual field, representing a major global cause of irreversible blindness. In 1997, the Myocilin gene (MYOC) was discovered, and mutations in this gene were involved in the development of primary open-angle glaucoma (POAG) and juvenile-onset of POAG (JOAG). In Brazil, 35.7% and 3.85% of patients with POAG and JOAG, respectively, are carriers of mutations in the MYOC gene. The gene cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), primarily associated with primary congenital glaucoma, has been related to phenotypic modulation of POAG in the presence of MYOC gene mutations. Recently, mutations in the CYP1B1 gene associated with POAG and JOAG were observed, regardless the presence of structural alterations in the MYOC gene in different populations. This project proposed to evaluate mutations in the MYOC and CYP1B1 genes using PCR and direct sequencing, in 100 individuals belonging to families with JOAG or POAG and 43 unrelated JOAG patients, and assess the possible role of the CYP1B1 gene in modulating the disease phenotype. A novel mutation in the MYOC gene, c.1187_1188insCCCAGA, was detected, segregating with the disease in three generations of one family. According to in silico analysis, this mutation can change the internal contacts of the protein, and has altered a phosphorylation site of casein kinase II. In the other three families the C433R mutation was detected, as described earlier. As family members presented with very different phenotypes, the CYP1B1 gene was evaluated as a possible modulator of the disease. However, the analysis showed no association of variants in CYP1B1 gene in modulating the glaucoma phenotype in these families. In unrelated cases of JOAG, the mutations P370L, Q368X and C433R were detected. No mutation in the CYP1B1 gene was observed in these cases, but only polymorphisms: R48G, A119S, V243V, V432L, A443G, D449D, and N453S. According to the present and other studies, we conclude that mutations in the MYOC gene play an important role in the development of POAG and JOAG, and the C433R mutation is the most frequent MYOC alteration in the Brazilian population. Furthermore, the CYP1B1 gene seems to have less contribution to the development of these types of glaucoma in our population / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Salvia genus is a widely cultivated genus and used in medicine for various purposes as having antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim was to investigate phenolic composition of Salvia absconditiflora and understand the possible effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of total phenolic content. Presence of important phenolic acids and flavonoids such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis. Cytotoxicity of Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were investigated through XTT and TBE assays both dose and time dependent manner. Cell proliferation was inhibited 50 % by different IC50 values calculated in different assays and different time intervals. This suggests that two breast cancer cell lines response in a different way to cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were investigated with qRT-PCR. CYP1A1 and CYP1B1 were upregulated in MCF-7 but down-regulated in MDA-MB-231 cells in response to Salvia absconditiflora treatment.

Salvia genus is a widely cultivated genus and used in medicine for various purposes as having antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim was to investigate phenolic composition of Salvia absconditiflora and understand the possible effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of total phenolic content. Presence of important phenolic acids and flavonoids such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis. Cytotoxicity of Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were investigated through XTT and TBE assays both dose and time dependent manner. Cell proliferation was inhibited 50 % by different IC50 values calculated in different assays and different time intervals. This suggests that two breast cancer cell lines response in a different way to cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were investigated with qRT-PCR. CYP1A1 and CYP1B1 were up-regulated in MCF-7 but down-regulated in MDA-MB-231 cells in response to Salvia absconditiflora treatment.

It is hypothesized that exposing endothelial cells to steady or non-reversing pulsatile shear stress produces a healthy, anti-atherogenic endothelium, whereas a reversing pulsatile shear stress promotes an unhealthy, pro-atherogenic endothelium. To further investigate this hypothesis, a novel parallel plate flow chamber system was used to expose human endothelial cells to a pro-atherogenic reversing shear stress waveform designed to simulate the wall shear stress at the carotid sinus, a region prone to atherosclerosis. Cells exposed to this reversing shear stress were compared to cells exposed to high levels of steady shear stress (15 dynes/cm²), low steady shear stress (1 dyne/cm², the time-average of the carotid shear stress), and static culture conditions. Functional analysis confirmed previous findings that reversing shear stress increases cell proliferation and monocyte adhesion. Microarray results indicate that although there are unique sets of genes controlled by both low average shear stress and by reversing flow, more genes were controlled by low average shear stress. We propose that low-time average shear stress, and not fluid reversal/oscillation, may be the more significant mechanical force. The reversing shear stress system was also used to investigate two shear stress-responsive genes, CYP1A1 and CYP1B1. Both were maximally up-regulated at arterial steady shear stresses of at least 15 dynes/cm² and reversing pulsatile shear stress attenuated expression of both genes. Furthermore, AhR nuclear localization and CYP1A1 protein expression correlate with the flow patterns in the mouse aortic arch. The data strongly suggest that the AhR/CYP1 pathway promotes an anti-atherogenic phenotype in the endothelium. Changes in free zinc were measured under different shear stresses. High steady shear stress dramatically increases the levels of free zinc in endothelial cells as compared to cells grown in static culture. This increase in free zinc is attenuated under reversing shear stress and low steady shear stress, which correlates with an increase in zinc-binding metallothinein proteins and zinc exporter Znt-1. Overall, the findings provide further insight into endothelial responses to mechanical forces and may be important in understanding mechanisms of atherosclerotic development and localization to regions of disturbed flow.

Depuis bon nombre d’années déjà, la question ne se pose plus ; l’utilisation des pesticides en agriculture est nécessaire puisque sans ces derniers les chances que les terres soient ravagées par les insectes, champignons ou petits rongeurs représenteraient un trop grand risque pour les agriculteurs. Cependant, cette utilisation massive de pesticides en agriculture apporte son lot de questionnements et d’inquiétudes face aux effets néfastes qu’ils pourraient avoir tant sur les humains que sur la faune et la flore. Bien que tous les pesticides utilisés au Canada soient homologués par Santé Canada, et sont donc jugés comme ne représentant pas de risques élevés ni pour l’environnement ni les humains, nul ne connait les effets d’un mélange de pesticides sur nos cellules, notre organisme. Ce projet de recherche vise donc à évaluer les effets d’un mélange de pesticides composé de cinq ingrédients actifs communément utilisés en agriculture sur les niveaux d’expression du gène CYP1A1, un gène cible du récepteur aux hydrocarbures d'aryle (AhR). Ce récepteur, bien qu’impliqué dans bon nombre d’autres réponses cellulaires, joue un rôle important dans la détoxification de l’organisme en activant la transcription de certains gènes dans la famille du cytochrome P450, dont le gène CYP1A1. Tel que mentionné précédemment, le but du présent projet de recherche est d’évaluer les effets d’un mélange composé d’au moins deux ingrédients actifs de pesticides sur l’activation de la voie de AhR. Les résultats du projet de recherche ont démontré que certaines combinaisons donnent lieu à une activation synergique de la voie AhR alors que d’autres donnent plutôt lieu à une activation additive. Dans le cas où la concentration des ingrédients actifs est élevée, on obtient plutôt un effet inhibiteur. N’est-ce pas paradoxal qu’à faibles doses, il y a un effet soit additif ou synergique alors qu’à de hautes concentrations, l’effet est plutôt inhibiteur ? Il ne faut alors pas croire que de fortes doses de pesticides sont bénéfiques puisque les effets sur les niveaux d’expression des gènes cibles de AhR ne signifient pas qu’il en sera de même pour les tous les autres gènes. Les résultats ont également démontré qu’en présence d’œstrogène, les ingrédients actifs seuls ou en combinaison ont le même effet que le 2,3,7,8-Tétrachlorodibenzo-para-dioxine (TCDD) sur l’interaction croisée entre AhR et le récepteur aux œstrogènes ; l’expression du gène CYP1A1 est réprimée alors que l’expression de CYP1B1 demeure inchangée. Maintenant qu’on comprend bien les effets que peuvent avoir une combinaison d’ingrédients actifs de pesticides sur l’activation AhR, il ne reste plus qu’à comprendre pourquoi certains mélanges donnent lieu à une activation synergique et d’autres additive. Une question bien simple, mais à laquelle il est si difficile de répondre.