Age-related macular degeneration (AMD) is a prevalent cause of vision loss and irreversible blindness that affects more than 11 million Americans. AMD is a multifactorial disease with a number of genetic, demographic, and environmental risk factors. Currently the etiology of AMD is still unclear and there are no effective cure for this devastating disease, but recent studies have demonstrated that RORA is a candidate gene involved in AMD pathophysiology. RORA is a critical regulator of multiple biological processes and has been implicated in various physiological processes including circadian rhythm, lipid metabolism, photoreceptor development, autism, and inflammation. Our current study will explore in depth the role of RORA in AMD. We will look at the effects of RORA in the retina of mice. Localization studies of retinal tissues obtained from mice with a conditional knockout of RORA in epithelial cells showed little effect of RORA on structural cells of the retina. However, there was a decrease in VEGF and TGF-B proteins in RORA knockout. This is an interesting finding because VEGF and TGF-B has an important function in angiogenesis and neovascularization which are pathophysiological effects of AMD. In addition, we will try to identify gene targets of RORA that have also been linked with AMD. By identifying the targets of RORA and discovering how RORA regulates these targets, we hope to better understand the role of RORA in AMD pathophysiology. ChIP-seq and software analysis of the data was performed to identify all genomic targets of RORA linked with AMD. A number of promising genes were found in both RORA and AMD networks. The next step of this study is to perform quantitative analysis of these genes and how their expression is affected by RORA. Also, we will perform additional conditional RORA knockout models in cone cells and developing retinal cells to further understand the role of RORA in the retina and AMD pathogenesis.

abstract: Crumb rubber use in asphalt mixtures using wet process technology has been in practice for years in the United States with good performance history; however, it has some drawbacks that include the need for special blending equipment, high rubber-binder temperatures, and longer waiting time at mixing plants. Pre-treated crumb rubber technologies are emerging as a new method to produce asphalt rubber mixtures in the field. A new crumb rubber modifier known as Reacted and Activated Rubber (RAR) is one such technology. RAR (industrially known as “RARX”) acts like an Enhanced Elastomeric Asphalt Extender to improve the engineering properties of the binder and mixtures. It is intended to be used in a dry mixing process with the purpose of simplifying mixing at the asphalt plant. The objective of this research study was first to perform a Superpave mix design for determination of optimum asphalt content with 35% RAR by weight of binder; and secondly, analyse the performance of RAR modified mixtures prepared using the dry process against Crumb Rubber Modified (CRM) mixtures prepared using the wet process by conducting various laboratory tests. Performance Grade (PG) 64-22 binder was used to fabricate RAR and CRM mixtures and Performance Grade (PG) 70-10 was used to fabricate Control mixtures for this study. Laboratory tests included: Dynamic Modulus Test, Flow Number Test, Tensile Strength Ratio, Axial Cyclic Fatigue Test and C* Fracture Test. Observations from test results indicated that RAR mixes prepared through the dry process had excellent fatigue life, moisture resistance and cracking resistance compared to the other mixtures. / Dissertation/Thesis / Masters Thesis Civil, Environmental and Sustainable Engineering 2018

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.

No description available.

O benzeno é um hidrocarboneto aromático produzido pela combustão de produtos naturais. A exposição ocupacional ao benzeno é caracterizada por ambientes industriais que o empregam em seus processos produtivos. Nos laboratórios de indústria do petróleo ele é utilizado em forma pura para análise, e está presente como contaminante em derivados, como gasolina, hexano, querosene, tolueno, entre outros. No Brasil o valor recomendado pela legislação como limite de exposição ambiental ao benzeno é de 1ppm. O câncer hematológico é considerado um dos principais fatores de risco para a saúde dos trabalhadores expostos ao benzeno e a utilização de biomarcadores no monitoramento destes profissionais tem sido sugerida em diferentes países. O presente trabalho teve como objetivo avaliar diferentes biomarcadores em sangue periférico de trabalhadores homens, cronicamente expostos ao benzeno em refinarias e siderurgia (18 deles com diagnóstico de intoxicação), que estavam afastados de suas funções por períodos que variaram de cinco meses a 27 anos, com idade média de 46,8 ± 9,33 anos comparado com um grupo controle composto também de 20 homens, selecionados em Bancos de Sangue (idade média de 45,7 ± 8,00 anos), com diferentes ocupações não correlacionadas ao agente em estudo. Em ambos os grupos foram realizados hemograma completo, teste do micronúcleo em linfócitos com bloqueio da citocinese (CBMN), teste de FISH em linfócitos para a translocação t(15;17), bem como testes moleculares para avaliação de polimorfismos em genes envolvidos na metabolização do benzeno (MPO, NQO1, CYP1A1 e CYP2E1) e na eliminação de xenobióticos (GSTM1/GSTT1). Quanto ao hemograma à análise estatística realizada pelo teste t-Student revelou que os expostos ao benzeno apresentavam leucopenia com diferença altamente significante na contagem de leucócitos (p<0,001), neutrófilos (p<0,001), segmentados (p<0,001), linfócitos (p=0,013) e monócitos (p=0,010). A avaliação da série eritrocitária também revelou diferenças estatísticas entre os grupos para os índices de RDW-CV (p= 0,031) e RDW-SD (p=0,008), assim como para o VPM (p=0,001). Não foi verificada diferença entre os grupos quanto à frequência de polimorfismos nos genes CYP1A1, CYP2E1, MPO, NQO1, GSTM1, GSTT1. No teste do CBMN a contagem de células com MN e pontes núcleoplasmáticas foi três vezes maior nos expostos (1,95 ± 2,37) que nos controles (0,65 ± 0,75), diferença essa considerada estatisticamente significante (t=2,33; 38gl; p=0,024). Na análise de FISH para o rearranjo PML/RAR? os trabalhadores expostos também apresentaram frequência três vezes maior de células com pelo menos uma fusão gênica (9,79 ± 9,54) em comparação aos controles (3,95 ± 3,17), diferença essa considerada estatisticamente significante pelo teste t-Student (p=0,019). Os resultados obtidos na presente investigação parecem estar de acordo com os dados da literatura que revelam alteração nas células primordiais da medula, decorrente da exposição ocupacional ao benzeno, bem como ação genotóxica, identificada pelos testes do CBMN e FISH em linfócitos de sangue periférico. Embora o número de trabalhadores estudados seja reduzido, e a exposição ocupacional possivelmente inclua outros agentes potencialmente genotóxicos que não só o benzeno é interessante ressaltar que os resultados positivos foram observados após em média nove anos de afastamento profissional. A utilização do teste do CBMN e o estudo de outras translocações, que não só a PML/ RAR?, em linfócitos de trabalhadores expostos pode representar um biomonitoramento importante e menos invasivo no acompanhamento destes trabalhadores. A análise de um grupo maior de trabalhadores, inclusive expostos a baixas concentrações de benzeno pode ser fundamental na validação destes biomarcadores. / Benzene is an aromatic hydrocarbon produced by the burning of natural products. The occupational exposure to benzene is characterized by industrial environments that use in their production processes. In the laboratories of Petroleum industry it is used in the pure form for analysis, and it is present as a contaminant in other products like gasoline, hexane, kerosene, toluene, etc. In Brazil the threshold limit value recommended by law in environmental exposure to benzene is 1 ppm. The hematological cancer is considered one of the main risk factors for the health of workers occupationally exposed to benzene and the use of biomarkers in monitoring these professionals has been suggested in different countries. The aim of this study was to assess different biomarkers in peripheral blood lymphocytes from 20 men workers chronically exposed to benzene in Petroleum Refinery and Steel Industry (18 workers with poisoning diagnosis), who were removed from workplace for periods ranging from five months to 27 years, with average age 46.8 ± 9.33 years old compared to a control group consisting also of 20 men, selected in Blood Banks (average age 45.7 ± 8.00 years old), with different occupations unrelated to the agent under study. In both groups were performed blood cell counts, cytokinesis-block micronucleus assay (CBMN), FISH assay in peripheral blood lymphocytes for translocation t(15;17), and molecular tests for evaluation of genetic polymorphisms in genes involved in benzene metabolism (MPO, NQO1, CYP1A1 e CYP2E1) and detoxification (GSTM1/GSTT1). Despite blood cell counts the statistical analysis performed by t-Student test revealed that workers exposed to benzene had leukopenia with a highly significant difference in leukocyte count (p<0.001), neutrophils (p<0.001), segmented (p<0.001), lymphocytes (p=0.013) and monocytes (p=0.010). The evaluation of red blood cells also revealed statistically significant differences between groups for RDW-CV (0.031), RDW-SD (0.008) and MPV (0.001). There were no differences between groups regarding the frequency of genetic polymorphisms in CYP1A1, CYP2E1, MPO, NQO1, GSTM1, and GSTT1. In the CBMN test the cell counts with MN and nucleoplasmic bridges was three times higher in exposed (1.95 ± 2.37) than in controls (0.65 ± 0.75), and the difference was considered statistically significant (t=2.33;38gl;p=0.024). In the FISH analysis for the PML/RAR? rearrangement the exposed workers also showed three times higher frequency of cells with at least one signal fusion (9.79 ± 9.54) in comparison to controls (3.95 ± 3.17), and the difference was statistically significant by the t-Student test (p=0.019). The results obtained in this study seem to agree with the literature data that show alterations in the stem cells of the bone marrow, resulting from occupational exposure to benzene, as well genotoxicity, identified by CBMN and FISH assay in peripheral blood lymphocytes. Although the number of subjects evaluated is reduced, and the occupational exposure includes other potentially genotoxic agents not only benzene, it is interesting to note that positive results were observed after an average of nine years of removal professional. The use of the CBMN test and the study of other translocations, not only PML/ RAR?, in lymphocytes of workers exposed may represent an important biomonitoring and less invasive monitoring of workers. The analysis of a major number of individuals, including those exposed to low concentrations of benzene, may be essential in validation of these biomarkers.

L'hormone thyroïdienne (T3) contrôle à la fois les processus développementaux et physiologiques. Elle agit via les récepteurs de l'hormone thyroïdienne (TR), membres de la famille des récepteurs hormonaux nucléaires. Ils agissent comme des facteurs de transcription dépendants du ligand. La méthylation de l'ADN en position 5 de la cytosine est une modification épigénétique importante qui affecte la structure de la chromatine et l'expression des gènes. Des études récentes ont établi un rôle important des protéines de la famille TET (Ten-eleven translocation) dans la régulation de la dynamique de la méthylation de l'ADN. Elles convertissent la 5-méthyl-cytosine (5mC) en 5-hydroxyméthylcytosine (5hmC). D’autres études ont démontré que les protéines TET (TET1, TET2 et TET3) possèdent des fonctions de régulation transcriptionnelle dépendantes et indépendantes de leur activité catalytique. Notre étude a identifié TET3 comme une nouvelle protéine interagissant avec TR. Le domaine AF2 de TR ainsi que le domaine catalytique et le domaine CXXC de TET3 sont responsables de cette interaction. Celle-ci permet la stabilisation de TR lié à la chromatine, entraînant une potentialisation de son activité transcriptionnelle. L'effet de modulation de TET3 sur TR présenté ici est indépendant de son activité hydroxylase de TET3. Ainsi, cette étude met en évidence un nouveau mode d'action de TET3 en tant que régulateur non classique de TR, modulant sa stabilité et son accès à la chromatine plutôt que son activité de transcription intrinsèque. Des mutations du gène codant pour TRα provoquent le symptôme RTHα dont la gravité varie en fonction de la mutation. Les différentes capacités d’interaction des mutants TRα, pertinents pour la maladie de RTHα humaine, avec TET3 pourraient expliquer les différences d’effet dominant négatif. La fonction de régulation de TET3 pourrait s’appliquer plus généralement aux facteurs de transcription des récepteurs nucléaires, car différents membres de la superfamille des récepteurs nucléaires présentent la même interaction avec TET3, tels que AR (récepteur des androgènes), ERR (récepteur des œstrogènes) et RAR (récepteur de l'acide rétinoïque). L'interaction entre TET3 et RAR implique le domaine de liaison ADN de RAR. La pertinence fonctionnelle de l'interaction TET3 / RAR a été étudiée plus en détail dans les cellules souches embryonnaire (cellules ES). L’absence combinée des trois TET a entraîné la diminution de 5hmC et la dérégulation des gènes impliqués dans la différenciation des cellules ES. Parmi les gènes dérégulés, nous avons identifié un sous-ensemble de gènes cibles de l’acide rétinoïque, suggérant que les RAR (récepteurs d'acide rétinoïque) et les TET pourraient travailler ensemble pour réguler la différenciation des cellules ES. Une étude supplémentaire a révélé que les protéines TET peuvent jouer un rôle dans la facilitation du recrutement de RAR aux régions promotrices de ses gènes cibles. En outre, nos résultats montrent un rôle potentiel de l'activité hydroxylase des protéines TET dans la modulation de l'activité transcriptionnelle des RAR. En conclusion, notre travail a identifié les protéines TET comme nouveaux régulateurs des récepteurs nucléaires. Les mécanismes exacts impliqués doivent être étudiés plus avant. / Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors, thyroid hormone receptors (TRs), are members of the nuclear hormone receptor family which act as ligand-dependent transcription factors. DNA methylation at the fifth position of cytosine is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (TET) family proteins in regulating DNA methylation dynamics by converting 5-methyl-cytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Studies demonstrated that TETs proteins (including TET1, TET2 and TET3) possess catalytic activity dependent and independent transcriptional regulatory functions. Our study identified TET3 as a new TR interacting protein. The AF2 domain of TR and the catalytic domain and CXXC domain of TET3 are responsible for their interaction. This interaction allows the stabilization of chromatin bound TR, resulting in a potentiation of its transcriptional activity. The modulation effect of TET3 on TR presented here is independent of its hydroxylase activity. Thus this study evidences a new mode of action for TET3 as a non-classical regulator of TR, modulating its stability and access to chromatin rather that its intrinsic transcriptional activity. Mutations in TR cause the RTH symptom which severity varies with the particular mutation. The differential ability of different TRα mutants, relevant for the human RTHα disease, to interact with TET3 might explain their differential dominant negative activity. The regulatory function of TET3 might be more general towards the nuclear receptor transcriptional factors since different members of the superfamily present the same interaction with TET3, such as AR (androgen receptor), ERR (Estrogen-related receptor) and RAR (retinoic acid receptor). The interaction between TET3 and RAR involves the DNA binding domain of RAR. The functional relevance of TET3/RAR interaction was further studied in ES cells. Combined deficiency of all three TETs led to depletion of 5hmC and deregulation of genes involved in ES differentiation. Among the deregulated genes, a subset of RA response genes was identified, suggesting that RARs (retinoic acid receptors) and TETs might work together to regulate ES cell differentiation. Further dissection revealed that TET proteins may have a role in facilitating RAR recruitment to the promoter regions of these RAR target genes. Moreover, our results indicated a potential role of the hydroxylase activity of TET proteins in modulating RAR transcriptional activity. Altogether, our work identified TET proteins as new regulators of NR (Nuclear Receptors). The exact mechanisms involved need to be further studied.

En mamíferos, así como en plantas, mutaciones en AND helicasas/ATPasas del la família SNF2, no solo afectan a la estructura de la cromatina, sino que también afectan al patrón global de la metilación del ADN. Sugiriendo una relación funcional entre la estructura de la cromatina y la epigenética. El complejo NuRD, el cual posee una ATPasa de la familía SNF2, está relacionado con la represión de la transcripción y en el remodelamiento de la cromatina. Nuestro laboratorio demostró que la proteína leucémica PML-RAR&#945; reprime la transcripción de sus genes diana por el reclutamiento de DNMTs y el complejo PRC2. En esta tesis, demostramos una relación directa del complejo NuRD en la represión génica y en los cambios epigenéticos en la leucemia promielocítica aguda (APL). Mostramos que PML-RAR&#945; se une y recluta NuRD a sus genes diana, incluyendo el gen supresor de tumores RAR&#61538;2, facilitando que el complejo de Polycomb se reclute y metile la lisina 27 de la histona H3. Tratamiento con Acido Retinóico (RA), el qual se utiliza en pacientes, reduce la ocupación de NuRD en células leucémicas. Eliminando NuRD no solo provoca que las histonas no se deacetilen y que la cromatina no se compacte, sino que también provoca que tanto la metilación del ADN y de las histonas no se produzca, así como la represión génica del gen RAR&#61538;2, favoreciendo la diferenciación celular. Nuestros resultados caracterizan un nuevo papel del complejo NuRD en el establecimiento de los patrones epigenéticos en APL, demostrando una relación esencial entre la estructura de la cromatina y epigenética durante el desarrollo de la leucemia, pudiéndose aplicar a la terapia de esta enfermedad. / In mammals, as in plants, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigentic marks. The SNF2-like containing NuRD complex is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PMLRAR&#945; represses target genes through recruitment of DNMTs and Polycomb complex. In this thesis, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leucemia (APL). We show that PML-RAR&#945; &#61472;binds and recruits NuRD to target genes, including to the tumor-suppressor gene RAR&#61538;2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment reduced the promoter occupancy of the NuRD complex. Knock-down of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction, but also impaired DNA and histone methylation as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.

O benzeno é um hidrocarboneto aromático produzido pela combustão de produtos naturais. A exposição ocupacional ao benzeno é caracterizada por ambientes industriais que o empregam em seus processos produtivos. Nos laboratórios de indústria do petróleo ele é utilizado em forma pura para análise, e está presente como contaminante em derivados, como gasolina, hexano, querosene, tolueno, entre outros. No Brasil o valor recomendado pela legislação como limite de exposição ambiental ao benzeno é de 1ppm. O câncer hematológico é considerado um dos principais fatores de risco para a saúde dos trabalhadores expostos ao benzeno e a utilização de biomarcadores no monitoramento destes profissionais tem sido sugerida em diferentes países. O presente trabalho teve como objetivo avaliar diferentes biomarcadores em sangue periférico de trabalhadores homens, cronicamente expostos ao benzeno em refinarias e siderurgia (18 deles com diagnóstico de intoxicação), que estavam afastados de suas funções por períodos que variaram de cinco meses a 27 anos, com idade média de 46,8 ± 9,33 anos comparado com um grupo controle composto também de 20 homens, selecionados em Bancos de Sangue (idade média de 45,7 ± 8,00 anos), com diferentes ocupações não correlacionadas ao agente em estudo. Em ambos os grupos foram realizados hemograma completo, teste do micronúcleo em linfócitos com bloqueio da citocinese (CBMN), teste de FISH em linfócitos para a translocação t(15;17), bem como testes moleculares para avaliação de polimorfismos em genes envolvidos na metabolização do benzeno (MPO, NQO1, CYP1A1 e CYP2E1) e na eliminação de xenobióticos (GSTM1/GSTT1). Quanto ao hemograma à análise estatística realizada pelo teste t-Student revelou que os expostos ao benzeno apresentavam leucopenia com diferença altamente significante na contagem de leucócitos (p<0,001), neutrófilos (p<0,001), segmentados (p<0,001), linfócitos (p=0,013) e monócitos (p=0,010). A avaliação da série eritrocitária também revelou diferenças estatísticas entre os grupos para os índices de RDW-CV (p= 0,031) e RDW-SD (p=0,008), assim como para o VPM (p=0,001). Não foi verificada diferença entre os grupos quanto à frequência de polimorfismos nos genes CYP1A1, CYP2E1, MPO, NQO1, GSTM1, GSTT1. No teste do CBMN a contagem de células com MN e pontes núcleoplasmáticas foi três vezes maior nos expostos (1,95 ± 2,37) que nos controles (0,65 ± 0,75), diferença essa considerada estatisticamente significante (t=2,33; 38gl; p=0,024). Na análise de FISH para o rearranjo PML/RAR? os trabalhadores expostos também apresentaram frequência três vezes maior de células com pelo menos uma fusão gênica (9,79 ± 9,54) em comparação aos controles (3,95 ± 3,17), diferença essa considerada estatisticamente significante pelo teste t-Student (p=0,019). Os resultados obtidos na presente investigação parecem estar de acordo com os dados da literatura que revelam alteração nas células primordiais da medula, decorrente da exposição ocupacional ao benzeno, bem como ação genotóxica, identificada pelos testes do CBMN e FISH em linfócitos de sangue periférico. Embora o número de trabalhadores estudados seja reduzido, e a exposição ocupacional possivelmente inclua outros agentes potencialmente genotóxicos que não só o benzeno é interessante ressaltar que os resultados positivos foram observados após em média nove anos de afastamento profissional. A utilização do teste do CBMN e o estudo de outras translocações, que não só a PML/ RAR?, em linfócitos de trabalhadores expostos pode representar um biomonitoramento importante e menos invasivo no acompanhamento destes trabalhadores. A análise de um grupo maior de trabalhadores, inclusive expostos a baixas concentrações de benzeno pode ser fundamental na validação destes biomarcadores. / Benzene is an aromatic hydrocarbon produced by the burning of natural products. The occupational exposure to benzene is characterized by industrial environments that use in their production processes. In the laboratories of Petroleum industry it is used in the pure form for analysis, and it is present as a contaminant in other products like gasoline, hexane, kerosene, toluene, etc. In Brazil the threshold limit value recommended by law in environmental exposure to benzene is 1 ppm. The hematological cancer is considered one of the main risk factors for the health of workers occupationally exposed to benzene and the use of biomarkers in monitoring these professionals has been suggested in different countries. The aim of this study was to assess different biomarkers in peripheral blood lymphocytes from 20 men workers chronically exposed to benzene in Petroleum Refinery and Steel Industry (18 workers with poisoning diagnosis), who were removed from workplace for periods ranging from five months to 27 years, with average age 46.8 ± 9.33 years old compared to a control group consisting also of 20 men, selected in Blood Banks (average age 45.7 ± 8.00 years old), with different occupations unrelated to the agent under study. In both groups were performed blood cell counts, cytokinesis-block micronucleus assay (CBMN), FISH assay in peripheral blood lymphocytes for translocation t(15;17), and molecular tests for evaluation of genetic polymorphisms in genes involved in benzene metabolism (MPO, NQO1, CYP1A1 e CYP2E1) and detoxification (GSTM1/GSTT1). Despite blood cell counts the statistical analysis performed by t-Student test revealed that workers exposed to benzene had leukopenia with a highly significant difference in leukocyte count (p<0.001), neutrophils (p<0.001), segmented (p<0.001), lymphocytes (p=0.013) and monocytes (p=0.010). The evaluation of red blood cells also revealed statistically significant differences between groups for RDW-CV (0.031), RDW-SD (0.008) and MPV (0.001). There were no differences between groups regarding the frequency of genetic polymorphisms in CYP1A1, CYP2E1, MPO, NQO1, GSTM1, and GSTT1. In the CBMN test the cell counts with MN and nucleoplasmic bridges was three times higher in exposed (1.95 ± 2.37) than in controls (0.65 ± 0.75), and the difference was considered statistically significant (t=2.33;38gl;p=0.024). In the FISH analysis for the PML/RAR? rearrangement the exposed workers also showed three times higher frequency of cells with at least one signal fusion (9.79 ± 9.54) in comparison to controls (3.95 ± 3.17), and the difference was statistically significant by the t-Student test (p=0.019). The results obtained in this study seem to agree with the literature data that show alterations in the stem cells of the bone marrow, resulting from occupational exposure to benzene, as well genotoxicity, identified by CBMN and FISH assay in peripheral blood lymphocytes. Although the number of subjects evaluated is reduced, and the occupational exposure includes other potentially genotoxic agents not only benzene, it is interesting to note that positive results were observed after an average of nine years of removal professional. The use of the CBMN test and the study of other translocations, not only PML/ RAR?, in lymphocytes of workers exposed may represent an important biomonitoring and less invasive monitoring of workers. The analysis of a major number of individuals, including those exposed to low concentrations of benzene, may be essential in validation of these biomarkers.

Le récepteur de l'acide rétinoïque RAR est une protéine de la superfamille des récepteurs nucléaires liant le ligand acide rétinoïque (AR). En présence de son ligand, RAR induit la transcription de ses gènes cibles alors qu'en son absence la transcription est inhibée. Le mécanisme de régulation de RAR est altéré dans les lignées cellulaires humaines de carcinome mammaire dû à une baisse de capacité de synthèse de l'AR. Aussi, l'expression des microARN (miR) est perturbée dans le cancer du sein et un grand nombre de gènes ont été identifiés, après une analyse in-silico, comme des cibles prédites des miRs. Ces derniers peuvent être régulés pas des facteurs de transcription et ils sont capables d'inhiber la prolifération cellulaire et d'induire l'apoptose via la régulation de leurs cibles. Ainsi, les miRs peuvent jouer un rôle dans le mécanisme de régulation de RAR et être impliqués dans des boucles de régulation avec ce récepteur. Dans le cadre de ce travail, nous décrivons une approche développée pour prédire et caractériser des circuits de régulation au niveau transcriptionnel et post-transcriptionnel dans le cancer du sein. Nous nous sommes intéressés aux boucles de régulation de type feed-forward où RAR régule un miR et en commun ils régulent un ensemble de gènes codants pour des protéines dans les cellules tumorales mammaires MCF7 et SKBR3. Ces circuits ont été construits en combinant des données de ChIP-chip de RAR et des données de micro-puces d'ADN tout en utilisant des outils in-silico de prédiction des gènes cibles de miRs. Afin de proposer le modèle approprié de régulation, une analyse in-silico des éléments de réponse de l'AR (RARE) dans les promoteurs des miRs est réalisée. Cette étape permet de prédire si la régulation par RAR est directe ou indirecte. Les boucles ainsi prédites sont filtrées en se basant sur des données d'expression de miR existantes dans des bases de données et dans différentes lignées cellulaires, en vue d'éliminer les faux positifs. De plus, seuls les circuits pertinents sur le plan biologique et trouvés enrichis dans Gene Ontology sont retenus. Nous proposons également d'inférer l'activité des miRs afin d'orienter leur régulation par RAR. L'approche a réussi à identifier des boucles validées expérimentalement. Plusieurs circuits de régulation prédits semblent être impliqués dans divers aspects du développement de l'organisme, de la prolifération et de la différenciation cellulaire. De plus, nous avons pu valider que let-7a peut être induit par l'AR dans les MCF7. / The retinoic acid receptor (RAR) is a type of nuclear receptor that is activated by the ligand retinoic acid (RA). In the presence of ligand, RAR induces the transcription of its targets whereas in the absence of ligand the transcription is blocked. The mechanism of regulation of RAR is altered in breast cancer cell lines due to a reduced capacity to synthesize RA. Also aberrant patterns of microRNA (miR) expression have been reported in human breast cancer and a number of genes involved in breast cancer progression have been identified by in-silico analysis to be targets of miRs. The miRs could be controlled by transcription factors and via the regulation of their mRNA targets, the miRs could promote apoptosis and even inhibit cell proliferation. Hence, the miRs may play a role in the mechanism of regulation of RAR and could be involved in regulatory loops with this receptor. In this work, we describe an approach developed for the prediction and characterization of mixed transcriptional and post-transcriptional regulatory circuits in breast cancer. We concentrated in particular on feed-forward loops, in which RAR regulates a miR, and together with it, a set of joint target protein coding genes in human breast cancer cell lines MCF7 and SKBR3. These loops are constructed by combining ChIP-chip datasets of RAR with datasets of DNA microarrays and by using miR target prediction tools. In order to predict the appropriate model of regulation, in-silico analysis was performed to look for retinoic acid response element (RARE) in miR promoter. This step could identify if the regulation by RAR is direct or indirect. The regulatory loops will be then filtered, in order to reduce the number of false positive, based on databases designed to represent human miR expression profiles in different tissues or cell types. Moreover, only biologically relevant circuits enriched in Gene Ontology were retained. Also, we propose to infer miR activity in order to detect their regulation by RAR. This approach was able to find some existing experimental data. Several regulatory circuits seem to be involved in various aspects of organism development, proliferation and cell differentiation. Furthermore, we were able to validate the induction of let-7a by RA in MCF7 cells.

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