Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 128-136. Publié aussi en version électronique.

Zugl.: Münster (Westfalen), Universiẗat, Diss., 2002.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-15T16:35:40Z No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) / Made available in DSpace on 2013-10-15T16:35:40Z (GMT). No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A maioria dos cães com leishmaniose visceral submetida à quimioterapia convencional apresenta recaída após a interrupção do tratamento, talvez, pelo fato desses animais desenvolverem resposta imune celular específica apenas transitoriamente. Uma vez que as citocinas IL-7 e IL-15 são descritas na literatura como sendo capazes de promover resposta imune celular de longa duração (memória), o presente trabalho teve como objetivo a clonagem, a expressão e a avaliação da atividade biológica de IL7 e IL-15 recombinantes caninas (rca-IL-7 e rcaIL-15). Diversos estudos têm demonstrado o papel essencial da IL-7 e IL-15 na homeostase de células-T. A Interleucina-7 é um fator de crescimento e anti-apoptótico de linfócitos T, sendo essencial para a sobrevida de células T maduras, naïve e células de memória, especialmente CD4+. IL-15 apresenta um amplo espectro de atividades biológicas. É crucial para o desenvolvimento, proliferação, sobrevivência e diferenciação de múltiplas células tanto da imunidade inata, quanto da imunidade adaptativa, e apresenta papel essencial na manutenção de células T CD8+ de memória. Nesse trabalho descreveu-se a clonagem do DNA complementar (cDNA) e a expressão de IL-7 e IL-15 recombinantes caninas biologicamente ativas em Escherichia coli. Para expressão em E. coli foram realizadas as clonagens do cDNA de IL-7 e de IL-15 no vetor pRSET, gerando as construções pRSET-caIL-7 e pRSET-caIL-15. O sucesso da clonagem em ambos os vetores de expressão foi confirmado a partir do sequenciamento. As proteínas foram expressas como proteína de fusão com seis moléculas de histidina na extremidade amina da cadeia polipeptídica e após serem solubilizadas com uréia, as proteínas foram purificadas por cromatografia de afinidade e renaturadas por diálise. A avaliação da atividade biológica de IL-15 canina purificada foi realizada em células CTLL-2 e foi demonstrada uma relação dose dependente na faixa de concentração proteica de 0,9 ng/mL até 1 µg/mL. O mesmo efeito foi demonstrado quando essas células foram cultivadas na presença de diferentes diluições de T-STIM (controle positivo do ensaio). Visando determinar as condições em que rca-IL-7 e rca-IL-15 são capazes de induzir efeitos biológicos in vitro, foram realizados experimentos com diferentes doses de ambas as citocinas e demonstrado pela primeira vez que tanto IL-7 quanto IL-15 são capazes de induzir a proliferação de CMNSP de cães pré-ativadas com PHA. Adicionalmente, as citocinas foram avaliadas quanto à capacidade de estimulação da proliferação de CMNSP de cães sem estímulo prévio ou concomitante e foi possível observar que rca-IL-7 foi capaz de manter a proliferação dessas células por até 12 dias de cultivo, enquanto rca-IL-15 foi capaz de fazê-lo por até 14 dias. Diante dos resultados obtidos a rca-IL-7 e rca-IL-15 poderão ser utilizadas no futuro em estudos que visam o estabelecimento de uma resposta de longa duração em cães com LVC submetidos à quimioterapia convencional. / Most dogs with LV subjected to conventional chemotherapy has relapsed after discontinuation of treatment, possibly due these animals develop specific cellular immune response transiently. Since IL-7 and IL-15 are described in the literature asbeing capable of promoting cellular immune response of long-term (memory), this study aimed at cloning, expression and biological activity evaluation of rca-IL7 and rca IL-15. Many studies have demonstrated the essential role of IL-7 and IL- 15 in the homeostasis of T-cells. The Interleukin-7 is a growth and anti-apoptotic factor of T lymphocytes, essential for the occurrence of mature, naive, and memory T cells, especially CD4+. IL-15 presents a wide spectrum of biological activities. It is crucial for the development, proliferation, survival, and differentiation of multiple cells of innate immunity as well as of adaptive immunity, and presents an essential role in the maintenance of CD8+ memory T cells. This work describes the cloning of complementary DNA (cDNA) and the expression of IL-7 and IL-15 canine recombinants biologically active in Escherichia coli. For expression in E. coli, we cloned cDNA of IL-7 and IL- 15 in the vector pRSET, generating the constructions pRSET-caIL7 and pRSETcaIL- 15. The success of the cloning in both the expression vectors was confirmed from the sequencing. The proteins were expressed as fusion protein with six molecules of histidine in the amine extremity of the polypeptide chain and afterwards were solubilized with urea, the proteins were purified by affinity chromatography and renatured by dialysis. The evaluation of the biological activity of purified canine IL-15 was achieved in CTLL-2 cells and a dose-dependent relation in the range of protein concentration of 0.9 ng/mL to 1 ƒÊg/mL was demonstrated. The same effect was demonstrated when these cells were cultivated in the presence of different dilutions of T-STIM (positive control of the test). With the goal of determining the conditions in which rca-IL-7 and rca-IL-15 are capable of inducing biological effects in vitro, we carried out experiments with different doses with both the cytokines and demonstrated for the first time that IL-7 as well as IL- 15 are capable of inducing proliferation of PBMC of dogs pre-activated with PHA. Also, the cytokines were evaluated as to the capacity of stimulation of the proliferation of PBMC of dogs without previous or concomitant stimulation and it was possible to observe that rca-IL-7 was capable of maintaining the proliferation of these cells for up to 12 days of cultivation, while rca-IL-15 was capable of doing it for 14 days. Considering the results obtained rca-rca-7 and IL-IL-15 may be used in future studies that aim to establish a long-termresponse in dogs with CVL undergoing conventional chemotherapy

A 3,4-metilenodioximetanfetamina (MDMA) é a principal substância psicoativa comercializada ilegalmente em comprimidos de ecstasy. O MDMA é uma droga de ação psicotrópica e uso proscrito, conforme lista F (grupo F2) da ANVISA, pois apresenta propriedades alucinógenas e estimulantes e seu uso/abuso pode gerar uma série de danos à saúde dos usuários. O desenvolvimento de eletrodos quimicamente modificados (EQMs) na eletroanalítica tem por finalidade a obtenção de sistemas de detecção mais sensíveis e seletivos para o analito de interesse. Também, considera-se necessário desenvolver novas técnicas e métodos para a detecção de compostos em amostras de interesse forense, a fim de obter ferramentas para auxiliar os cientistas forenses no combate ao comércio ilícito de substâncias. Conforme problemática exposta, este trabalho teve por finalidade o desenvolvimento de eletrodos quimicamente modificados utilizando como modificadores da superfície eletródica de carbono vítreo o Nafion e Nafion/CB[7], utilizando deposição por drop coating e spin coating para a detecção de MDMA através das técnicas de voltametria cíclica e onda quadrada. Conforme o sistema empregado, os melhores EQMs desenvolvidos foram de Nafion (1,5% v/v) e Nafion (1,5% v/v)/CB[7] (10,0 µg.mL-1). Os EQMs desenvolvidos apresentaram limite de detecção e quantificação na faixa de traços e menores que aqueles reportados em outros trabalhos da literatura. Considerando a aplicação dos EQMs para a detecção de MDMA em amostras de ecstasy, verificaram-se as respostas voltamétricas de outras substâncias: cafeína, metanfetamina, teobromina, lidocaína, cloridrato de procaína, (±)-metanfetamina e cloridrato de cocaína. Nas condições experimentais empregadas, observou-se que as substâncias estudadas não atuam como falsos positivos para o MDMA. Paralelamente, obtiveram-se onze lotes de comprimidos de ecstasy (apreendidos e cedidos pela Polícia técnico-científica de Ribeirão Preto-SP) e realizaram-se análises qualitativas e quantitativas nos mesmos, utilizando técnicas colorimétricas (Marquis, Ácido sulfúrico, Simon e Simon com acetona) e cromatográficas (CG-EM E CLAE-EM). Considerando o melhor EQM desenvolvido, quantificaram-se 11 lotes de ecstasy pela técnica voltamétrica e cromatográfica, dentre os lotes estudados, dois não continham MDMA, um apresentou uma mistura de MDMA e cafeína e os demais continham MDMA. A concentração de MDMA presente nos lotes variou de 0 até 61 % em massa. A detecção de MDMA em ecstasy pelo método voltamétrico desenvolvido se mostrou viável e sensível para o analito de interesse. / The 3,4-methylenedioxymethamphetamine (MDMA) is the main psychoactive component of ecstasy tablets, that have an illicit trade. MDMA has been an illicit psychotropic drug, and it has a prohibited use (group F2, in ANVISAs F list), because of its hallucinogenic and stimulating effects, and the use/abuse can poses serials health risks. The development of chemically modified electrodes (CME) in electroanalytical methods aims to get more sensitive and selective systems to detect the analytes. In this context, it is necessary to develop new techniques and methodologies to the detection of illicit samples; it provides more tools to help the forensic scientists to combat the illicit drug trade. So, this work focused in the development of chemically modified electrodes (CMEs) with modifications on the glassy carbon surface by drop coating and spin coating using Nafion and Nafion/CB[7] solutions. The CMEs were tested using cyclic, and square wave voltammetry to detect MDMA. Considering the employed system, the best CMEs were made by Nafion (1.5% v/v), and Nafion (1.5% v/v)/CB[7] (10.0 µg.mL-1) thin films. It was possible to observe better sensitivities for these sensors, in comparison to other MDMA studies reported in the literature. The specificity of the proposed sensors was checked in relation to other drugs: caffeine, methamphetamine, theobromine, lidocaine, procaine hydrochloride, and cocaine hydrochloride. These drugs do not interfere in this voltammetric method. Additionally, we studied eleven lots of ecstasy samples, allowed by the Scientific Police - Ribeirão Preto-SP, and we provide qualitative and quantitative studies using colorimetric techniques (Marquis, Sulfuric acid, Simon, and Simon with acetone), and chromatografic techniques (GC-MS and HPLC-MS). The MDMA quantification in real samples was obtained by high performance liquid chromatography with a mass spectrophotometer, and we compared with the voltammetric technique, using the developed CME. Between the analyzed lots, two of them didnt present in their composition, one lot had a mix of caffeine and MDMA, and another presented MDMA. The MDMAs concentration in lots had a large variation, with 0 to 61 % w/w. The MDMAs voltammetric detection in ecstasy lots was viable. And, it is also possible to apply this methodology to analyze MDMA traces.

Di-2-piridil cetona saliciloilhidrazona (DPKSH) é uma hidrazona que forma compostos de coordenação com diversos íons metálicos. As resinas Amberlite XAD-2 e XAD-7 são polímeros não-iônicos que podem ser usados para a pré-concentração de íons metálicos. A adsorção de DPKSH nessas matrizes poliméricas foi etudada utilizando a espectrofotometria. A quantidade de DPKSH adsorvida nessas resinas foi calculada a partir da diferença entre a concentração inicial a concentração remanescente na solução sobrenadante, após o contato com a fase sólida em diferentes intervalos de tempo. Modelos cinéticos de pseudo-primeira e pseudo-segunda ordens foram aplicados aos dados experimentais coletados no estudo cinético. Outros experimentos realizados com tempo constante, mas variando a concentração inicial de DPKSH, forneceram dados experimentais que foram aplicados a três modelos de isotermas (Langmuir, Freundlich and Dubinin-Radushkevich) e alguns parâmetros termodinâmicos foram calculados para descrever a adsorção. Para a XAD-7, um estudo cinético mais completo foi realizado incluindo concentrações iniciais de DPKSH. A resina XAD-7 modificada com DPKSH foi então utilizada para estudar a retenção de íons Cu(II) usando três sistemas diferentes: (a) espectrofotometria direta; (b) uma coluna de vidro parcialmente preenchida com a resina modificada e (c) usando a técnica de análise por injeção em fluxo (FIA) com uma mini-coluna preenchida com a fase sólida. Finalmente, Cu(II) foi determinado em amostras sintéticas e comerciais de aguardente. / Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) is a hydrazone which forms coordination compounds with several metallic ions. Amberlite XAD-2 and XAD-7 resins are non-ionic polymers which can be used to pre-concentrate metallic ions. Adsorption of DPKSH onto these polymeric matrices was investigated using the spectrophotometry. The amount of DPKSH adsorbed onto the resins was calculated as the difference between the initial concentration and the remained concentration in the supernatant solution, after the contact with the solid phase at different intervals of time. Kinetic models of pseudo-first and -second orders and intra-particle diffusion model were applied on experimental data collected from the kinetic study. Other experiments carried out under a constant time, but changing the initial DPKSH concentration, led to experimental data which were applied to three different isotherm models (Langmuir, Freundlich and Dubinin-Radushkevich) and some thermodynamic parameters were calculated and used to describe the adsorption. For XAD-7 a more complete kinetic study was carried out including different initial concentrations of DPKSH. The resin XAD-7 modified with DPKSH was then applied to study the retention of Cu(II) ions using three differents systems: (a) direct spetrophotometry; (b) a glass column filled with the modified resin and (c) using the flow injection analysis (FIA) with a mini-column partially filled with the solid phase. Finally, Cu(II) was determined in commercial and synthesized samples of sugar cane brandy.

Bisfenol A (BPA) é um insumo largamente utilizado na produção de plástico policarbonato e amplamente difundido no meio ambiente, levando o ser humano à exposição crônica desde o período intrauterino. A literatura aponta a possibilidade de BPA aumentar o risco de diversos tipos de câncer, mas são necessários estudos que possibilitem o entendimento de mecanismos pelos quais isso pode ocorrer. Neste trabalho foram investigados os efeitos do BPA ou nitro-BPA em células HL-60, MCF-7 e tecidos de ratos. Células HL-60 foram expostas ao BPA ou nitro-BPA nas concentrações de 25, 100 e 250 µM (0,1 % DMSO v/v) por 2, 24 ou 48 horas na presença ou ausência de H2O2 (40 nmol/5 x 104 células). Células MCF-7 foram expostas da mesma forma, sem o uso de H2O2, mas na presença e ausência de agonista (PCB) de receptor Ah. Ratos Sprague-Dawley machos receberam BPA diariamente ao longo de 4 semanas (50 mg/kg de peso corpóreo) por gavagem, na vigência e ausência de diabetes, com subsequente coleta de urina, fígado, rins, medula óssea e sangue. Nos experimentos com as células, a viabilidade, ciclo celular, fragmentação do DNA e a produção intracelular de espécies reativas de oxigênio (ROs) foram avaliadas por citometria de fluxo, a atividade da cadeia respiratória mitocondrial pelo ensaio do XTT, e a atividade de MPO de células HL-60 por ensaio de fluorescência, bem como a produção de •NO. A metilação e hidroximetilação global do DNA e os adutos 8-oxodG, CEdG, 1,N6-εdA, 1,N2-εdG e BPA-Gua no DNA das células, tecidos, meio de cultura e urina foram analisados por HPLC-ESI-MS/MS. O hemograma e mielograma dos animais foram obtidos no Laboratório de Hematologia Experimental da FCF USP. Observou-se que tanto BPA quanto nitro-BPA induziram a geração de ROS em células HL-60 logo após 2h de incubação. BPA levou subsequentemente à perda de atividade da cadeia respiratória mitocondrial, aumento da permeabilidade da membrana plasmática, fragmentação do DNA, parada na fase G2/M do ciclo celular e hipermetilação acompanhada de hipohidroximetilação global do DNA. A citotoxicidade induzida pelas mesmas concentrações de nitro-BPA em células HL-60 foi menos pronunciada, sem perda de atividade da cadeia respiratória mitocondrial, com pouca fragmentação do DNA, mas com parada na fase G0/G1 do ciclo celular e indução de hipohidroximetilação global do DNA na presença de H2O2. Não foi observada a indução de adutos de DNA nas células HL-60 incubadas com BPA, mas sim de CEdG nas células incubadas com nitro-BPA. Os dados obtidos a partir da exposição das células HL-60 a BPA e nitro-BPA nos indicam que as duas moléculas provocam alterações metabólicas distintas nesse tipo celular, independentes da via estrogênica, que levam a alterações predominantemente epigenéticas (BPA) ou genéticas e epigenéticas (nitro-BPA), que podem ter consequências fenotípicas, como progressão maligna, que precisam ser investigadas. Foi observado que as células MCF-7 são mais resistentes que as células HL-60 à citotoxicidade induzida por BPA e nitro-BPA. Como resultado da exposição das células MCF-7 a BPA, houve pequeno aumento da permeabilidade da membrana plasmática (250 µM), indução dos níveis de ROS após 24 h (25 µM) e aumento da população de células em sub G1, ou seja, com DNA fragmentado (100 µM e 250 µM), mas sem alteração do ciclo celular. No caso de nitro-BPA, foi observada parada do ciclo celular em G2/M (25 µM, 100 µM e 250 µM), assim como aumento de permeabilidade da membrana plasmática após 24 h de incubação (25 µM, 250 µM), sem indução de ROS ou aumento de células em sub G1. Entretanto, observou-se aumento dos níveis de CEdG e 8-oxodG no DNA das células incubadas com BPA (100 µM, 250 µM) sem a ativação prévia de receptores Ah. A ativação dos receptores Ah com PCB levou a menor aumento do nível das lesões após as incubações com BPA. A maior resistência das células MCF-7 aos efeitos citotóxicos do BPA está provavelmente relacionada à ação estrogênica desse xenobiótico. A sinalização estrogênica juntamente com o aumento dos níveis de lesões no DNA aumenta a chance de mutações e de transformação maligna. Nas células com ativação do receptor Ah, BPA levou ainda ao aumento da hidroximetilação global, sem alteração da metilação global do DNA. Os animais não diabéticos expostos ao BPA apresentaram quantidades diminuídas de promielócitos, blastos e bastonetes na medula óssea (aplasia medular), sem alteração no hemograma. Houve aumento dos níveis de CEdG no fígado, da metilação e hidroximetilação global do DNA hepático, e não foi observada alteração das marcas epigenéticas e adutos de DNA no rim ou na urina. Os animais diabéticos expostos ao BPA apresentaram aumento do número de eosinófilos e linfócitos na medula óssea, podendo-se sugerir a indução de um estado inflamatório alérgico, e aumento do número total de hemácias circulantes e do hematócrito. Houve aumento dos níveis de CEdG, da metilação e hidroximetilação global do DNA hepático, aumento dos níveis de 8-oxodG no DNA renal, sem alteração das marcas epigenéticas no rim, e não foi observada alteração dos adutos de DNA na urina. Os dados obtidos apontam para a geração de ROS como uma importante via de cito- e genotoxicidade induzidas por BPA. Sua biotransformação para BPA-3,4- quinona nos modelos utilizados parece ter menor importância para os efeitos, uma vez que não foi detectada a lesão BPA-Gua em nenhuma amostra de DNA, meio de cultura das células ou urina dos animais. Alterações metabólicas induzidas por BPA e ROS podem favorecer as alterações das marcas epigenéticas observadas no DNA das células HL-60, MCF-7 e fígado dos animais. Todas essas alterações podem contribuir para a transformação maligna de células expostas ao BPA. / Bisphenol A (BPA) is a compound widely used in polycarbonate plastic production and widespread in the environment, humans are chronic exposed to BPA in intrauterine period and entire life. The literature suggests the possibility of BPA increase the risk of developing cancers, but studies are required to enable the understanding of mechanisms by which this can occur. HL -60 cells were exposed to BPA or nitro-BPA at concentrations of 25, 100 and 250 uM (0.1% DMSO v/v) for 2, 24 or 48 hours in presence or absence of H2O2 (40 nmol/5x104 cells), MCF-7 cells followed a similar profile of exposure without the use of H2O2, but in presence or absence of Ah agonist receptor (PCB126). Male Sprague-Dawley rats received BPA daily over 4 weeks (50 mg/kg body weight) by gavage in presence and absence of diabetes, with subsequent collection of urine, liver, kidney, bone marrow and circulating blood. The viability, cell cycle, DNA fragmentation and the intracellular production of reactive oxygen species (ROS) was evaluated by flow cytometry, MPO activity and NO production was evaluated by fluorescence assay for HL- 60 cells, mitochondrial activity by XTT assay, and the global DNA methylation was checked by HPLC-PDA. DNA adducts 8-oxodG, CEdG, 1,N6-εdA, 1,N6-εdG and BPA-Gua were quantified by HPLC- ESI-MS/MS in DNA of cells, culture medium, urine and tissue collected from Sprague-Dawley rats. Blood count and bone marrow examination were obtained in collaboration with Experimental Hematology Laboratory of University of Sao Paulo We observed that both BPA and BPANO2 induced ROS generation in HL- 60 cells after 2 hours of incubation. BPA subsequently led to failure of mitochondrial respiratory chain activity, increased permeability of the plasma membrane, DNA fragmentation, arrest in G2/M phase of cell cycle, DNA hypermethylation with global hipohydroxymethylation. We saw low cytotoxicity in HL-60 cells induced by nitro-bpa n the same concentration, without loss of mitochondrial respiratory chain activity, discrete DNA fragmentation, but leading cell cycle to stopping at G0/G1 phase, and induction of DNA global hypermethylation. No lesions were observed in the DNA of HL-60 cells.The results obtained from the exposure of HL-60 cells to BPA and nitro-BPA indicate that these two molecules induce different metabolic abnormalities in this cell line, independent of estrogen pathway, leading to changes in epigenetic (BPA) or genetic and epigenetic (nitro-BPA) profile, that can induce phenotypic consequences such as malignant progression. It was observed along the study that MCF-7 cells are more resistant than HL-60 cells to cell damage induced by BPA and nitro-BPA. As a result of MCF-7 cells exposure to BPA, we saw a slight increase in membrane permeability (250 mM), ROS generation after 24h (25 mM) and increase in cell population in sub G1, so we had DNA fragmentation (100 uM and 250 uM), but with no effect on cell cycle. However, we observed increased levels of CEdG and 8-oxodG on DNA of cells incubated with BPA (100 uM, 250 mM) without prior activation of Ah receptors. The activation of Ah receptors with PCB took a small increase in the level of DNA lesions after incubations with BPA. MCF-7 cells resistance to the cytotoxic effects of BPA is probably related to estrogen action of this compound. Estrogen signaling in addition with the increased levels of DNA damage increases the chance of mutations and malignant transformation. In cells with Ah receptor activation, BPA also led to increased DNA global hydroxymethylation, without changing the global DNA methylation. Nondiabetic animals exposed to BPA had decreased amounts of promyelocytes, blasts and rods in the bone marrow, with any change in blood count. There were an increase of CEdG levels in liver, methylation and global hydroxymethylation on hepatic DNA, and was observed any alteration on epigenetic markers and DNA adducts in kidney or urine. On the other hand diabetic animals exposed to BPA showed increased numbers of eosinophils and lymphocytes in bone marrow, suggesting the induction of an allergic inflammatory state. There were increased levels of CEdG, methylation and global hydroxymethylation on hepatic DNA, increased 8-oxodG levels on kidney DNA without changing epigenetic markers, was not observed DNA adducts in urine. The data obtained indicate that the generation of ROS could be the major route of cytotoxic and genotoxic induced by BPA exposure. BPA biotransformation to BPA-3,4-quinone used in the models seem to have poor effects, since we was not detected BPA-Gua lesion in any DNA sample, culture medium of cells or urine of the animals. Metabolic changes induced by BPA and ROS can enable changes in epigenetic markers observed in the DNA of HL-60 cells, MCF-7 and liver tissue. All these changes may contribute to malignant transformation of cells that were exposed to BPA.

O câncer de mama representa problema mundial de saúde pública e a causa mais frequente de morte por câncer entre as mulheres. A identificação de agentes moduladores de marcas epigenéticas, tais como metilação global do DNA e modificações pós-tradução em histonas, compreende alternativa promissora para estabelecimento de estratégias de controle da carcinogênese mamária. Dentre os nutrientes, o elemento traço essencial selênio (Se) pode ser destacado como agente dietético com potencial anti-câncer de mama e que poderia atuar modulando processos epigenéticos. Entretanto seus mecanismos de ação são pouco elucidados. Este estudo objetivou, assim, identificar efeitos do tratamento com selênio no crescimento e marcas epigenéticas de células de adenocarcinoma mamário humano MCF-7. Células MCF-7, positivas para o receptor de estrógeno, foram tratadas com ácido metilselenínico (MSA) ou selenito de sódio (ST) por diferentes tempos e em diferentes concentrações. Foram avaliados: padrão de proliferação (ensaio cristal violeta) e viabilidade celular (método de exclusão azul de tripan); integridade de membrana plasmática (citometria de fluxo); níveis de fragmentação do DNA (citometria de fluxo), distribuição das fases do ciclo celular (citometria de fluxo); apoptose (citometria de fluxo/ marcação dupla com Anexina V - Iodeto de propídio); níveis de lisina 9 acetilada (H3K9ac) e trimetilada (H3K9me3) em histona H3; níveis de lisina 16 acetilada (H4K16ac) em histona H4 (Western blot); padrão de metilação global do DNA (HPLC-DAD); expressão de gene supressor de tumor (RASSF1a; qPCR) e padrão de metilação da região promotora (RASSF1a e RAR&#946;; MS-PCR); expressão da enzima DNA metilstransferase 1 (DNMT1) (Western Blotting). Comparado ao grupo controle de células não tratadas (GC), ambos os tratamentos com MSA ou ST inibiram a proliferação e viabilidade de células MCF-7 de forma dose e tempo dependente. Ambas as formas químicas de Se induziram a parada do ciclo celular, aumentando (p< 0,05) a proporção de células na fase G2/M e reduzindo (p< 0,05) a proporção daquelas nas fases G0/G1 e S. Os tratamentos com MSA favoreceram a morte celular por apoptose, que foi associada com nível de fragmentação de DNA aumentado (p< 0,05), e reduzida ruptura da membrana plasmática associada com a exposição aumentada (p< 0,05) de fostadilserina. Por outro lado, o ST aumentou (p< 0,05) a fragmentação do DNA e (p< 0,05) a positividade ao iodeto de propídio associado à indução de necrose (p< 0,05). Dentre os mecanismos epigenéticos investigados, 1,6&#181;M e 2&#181;M reduziram a acetilação de H3K9ac (72h; p< 0,05) e aumentaram a de H4K16ac (96h; p< 0,05). O tratamento por 96h com 2&#181;M de MSA reduziu (p< 0,05) a metilação de H3K9me3. Ambos MSA e ST não alteraram o padrão de metilação global do DNA, mas reduziram a expressão de DNMT1, após 96h com 2&#181;M de MSA (p< 0,001; 88%) e após 120h com 10&#181;M de ST (p< 0,001; 96%). ST, mas não o MSA, aumentou (p< 0,05; 45%) a expressão do gene RASSF1a. Em ambos os grupos tratados com MSA ou ST, bem como no GC, a região promotora dos genes RASSF1a e RAR estavam predominantemente metiladas. Estes resultados fornecem evidências de que as ações anti-câncer de mama de compostos do selênio dependem de sua forma química. Além disso, a modulação de processos epigenéticos parecem ser relevantes para as ações inibitórias do MSA em células de câncer de mama. / Breast cancer is a global public health problem and the most frequent cause of cancer death among women. The identification of agents able to modulate epigenetic marks, such as global DNA methylation and histone post-translational modifications, comprises promising alternative for establishing control strategies on mammary carcinogenesis. Among the nutrients, the essential trace element selenium (Se) can be highlighted as a dietary agent with potential anti-breast cancer and could act by modulating epigenetic processes. However its mechanisms of action are poorly understood. This study aimed, therefore, to identify the effects of selenium treatment on growth and epigenetic marks of MCF-7 human breast adenocarcinoma cells. MCF-7 cells, positive for estrogen receptor, were treated with methylseleninic acid (MSA) or sodium selenite (ST) for different times and in different concentrations. Evaluated parameters included: cell proliferation (crystal violet assay) and cell viability (trypan blue exclusion assay); plasma membrane integrity (flow cytometry); levels of DNA fragmentation (flow cytometry), apoptosis (flow cytometry - double labeling with Annexin V - propidium iodide); distribution of cell cycle phases (flow cytometry); acetylated (H3K9ac) and trimethylated (H3K9me3) lysine 9 levels on histone H3; acetylated (H4K16ac) lysine 16 level on histone H4 (Western blot); global DNA methylation (HPLC-DAD); tumor suppressor gene expression (RASSF1a; qPCR) and promoter methylation (RASSF1a, RAR&#946;; MS-PCR); DNA methyltransferase 1 (DNMT1) expression (Western blot). Compared to untreated cells (controls), both MSA and ST inhibited (p< 0.05) MCF-7 cell proliferation and viability in a dose- and time-dependent manner. Treatments with MSA favored cell death by apoptosis, that was associated with increased (p< 0.05) DNA fragmentation level, reduced plasma membrane rupture associated with high (p< 0.05) phosphatidylserine exposure. On the other hand, ST increased (p< 0.05) DNA fragmentation, enhanced (p< 0.05) propidium iodide positivity associated to necrosis induction (p< 0,05). Both chemical forms of Se induced nduced cell cycle arrest, increasing (p< 0.05) the proportion of cells in G2/M phase and reducing (p< 0.05) the proportion of those in G0/G1 and S phases. Among the epigenetic mechanisms investigated, 1.6&#181;M and 2&#181;M of MSA reduced acetylation of H3K9ac (72h, p< 0.05) and increased the H4K16ac (96h, p< 0.05). The treatment for 96h with 2&#181;M of MSA reduced (p< 0.05) the H3K9me3 methylation. Neither MSA nor ST altered (p> 0.05) global DNA methylation, while both compounds reduced (p< 0.05) DNMT1 protein expression, after 96h with 2&#181;M of MSA (p< 0.001; 88%) and after 120h with 10&#181;m of ST (p< 0.001; 94%). ST, but not MSA, increased (p< 0.05; 45%) RASSF1a gene expression. In control and Se-treated cells promoter regions of RASSF1a and RAR&#946; were predominantly methylated. These results provide evidence that the anti-breast cancer actions of selenium compounds depend on its chemical form. Additionally, modulation of epigenetic processes seems to represent a relevant feature of MSA inhibitory effects in breast cancer cells.

Malgré leur efficacité pour réduire la réplication du VIH, les traitements antirétroviraux ne s’accompagnent pas systématiquement de la restauration du compartiment des lymphocytes T CD4+ périphériques. L’espérance et la qualité de vie des individus en échec immunologique sont grandement impactées. Concomitante avec les anomalies périphériques, une atteinte des progéniteurs hématopoïétiques rend compte des fréquentes cytopénies observées au cours des stades tardifs de l’infection. Si l’infection directe des progéniteurs CD34+ reste marginale, les études qualitatives menées in vitro évoquent la perturbation du potentiel de différenciation de ces cellules.Nous avons sélectionné et apparié les patients infectés par le VIH à chaque extrême quand au profil de leur restauration immunitaire et un groupe de donneurs non infectés. Les répondeurs (IR) et non répondeurs immunologiques (INR) traités depuis plus de 8 ans présentaient les caractéristiques similaires pour chaque paramètre pouvant impacter la magnitude de la reconstitution du système immunitaire. Les INR montraient l’activation chronique du système immunitaire, l’inflammation persistante et les signes de l’atteinte de la thymopoïèse. La fréquence des progéniteurs hématopoïétiques circulants n’étant pas différente entre les deux groupes de patients, nous avons analysé le potentiel de ces cellules aux stades pré-thymiques de la différenciation.En utilisant un système de co-culture des progéniteurs hématopoïétiques avec une lignée stromale OP9-DL1 (différenciation T) ou MS5 (différenciation B et NK) avec un cocktail cytokinique approprié, nous avons mis en évidence l’altération du potentiel de différenciation T des cellules issues de patients INR impactant leur restauration périphérique. Ce n’était pas le cas chez les patients IR qui étaient similaires aux donneurs non-VIH. En revanche, le potentiel NK était impacté chez tous les patients infectés en comparaison aux donneurs. Finalement aucune anomalie de potentiel B n’était révélée.En étudiant les voies moléculaires de l’engagement des précurseurs T (Notch), de leur prolifération (IL-7/IL7R) et leur survie (Fas/FasL, TNFR, caspase-1, P2X7) nous avons constaté une diminution de la viabilité des progéniteurs hématopoïétiques chez les patients VIH+ qui présentaient d’avantage d’activation de la caspase-1 qui orchestrait la mort cellulaire par pyroptose. De plus, l’expression de certains gènes-cibles de Notch était clairement Notch-indépendante. Néanmoins, les différences dans le profil transcriptionnel de BCL11B entre les patients IR et INR nous ont permis de proposer un modèle selon lequel la différenciation T était promue chez les patients IR au dépit de celle des précurseurs NK. Enfin, les progéniteurs CD34+ de patients INR présentait la surexpression du P2X7 (récepteur à l’ATP extracellulaire) et l’absence de l’ectonucléotidase CD73 (hydrolyse de l’ATP) ce qui suggérait leur susceptibilité accrue aux nucléotides extracellulaires.L’ensemble de données nous permet de postuler qu’il existe un microenvironnement hautement inflammatoire dans la niche médullaire des progéniteurs hématopoïétiques chez les patients VIH+ qui perturbe leur survie et différenciation. Cette mortalité accrue des cellules CD34+ et probablement des cellules voisines amplifie l’inflammation locale. Ce processus est compensé chez certains patients par la meilleure différenciation T des progéniteurs CD34+ et la réponse immunologique qui s’en suit. Quand ce n’est pas le cas l’atteinte de la lymphopoïèse est importante et l’absence de la reconstitution de la population des lymphocytes T CD4+ périphériques est observée. Ainsi, nous pensons avoir identifié une population de patients infectés par le VIH pour qui les interventions ponctuelles avec les médicaments anti-inflammatoires (par exemple, les antagonistes du P2X7) peuvent s’avérer d’un bénéfice clinique irréfutable. / Despite the efficient reduction of the HIV replication, the administration of combination antiretroviral therapy (c-ART) is not systematically accompanied by the restoration of the peripheral T CD4+ lymphocyte compartment. The life expectancy and quality are severely impacted in individuals with immunological failure. Together with peripheral abnormalities, an alteration of CD34+ hematopoietic progenitor may explain the frequency of the cytopenia observed in the latest stages of the disease. While a direct infection of CD34+ progenitors is thought to be extremely rare, quantitative studies performed in vitro have highlighted the impairment of the differentiation potential of these cells.We selected and matched individuals infected with HIV presenting extremely opposite immunological profile in response to c-ART as well as non-infected donors. The Immune Responders (IR) and Immune Non Responders (INR) treated since more than 8 years, presented similar characteristics for each parameter known to be involved in poor reconstitution of immune system. INR patients showed chronic immune activation, persistent inflammation and thymic regeneration failure. The frequency of circulating CD34 hematopoietic progenitors being not different between both groups of patients, we analyzed the differentiation potential of these cells at pre-thymic stages of lymphopoiesis.Using a co-culture system of hematopoietic progenitors with stromal cell lines OP9-DL1 (T-cell assay) or MS5 (B- and NK-cells assay) with appropriate cytokines, we highlighted an alteration of T-cell differentiation potential in INRs impacting their peripheral restoration. This was not observed in IRs who were similar to non-HIV donors. On the other hand, NK-cell differentiation potential was impaired in both groups of patients in comparison to non-HIV donors. Lastly, no abnormalities in B-cell potential were revealed.Studying molecular pathways involved in T-cell specification (Notch), proliferation (IL-7/IL7R) and survival (Fas/FasL, TNFR, caspase-1, P2X7) we observed the decreased viability of hematopoietic progenitors in HIV patients with increased caspase-1 activation involved in cellular death by pyroptosis. Moreover, expression of some Notch target genes was clearly Notch-independent. However, differences in transcriptional profile of BCL11B between IRs and INRs allowed us to postulate that T-cell differentiation is promoted over NK-cell differentiation in IR patients. Finally, CD34+ cells from INRs presented P2X7 overexpression (extracellular ATP receptor) and absence of CD73 ectonucleotidase (ATP hydrolysis) pointing out their increased susceptibility to extracellular nucleotides.Taken together our data, we postulate that highly inflammatory microenvironment of hematopoietic progenitor’s bone marrow niche disturbs their survival and differentiation in HIV patients. Thus, increased cellular death of CD34+ cells and probably neighboring cells amplifies the local inflammation. This is compensated in some patients by enhanced T-cell differentiation of CD34+ progenitors and results in immunological success. When it is not the case, the alteration of lymphopoiesis is important and the absence of reconstitution of peripheral T CD4+ lymphocyte compartment is noted. We believe have identified the population of HIV-infected individuals who will benefit from occasional administration of anti-inflammatory drugs (such as P2X7 antagonists).

Parmi les huiles naturelles, l'huile d'argan suscite beaucoup d’intérêt. Elle a été utilisée en médecine traditionnelle, en usage externe par les femmes berbères pour les soins corporels et capillaires, et en usage interne pour prévenir certaines maladies cardiovasculaires. L’huile d’argan se caractérise par ses fonctions hypocholestérolémiantes, anti-diabètique et antiproliférative sur des lignées cancéreuses humaines de prostate. Riche en acides gras insaturés, l’huile d’argan est caractérisée également par ses composés mineurs : polyphénols, tocophérols et stérols qui lui confèrent des propriétés antioxydantes. Le rôle des antioxydants alimentaires dans la fonction neurologique et dans certaines maladies liès à l’âge est centrée sur la vitamine E qui est une molécule majoritaire de l’huile d’argan dotée. L’objectif de ce travail a consisté dans un premier temps en une étude phytochimique et en une évalutation du pouvoir antioxydant des extraits d’arganier de deux régions marocaines. Une caractérisation biochimique de la composition de l’huile d’argan a aussi été réalisée. Dans un second temps, une évaluation des effets protecteurs de l’huile d’argan ou certains de ces composés majoritaires a été réalisée in vitro sur des oligodendrocytes murins 158N et in vivo sur des rats. ces effets ont été spécifiés dans un environnement pro-oxydant et cytotoxique mimé par le 7-cétocholestérol (7KC) et l’AlCl3. La cytotoxicité du 7KC induit divers effets au niveau de la mitochondrie (modification fonctionnelle), ce qui conduit à une rupture de l’équilibre RedOx (surproduction d’espèces radicalaires de l’oxygène), à une induction d’apoptose et d’autophagie, à une perte d’adhésion, à une diminution de la prolifération et à des dysfonctions du lysosome. Le 7KC affecte aussi l’expression des marqueurs peroxysomaux (ABCD1, ABCD2, ACOX1) ainsi que l’expression de facteur de transcription PPARα au niveau cellulaire. L’étude in vivo réalisée sur des rats Wistar traités à 100 mg/kg d’AlCl3, a montré qu’AlCl3 induit une rupture de l’équilibre RedOx en diminuant les activités des enzymes anti-oxydantes (catalase, superoxyde dismutase, les glutathions) au niveau du cortex, une altération des fonctions cognitives qui ont été étroitement liées aux maladies neurodégénératives, et des modifications des paramètres plasmatiques. AlCl3 induit une légère perte du poids corporel des rats tout au long de la période de traitement. Dans son ensemble, le travail réalisé montre que l’huile d’argan est capable de s’opposer au stress oxydatif induit par 7KC et AlCl3. L’huile d’argan pourrait être potentiellement être intéressante pour traiter les maladies neurodégénératives. Elle pourrait exercer des effets cytoprotecteurs et antioxydants ce qui permet d’envisager de nouveaux domaines d’applications pour cette huile. / Argan oil has lot of interest for human health. It has been used in traditional medicine for several centuries. The rich composition of argan oil in term of tocopherols and unsaturated fatty acid makes it a very interesting oil regarding its potential actions on risk factors for cardiovascular diseases, hyperlipidemia, hypercholesterolemia and hypertension. Argan oil is also used for skin infections. Previous studies have shown the antiproliferative and pro-apoptotic effects on prostate cancer cell lines. The role of dietary antioxidants in neurological function and in some aged related diseases is centered on vitamin E which is a major molecule of argan oil. The objective of this work was 1) to do a phytochemical study and to evalutate the argan extracts antioxidant properties of argan oils from two Moroccan regions, and 2) to characterize the biochemical composition of argan oils used in this study. Third, we evaluated the protective effects of argan oil and of some of its major compounds in vitro on 158N murine oligodendrocytes, and in vivo in the rat. The effects have been specified in a pro-oxidant environment and cytotoxic mimicked by the 7-ketocholesterol (7KC) and AlCl3. Different methodologies were used: phase contrast microscopy, fluorescence microscopy, histochemistry, flow cytometry, western blotting, and several biochemical methods. Furthermore, 7KC affect the expression of peroxisomal markers (ABCD1, ABCD3, ACOX1) as well as expression of PPARα at the cellular level. The results show that argan oil has cytoprotective and antioxidant effects on 158N murine oligodendrocytes. Argan oil is able to attenuate the overproduction of reactive oxygen species, mitochondrial and lysosomal dysfunctions, loss of cell adhesion and decreased proliferation, cell death by apoptosis and autophagy. We investigate also the effect of aluminum chloride (AlCl3) on brain function and neuronal oxidative stress. Male Wistar rats received daily AlCl3 100 mg/kg for 42 days. AlCl3 exposed group showed a significant reduction in spatial memory performance. Moreover, AlCl3 – treated rats exhibited a marked deterioration of oxidative markers; SOD, CAT, GSH, and changes in the plasma parameters. AlCl3 slightly affects the body weight of rat. Argan oils (or some of their compounds) have cytoprotective and antioxidant effects on 158N murine oligodendrocytes. These different molecules attenuate oxidative stress and could be potentially useful for treating neurodegenerative diseases. Argan oil could exert cytoprotective and antioxidant effects which allows to consider new fields of application for this oil. It is possible to envisage that a controlled diet or functional foods with argan oil could help to prevent some forms of neurodegeneration. / من بين الزيوت الطبيعية أثار زيت أركان الكثير من الاهتمام. فقد استخدم زيت الأركان في الطب التقليدي، من قبل نساء البربرللعناية بالجسم و الشعر، و لمنع بعض الأمراض القلب و الأوعية الدموية . يتميز زيت الأركان بوظائفه التي تخص خفض الكوليسترول، مكافحة داء السكري ومكافحة التكاثر في خطوط الخلايا السرطانية البشرية للبروستات. يعتبر زيت اركان غنيا بالأحماض الدهنية غير المشبعة، البوليفينول، التوكوفيرول و الستيرول .هذه المواد تجعله مادة مضادة للأكسدة.يتركز دور مضادات الأكسدة الغذائية في وظيفة الجهاز العصبي وبعض الأمراض المرتبطة بالعمر على فيتامين E الذي يعتبر المكون الرئيسي لزيت أركان . الهدف من هذا العمل هو أولا دراسة كيميائية لنبتة اركان وتقييم القوة المضادة للأكسدة لمقتطفات هذه النبتة مع توصيف شامل لتركيبة زيت أركان المستخدم في دراستنا.وثانيا تقييم للآثار الوقائية لهذا الزيت ولبعض مركباته الرئيسية في المختبر على الخلايا العصبيةN158 وعلى فئران من فئة وينستار. وقد تم تحديد فوائد زيت اركان في بيئة سامة بواسطة KC7 و AlCl3 النتائج المحصل عليها تكمن في تأثيرات مختلفة على الميتوكوندريا، الأمر الذي يؤدي إلى تمزق في توازن الأكسدة , استحثاث موت الخلايا المبرمج والالتهام الذاتي وفقدان الإلتصاق. يؤثر KC7 أيضا على الناقلات البيروكسيزومية (ABCD1,ABCD3,ACOX1) على المستوى الخلوي PARaP. الدراسة المجراة على الفئران بجرعة 100 ملغم/كغم من AlCl3 أظهرت أن AlCl3 يؤدي الى تمزق التوازن الأكسدة عن طريق الحد من أنشطة الإنزيمات المضادة للأكسدة (الكاتلاز,الجلوتاثيون،سيبيروكسيد) على مستوى المادة الرمادية بالمخ وضعف الادراك الذي ارتبط ارتباطا وثيقا بالأمراض العصبية والتغيرات في معالم البلازما. وبشكل عام، تشير الدراسة ان زيت أركان أو البعض من مكوناته قادرون على مكافحة الأكسدة الناجمة عن KC7 و AlCl3 .

The thesis entitled “Enantiospecific synthesis of bio-active stryllactones” comprise an introduction about stryllactone and two chapters describes the synthesis of stryllactones. Trees of the genus Goniothalamus of the plant family Annonaceae in South East Asia has been known for a long time for their proven as folkloric medicine. Stryryllactones were found to exhibit moderate to significant biological activity including antitumour, antifungal as well as antibiotic properties. Because of their unique and intriguing structures and the activity associated much effort has been centered on the development of methodology for the synthesis of these compounds. The structures and relative configurations of these compounds were determined either by X-ray crystallography or by extensive NMR spectral analysis and by mass spectroscopic techniques. The research group of McLaughlin isolated and characterized a series of styryllactones, possessing significant to marginal cytotoxic activity against human tumor cell lines. The structures and relative configurations of these compounds were determined either by X-ray crystallography or by extensive NMR spectral analysis. Classification of these styryllactones is based on the structural characteristics of the six different skeletons as shown in Figure 1. Figure 1:features styryllactone the genus In this thesis, enantioselective total synthesis of styryllactones ()-9-deoxygoniopypyrone 1, ()-goniopypyrone 2, ()-7-epi-goniofufurone 3, ()-7-epigoniodiol 4 and the putative structure of ()-etharvendiol 5 is presented. a) Total synthesis of ()-9-deoxygoniopypyrone, ()-goniopypyrone, ()-7-epigoniofufurone and ()-7-epi-goniodiol: Synthesis of the styrylalctones is relied on elaboration of the trihydroxy ester 11 derived from tartaric acid. Appropriate protection of the hydroxy groups and further modifications of the ester functionality (which can be transformed into the corresponding alcohol or aldehyde) is planned for the synthesis of the styryllactones 1-5. Accordingly, the bis-dimethylamide 9 derived from D-()-tartaric acid, was transformed to the -hydroxy amide 10 using a combination of Grignard reagent addition followed by reduction of the resultant ketone. Acid mediated deprotection of the acetonide with concomitant hydrolysis of the amide to the ester is accomplished in one pot by treating 10 with p-TSA in benzene/MeOH mixture Treatment of the trihydroxy ester with 2,2-dimethoxy propane in presence of p-TSA afforded the hydroxy ester 12 which was elaborated to the styrylalctones 9deoxygoniopypyrone, 7-epi-goniodiol, 7-epi-goniofufurone and goniopypyrone (Scheme-2). (Part of this work is published: Prasad, K. R.; Dhaware, M.G. Synlett. 2007, 11121114.; Prasad, K.R.; Dhaware, M.G. Synthesis 2007, 3697) b) Stereoselective synthesis of the putative structure of (+)-etharvendiol: In 1997, Bermejo et al isolated the styryl pyrone etharvendiol 5 from the ethanolic extract of stem bark from Goniothalamus arvensis. Hitherto, no synthesis of etharvendiol is reported in the literature. In this section, approach towards the synthesis of putative structure of etharvendiol will be discussed. Synthesis of etharvendiol 5 is anticipated by the elaboration of masked tetrol 15, comprising an alkene tether and four contiguous hydroxy groups installed with definite configuration. It is relied on exploiting the hydroxy directed lactonization via the oxidation of alkene in 15, and subsequent elaboration to 7. Bis-dimethylamide 9, derived from D-()-tartaric acid was identified as the suitable precursor for the synthesis of 15. Synthesis of masked tetrol 15 is accomplished from 9 involving a combination of selective Grignard additions and stereoselective reduction (Scheme 3). (For structural formula pl see the pdf file)