Orientadores: João Ernesto de Carvalho, Wanda Pereira Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T13:14:28Z (GMT). No. of bitstreams: 1 Kohn_LucianaKonecny_D.pdf: 3966647 bytes, checksum: 884bfab3a168fe2feddf628f0cc13684 (MD5) Previous issue date: 2005 / Resumo: Os adutos de Baylis-Hillman apresentam em sua estrutura grupos funcionais passíveis de interação com biomoléculas, tornando-os potentes protótipos para o desenvolvimento de novas drogas antineoplásicas. O objetivo deste estudo é avaliar a atividade anticâncer de alguns b-hidroxiacrilatos e alguns derivados hidrogenados e protegidos. As substâncias contendo ligação dupla conjugada a carbonila foram obtidas através da reação de Baylis-Hillman, catalisada por DABCO, entre acrilato de metila e o aldeído correspondente. Os derivados hidrogenados e os protegidos foram obtidos pelo tratamento do aduto correspondente com hidrogênio (H2, Pd/C) e cloreto de trietilsilila, respectivamente. Os testes in vitro foram realizados com sete linhagens: UACC-62 (pele tipo melanoma), MCF-7 (mama), NCI-H460 (pulmão), OVCAR-3 (ovário), PC-03 (próstata), 786-0 (rim) e NCI-ADR (mama resistente) em diferentes concentrações (0,25 a 250mg/mL). Os resultados foram obtidos através do ensaio do doseamento protéico pela sulforrodamina B.Os adutos aromáticos apresentaram atividade antiproliferativa significativa, enquanto seus derivados hidrogenados não inibiram o crescimento celular. Em relação aos diferentes substituintes do anel aromático, o aduto 9 apresentou maior efeito citocida, porém sem seletividade. Já o aduto 10, com substituinte doador de elétrons, apresenta seletividade para a linhagem de carcinoma ovariano. O aduto 4 com substituinte retirador de elétrons, apresentou o melhor perfil de atividade com potência elevada, concentração-dependente e seletividade. O fato do grupo NO2 ser fortemente retirador de elétrons parece ter sido decisivo para a atividade, visto que é o único fator diferencial do aduto com substituintes doadores.O aduto 4 foi selecionado para verificação de sua toxicidade e possível(is) mecanismo(s) de ação. A dose letal 50 (DL50) em camundongos Swiss foi de 1,3g/Kg de peso corporal animal, sendo considerado de baixa toxicidade segundo Loomis em ¿Fundamentos de Toxicologia¿. As alterações morfológicas desta substância sobre a linhagem de ovário (OVCAR-3) estão em concordância com o resultado obtido no teste de atividade antiproliferativa, uma vez que manteve uma resposta concentração dependente, com um aumento da citotoxicidade em relação à dose utilizada e a morte celular pelo processo de apoptose. A possibilidade de o aduto estar inibindo a proliferação celular através de intercalação na dupla fita de DNA foi descartada através do ensaio DNA methyl green no qual o aduto 4 não apresentou atividade quando comparado a doxorrubicina, a qual apresentou intercalação concentração dependente na faixa de 32 a 82%, confirmando assim nossa hipótese que o aduto não intercalaria com a dupla fita de DNA. Quando foi avaliada a interferência do óxido nítrico (NO) na atividade antitumoral pode-se observar claramente a perda de atividade da substância quando a cultura foi pré-tratada com L-NAME para a linhagem NCI-ADR e diminuição do potencial da atividade para as demais linhagens; com o pré-tratamento com AMG só ocorreu a diminuição da atividade. A quantificação enzimática permitiu concluir que a atividade do aduto 4 tem correlação com a NOs constitutiva dependente de cálcio (nNOs e eNOs). Os resultados interessantes com os inibidores da NOs sobre a linhagem de mama (NCI-ADR) que expressa o fenótipo de resistência a múltiplas drogas (MDR) sugere a hipótese dessa substância ter alguma correlação com o processo de resistência. Dessa forma, tanto o derivado 4 como a doxorrubicina tiveram sua atividade anticâncer avaliada na presença de um bloqueador de canais de Ca+2, o verapamil. Verificou-se que a atividade da doxorrubicina aumenta significantemente na presença do verapamil sem alterações na atividade do aduto 4. Esse resultado descarta a hipótese de que esta substância tenha envolvimento com o processo de resistência.Conclui-se que os adutos de Baylis-Hilman apresentam atividade antiproliferativa ¿in vitro¿ em células tumorais humanas sendo esta dependente o sistema a-b insaturado e anel aromático presente na estrutura. O padrão toxicológico observado na análise permite dizer que a morte celular ocorre por apoptose e possui baixa atividade em fibroblastos normais. A toxicidade ¿in vivo¿ foi avaliada como ligeiramente tóxico / Abstract: Baylis-Hillman adducts present in their structures functional groups capable to interact with biomolecules. This capacity makes them good lead compounds for the design of anti-neoplastic drugs. This study is focusing on anticancer activity evaluating for some b-hydroxyacrylates and their hydrogenated or O-protected derivatives. Baylis-Hillman adducts of this study were obtained from reaction between an aldehyde and methyl acrylate in the presence of 1,4-diazabicyclo-2,2,2-octane (DABCO). Hydrogenated or O-protected BHAs-derivatives were produced from the hydrogenation (H2, Pd/C) or treatment of correspondent BHA-precursor with triethylsilil chloride and triethylamine. In vitro tests were performed against the following human tumor cell lines: UACC62 (melanoma), MCF-7 (breast), NCI-460 (lung), OVCAR-3 (ovarian), PC-03 (prostate), 786-0 (renal), NCI-ADR (multi-resistant breast). BHAs concentration were employed in the range of 0.25 - 250 mg/mL and doxorubicin (DOX, at the sameconcentration range), as positive control. Cell proliferation was determined by spectrophotometric assay using sulforhodamine B as protein-binding dye. Aromatic adducts displayed significant antiproliferative activity while their hydrogenated derivatives were not able to inhibit cell growth. Concerning to different groups attached in the aromatic ring, adduct 9 presented the highest cytotoxic activity, however with no selectivity. On the other hand, adduct 10, which possess an electron-donating group in the aromatic ring, show to be selective for OVCAR-3 (ovarian cancer cell). Electron-withdrawing group (-NO2 in adduct 4) improved both potency and selectivity of Baylis-Hillman adduct. These results showed that the presence of electron-withdrawing groups in the aromatic is very important for determining high activity of this Baylis-Hillman adduct. Based on this, adduct 4 was chosen for verifying of its toxicity on mice swiss as well as the possible mechanism of action of this molecule. Lethal dose (LD50) was shown to be 1.3 g of adduct 4/Kg of corporeal weight mouse, demonstrating that this compound has low toxicity according to Loomis (Fundamentos de Toxicologia). In addition to this, morphological modifications caused by adduct 4 on OVCAR-3 are in agreement with results obtained in the antiproliferative activity assays where it was observed a concentration-dependent effect of this compound. DNA methyl green assay demonstrated that the adduct 4 is not able to bind DNA as compared to doxorubicin a positive control. Interestingly, pre-treatment of NCIADR with Nitric Oxide Synthase (NOS) inhibitor NG-Nitro-L-arginine methyl ester (LNAME) abolished the antiproliferative activity of adduct 4. No significant loss of activity for adduct 4 was observed on the other cancer cell lines when they were pre-treated with LNAME. Only slight decrease of adduct 4 activity studied was observed on all cancer cell lines under pre-treatment with NOS-inducible inhibitor aminoguanidine (AMG) suggesting that the antiproliferative activity of adduct 4 on NCI-ADR is related to the increase of constitutive NOS activity. However, measurement of NOS activity using substrate labeled with 14C showed decreasing in the activity of constitutive NOS (nNOS and eNOS) according to the time of NCI-ADR cells exposure to adduct 4. Although these results are not conclusive, it is noteworthy the involvement of NOS enzyme in the antiproliferative activity of adduct 4 on NCI-ADR cells. Since NCI-ADR cells present multiple drugs resistance (MDR) and considering the participation of NOS enzymes it seems that the effect of adduct 4 may be related to the resistance process of these cells. Nevertheless, the antiproliferative activity of adduct 4 on NCI-ADR in the presence of the calcium channels blocking verapamil revealed that no alteration on adduct 4 activity was observed suggesting that calcium channels are not involved in the mechanism of action of this adduct. Overall, these results indicate that Baylis-Hillman adducts present in vitro antiproliferative activity against different human cancer cell lines. This activity is associated to the presence of a,b-unsaturated system and aromatic ring. Among the compounds studied, adduct 4 was the most active and selective compound. Besides this, the high cytotoxic activity of adduct 4 against cancer cells appears to be specific for tumor cells since in vivo assay demonstrated low toxicity of this compound (1.3 g/Kg) on mice as well as low in vitro activity on normal fibroblasts. Cell death caused by adduct 4 presents hallmarks of apoptotic process / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

La fluorescence induite par laser multibande est une technique non intrusive permettant d’accéder à la température de la phase liquide dans des milieux complexes. L’application de cette technique dans des liquides monophasiques où le chemin optique est variable (produit de la concentration moléculaire du traceur et de la distance dans le milieu liquide entre le volume de mesure et l’optique de détection) a été considérée. Le rapport des intensités de fluorescence collectées sur deux bandes spectrales permet de s’affranchir de la concentration en traceur fluorescent, de l’intensité laser incidente et du volume de mesure. Une troisième bande spectrale de détection permet de prendre en compte le terme de ré-absorption de la fluorescence dans le cas de chemins optiques non-négligeables et variables. Puis l’application de la technique à la mesure de la température de la phase liquide d’un spray est présentée. Un traitement spécifique du signal a été développé afin de tenir compte de la nature aléatoire du signal de fluorescence liée à la présence de gouttes dans le volume de mesure. De plus, ce traitement a été adapté afin de rendre possible le couplage des données de fluorescence avec des mesures granulométriques effectuées par la technique phase Doppler dans la perspective finale d’obtenir des températures de goutte par classe de taille. Cependant plusieurs phénomènes perturbateurs ont été mis en évidence : - une dépendance inattendue et non linéaire à la taille de goutte du rapport des intensités de fluorescence collectées sur deux bandes spectrales. - une forte diffusion de la lumière laser incidente par le nuage de gouttelettes induit une fluorescence bien au delà de la zone d’excitation laser. Cette fluorescence parasite est néanmoins détectée dans la profondeur de champ du dispositif optique et se conjugue avec l’effet non-linéaire de la taille des gouttes. Une stratégie de correction de ces différents phénomènes est proposée et une expérience de validation est réalisée sur un spray chauffé injecté dans une cellule saturée en vapeur / Multiband laser-induced fluorescence is a non-intrusive technique able to provide a measurement of the liquid phase of complex media. The application of this technique in single phase liquids, with a variable optical path (product of the fluorescent tracer molecular concentration by the distance between the probe volume and the collection optics in the liquid) was considered. The ratio of the fluorescence intensities collected on two spectral bands allows removing the influence of the fluorescence tracer concentration, incident laser intensity and probe volume. A third spectral band of detection is used to take into account the re-absorption of the fluorescence in the case of non negligible and variable optical paths. Then, the application of this technique to the measurement of the temperature of the liquid phase of a spray is presented. A specific data processing was developed in order to take into account the random presence of droplets in the probe volume. Moreover, the processing was adapted to achieve combined fluorescence and droplet size measurements using the phase Doppler technique. The overall foreseen goal is to measure temperature per droplet size class. However, several disturbing phenomena were highlighted: - an unexpected non-linear dependence on the droplet size of the ratio of the fluorescence intensities collected on two spectral bands, - a strong incident laser light scattering by the droplets cloud, which induces a fluorescence beyond the excitation zone. This fluorescence is also collected in the depth of field of the optical device and combines with the non-linear size dependence. A correction strategy of these phenomena was implemented and a validation experiment on a heated spray injected in a vapour-saturated cell was performed

The discovery of a method by which proteins of interest can selectively be labeled with a probe of choice intracellularly is a longstanding goal in chemical biology research. Conventional labeling techniques have utilized large domain tags but despite the development of small labeling molecules there have been no short peptide sequences known to covalently label a small molecule without the aid of an enzymatic process or metal chelation. We aimed to find a sequence of nucleophilic peptides that reacted covalently and specifically with electrophilic small labeling molecules using mRNA display. Our goal was to show that an electrophilic small labeling molecule that is brought in proximal distance to a protein of interest via affinity can result in nucleophilic attack by a neighboring nucleophilic amino acid to covalently label the protein of interest. Utilizing affinity between a small labeling molecule and a protein of interest to bring them spatially close to one another maximizes the chance that a covalent reaction can take place and provides selectivity between two components in a complex mixture. Towards this goal, we developed several electrophilic fluorescent small molecules. Covalent labeling was achieved using electrophilic bait in the form of sulfonate esters, a polyethylene oxide linker provided structural flexibility, and a fluorescent affinity tag containing a rhodamine backbone served as the potential binding site to a key peptide sequence encoded within a protein of interest. The synthetic routes to access our electrophilic rhodamine B and sulforhodamine 101 fluorophores were optimized. Key intermediates were produced and served as flexible points of modification to make various analogs of our desired electrophilic fluorophores. The affinity between proteins containing the peptide sequence and the fluorescent electrophiles were determined by fluorescence polarization. Covalent labeling was determined to be both time and concentration dependent. The expected published affinity between the peptides and fluorophore was not high enough to produce selective labeling. However, our small labeling molecules were found to be effective at labeling various proteins in vitro. In addition, our electrophilic fluorophores have been found superior to sulforhodamine 101 in live cell imaging of astrocytes.

Since ancient times, indigenous plants have been used by traditional healers for treating various ailments. Sutherlandia frutescens is one of the most commonly used medicinal plants of southern Africa. This widely distributed plant has been traditionally used to treat cancer and HIV patients; however scientific validation is still in high demand. This research aimed to phytochemically characterise the various extracts prepared and to determine if any chemotypes were present. Subsequent biological characterisation was carried out to preliminary ascertain whether this medicinal plant could have anti-cancer and/or immunemodulating properties and which compounds might be responsible for these actions. Various traditional and organic extracts were prepared. Extracts, fractions and compounds generated were analysed and chemical profiles obtained. Column chromatographic techniques were used to isolate and purify compounds and structure elucidation was carried out using various analytical techniques. Sulforhodamine B and cytometric bead array assays were performed to determine the biological activities of samples generated. / Life and Consumer Sciences / (M. Sc. (Life Sciences)

Since ancient times, indigenous plants have been used by traditional healers for treating various ailments. Sutherlandia frutescens is one of the most commonly used medicinal plants of southern Africa. This widely distributed plant has been traditionally used to treat cancer and HIV patients; however scientific validation is still in high demand. This research aimed to phytochemically characterise the various extracts prepared and to determine if any chemotypes were present. Subsequent biological characterisation was carried out to preliminary ascertain whether this medicinal plant could have anti-cancer and/or immunemodulating properties and which compounds might be responsible for these actions. Various traditional and organic extracts were prepared. Extracts, fractions and compounds generated were analysed and chemical profiles obtained. Column chromatographic techniques were used to isolate and purify compounds and structure elucidation was carried out using various analytical techniques. Sulforhodamine B and cytometric bead array assays were performed to determine the biological activities of samples generated. / Life and Consumer Sciences / (M. Sc. (Life Sciences)

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