Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of genomes allowing lineage-tracing studies, temporal and spatial misexpressions, and in particular the generation of conditional knockout alleles. Previously, we and others showed that Cre and its ligand-inducible variant CreERT2 are also highly efficient in the developing and adult zebrafish. The number of Cre driver and effector lines is currently still limited in zebrafish. However, the recent advent of novel genome editing tools such as TALEN and CRISPR/Cas will significantly increase interest in the conditional Cre/lox-technology in this organism. The considerations of basic transgene design and subsequent transgenesis have been addressed elsewhere. Here we outline practical experimental steps for transient functionality tests of CreERT2 driver and effector constructs. In addition, we introduce detailed protocols to elicit CreERT2-mediated recombination in vivo at embryonic as well as adult stages.

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Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase is the cytochrome P450 enzyme that converts androgens to estrogens. Aromatase is the target of the aromatase inhibitor class of drugs widely used to treat estrogen-mediated conditions including breast cancer. Little is known about the role of this enzyme in drug metabolism or in drug interactions. Since this lack of knowledge has been an impediment to optimal therapy, it is important to understand these roles of aromatase. Therefore, a comprehensive series of studies was carried out to characterize its ability to metabolize drugs and its susceptibility to inhibition by xenobiotics. The overall objective of this work was to better understand the interactions of small molecules with aromatase and to use this new knowledge to predict aromatase-mediated drug interactions and anticipate novel molecular structures that interact with the enzyme. Aromatase was shown to be a drug metabolizing enzyme able to metabolize methadone both in vitro (Km of 314 μM) and in vivo (22% of methadone clearance). A number of novel aromatase inhibitors that employ diverse kinetic mechanisms were identified. These include a potent competitive inhibitor: norendoxifen (Ki of 35 nM), two non-competitive inhibitors: endoxifen (Ki of 4.0 μM) and N-desmethyl-tamoxifen (Ki of 15.9 μM), a mechanism-based inhibitor: methadone (KI of 40.6 ± 2.8 μM; kinact of 0.061 ± 0.001 min-1), and a stereoselective inhibitor: naringenin (IC50s of 2.8 μM for (R)-enatiomer and 1.4 μM for (S)-enatiomer). Through investigation of the structure-potency relationships so discovered, a series of new biochemical structures to be exploited as aromatase inhibitors were identified. These studies have identified new roles for aromatase as a catalyst for methadone metabolism and as a mediator of the effects of tamoxifen by demonstrating that a number of its metabolites can act as aromatase inhibitors. This work also provides a new mechanistic framework for the design of novel aromatase inhibitors that can be used in breast cancer. Overall, the data suggest ways to more consistently treat breast cancer with current medications, to better anticipate drug interactions, and therefore to improve the quality of life of patients in ways that minimize side effects, while optimizing therapeutic benefits, in each person treated.

Tamoxifen is an effective anti-estrogen for treatment of women with hormonedependent breast cancer but acquired drug resistance limits its therapeutic benefit. We have previously reported that expression of active Akt3 in MCF-7 breast cancer cells results in estrogen-independent tumors that are actually stimulated to grow after tamoxifen treatment. We hypothesize that this tamoxifen resistance may be attributed to binding of different co-regulator proteins and/or different binding affinity of these proteins to the estrogen receptor in M CF-7 cells overexpressing active Akt3 as compared to parental MCF-7 cells. We have immuno-precipitated the estrogen receptor along with bound co-regulator proteins in both cells lines after tamoxifen, estradiol, or vehicle treatment. After 2-D gel electrophoresis separation of these immuno-precipitated proteins and comparing them using PDQuest 2-D analysis software, we identified protein spots that were statistically different under the treatment conditions between the two cell lines. The isolated protein spots were subjected to MALDI-TOF mass spectrometry. By searching protein databases through the MASCOT website for protein identification, we have identified estrogen receptor co-regulator proteins that may play a potential role in tamoxifen resistance. Current studies are focused on addressing the role of differential protein binding as a possible mechanism of tamoxifen resistance in Akt3 over-expressing breast cancer cells.

[pt] A partir da associação de condensações aldólicas e acoplamentos cruzados em blocos de construção arilcarbonílicos já conhecidos, é possível modular as estruturas conjugadas dos produtos, direcionando-os a diferentes aplicações. Sendo assim, na presente tese, buscou-se realizar a síntese de todos os compostos por meio de associações de condensações aldólicas e acoplamentos. Este trabalho está dividido em quatro capítulos que dizem respeito a: (i) síntese e avaliação fotofísica de híbridos de chalconas e benzotiadiazolas (BTDs); (ii) síntese e aplicação de híbridos BTD-chalconas como dispositivos OLEDs; (iii) síntese de chalconas e BTDs e avaliação de potenciais fotossensibilizadores para DSSCs (células solares sensibilizadas por corante); (iv) síntese de beta-arilchalconas com potencial atividade antiestrogênica. Levando em consideração as reconhecidas propriedades fotofísicas de chalconas e derivados de BTDs, é descrita a síntese de novos híbridos BTDchalcona fotoativos. Os produtos foram obtidos em rotas de duas etapas (reação de Suzuki e posterior condensação aldólica) com rendimentos reacionais variando entre 32 e 64 por cento. As modulações nas estruturas foram realizadas de tal forma que os compostos fossem emissivos no estado agregado. Sabendo das limitações do desenvolvimento de filmes luminescentes relacionadas ao efeito de quenching causado por agregação (ACQ), buscou-se sintetizar compostos com propriedades do tipo AIEE (emissão aumentada induzida por agregação). Os estudos de suas propriedades fotofísicas demonstraram que o composto com arquitetura arilóxiBTD-chalcona apresentou os melhores resultados nos quais foram observados aumentos expressivos na intensidade de fluorescência (30x) e no rendimento quântico de fluorescência (de 0,0070 a 0,143) em estado agregado. Com estes dados, novos análogos foram sintetizados respeitando o esqueleto do tipo arilóxiBTD-chalcona (ou -fluoreno) com rendimentos que variaram de 42 a 79 por cento. Os novos análogos também apresentaram AIEE e foram aplicados como camadas emissoras em OLEDs de estrutura bicamada para avaliação e comparação dos seus desempenhos eletroluminescentes. A associação de acoplamento cruzado e condensação aldólica também foi utilizada como estratégia para a produção de novos fotossensibilizadores para DSSCs. Para tal, buscou-se sintetizar compostos do tipo D-pi-A-pi-A, nos quais os grupos aceptores terminais têm a função de ancoradores de TiO2. O uso deste tipo de estrutura facilita a injeção de elétrons devido à transferência direcional de elétrons do doador para a porção aceptora. Com base nisso, foram sintetizados novos derivados de chalconas e BTDs. As sínteses foram realizadas em duas etapas resultado em rendimentos de 35 a 44 por cento. Estudos eletroquímicos apontaram bons valores de HOMO, LUMO e band gap para todos os compostos. Os valores de LUMO se mostraram acima da faixa de condução do semicondutor TiO2 (-4,0 eV), garantindo a força motriz necessária para o direcionamento dos elétrons. Além disso, os valores de HOMO de todos os compostos se mostraram inferiores ao potencial redox do eletrólito (-4,8 eV) gerando uma alta força motriz para regeneração do corante. Por meio de Espectrometria de UV-Vis, foi observado que os compostos absorvem em região próxima a 350 nm em diferentes solventes, o que dificulta o uso destes como DSSCs. Por fim, no último capítulo foi apresentada uma nova estratégia para a obtenção de moléculas potencialmente bioativas a partir da associação entre condensações aldólicas e reações de acoplamento. O sistema alfa-beta insaturado da chalcona torna essa uma interessante plataforma para inserção régio- e estereosseletiva de substituintes arílicos na porção olefínica do substrato. A partir de uma estratégia de inserção de porções arílicas já descrita por nosso grupo, aplicamos estas para a síntese de análogos do tamoxifeno, fármaco utilizado atualmente no tratamento de alguns tumores de mama. Seis produtos foram sintetizados por meio de uma reação de Heck, resultando em misturas E/Z de aproximadamente 50:50 e rendimentos de 29 a 57 por cento. / [en] It is possible to modulate the conjugated structures of the products, directing them to different applications from the association of aldol condensations and crosscouplings in known arylcarbonyl building blocks,. In this sense, this thesis is divided into four chapters concerning: (i) synthesis of analogues and hybrids of chalcones and BTDs; (ii) synthesis and application of chalcone-BTD hybrids as OLED devices; (iii) synthesis and evaluation of potential photosensitizers for DSSCs (dye-sensitized solar cells); (iv) synthesis of compounds with potential antiestrogenic activity. For the synthesis of all compounds, associations of aldol condensations and cross couplings were performed. Considering the recognized photophysical properties of chalcones and BTD derivatives, the synthesis of new photoactive BTD-chalcone hybrids is described. The products were obtained in two-step routes (Suzuki reaction and aldol condensation) and reaction yields ranged from 32 to 64 percent. The modulations in the structures were performed in such a way that the compounds were emissive in the aggregated state. Knowing the limitations of the development of luminescent films related to the aggregation-caused quenching effect(ACQ), compounds were synthesized with AIEE-like properties (aggregation-induced increased emission). The studies of its photophysical and electrochemical properties indicated that a compound with aryloxy-BTD-chalcone architecture presented the best results in which expressive increases were observed in fluorescence intensity (30x) and in fluorescence quantum yield (from 0.0070 to 0.143) with the aggregation. With these data, new analogs were synthesized respecting the aryloxy-BTD-chalcone (or -fluorene) with yields ranging from 42 to 79 percent. The new analogues also presented AIEE and were applied as emitting layers in bilayer structure OLEDs for evaluation and comparison of their electroluminescent performance. The association of cross-coupling and aldol condensation was also used as a strategy to produce new photosensitizers for DSSCs. To this end, D-pi-A-pi-A compounds were synthesized in which the terminal acceptor groups act as TiO2 anchors. The use of this type of structure facilitates the injection of electrons due to the directional transfer of electrons from the donor to the acceptor portion. Based on this, new derivatives of chalcones and BTDs were synthesized. The syntheses were performed in two steps resulting in yields of 35 to 44 percent. Electrochemical studies showed good HOMO, LUMO and band gap values for all compounds. The LUMO values were above the TiO2 semiconductor conduction range (-4.0 eV) which guarantees the necessary driving force for the directioning of electrons. In addition, the HOMO values of all compounds were lower than the redox potential of the electrolyte (-4.8 eV) generating a high driving force for dye regeneration. It was observed that the compounds absorb in a region close to 350 nm in different solvents, which makes their use as DSSCs difficult. Finally, in the last chapter, a new strategy was presented to obtain potentially bioactive molecules from the association between aldol condensations and coupling reactions. The alpha-beta unsaturated system of chalcone makes it an interesting platform for regio and stereoselective insertion of aryl substituents in the olefinic portion of the substrate. Based on a strategy of insertion of aryl moieties already described by our group, tamoxifen, a drug currently used in therapy for the treatment of some breast tumors, analogues were synthesized. Six products were synthesized via a Heck reaction, resulting in E/Z mixtures of approximately 50:50 and yields of 29 to 57 percent.

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