Telomeres are regions of tandem repeats at the ends of chromosomes ensuring chromosome stability or inducing replicative senescence when critically short. Telomerase extends telomeres and its catalytic subunit, telomerase reverse transcriptase is tightly regulated at multiple levels. Cancerous cells prevent telomere-mediated senescence to attain unlimited proliferation, in most cases by enhancing telomerase activity. Chronic myeloid leukaemia is characterised by the translocation, t(9;22), in haematopoietic stem cells. The resulting fusion protein exhibits constitutive tyrosine kinase activity in the cytoplasm, promoting cellular proliferation, inhibiting apoptosis and impeding cell adhesion. Changes in telomere biology have been observed in chronic myeloid leukaemic cells. The current study aimed to investigate telomere biology in 18 chronic myeloid leukaemia patients at various time intervals from date of diagnosis. Although telomeres were significantly shorter in patients compared to controls, results point to complex telomere dynamics in the malignancy. Increased telomerase activity did not necessarily accompany telomere lengthening and increased transcription of the telomerase catalytic subunit was not necessarily indicative of telomerase activity. Ultimately the current study could not detect any trends between telomere length, telomerase activity and telomerase catalytic subunit expression in chronic myeloid leukaemia patients. Together with inherent patient-to-patient variation and the high cost per assay, measurement of telomere biology does not appear to hold prognostic value in chronic myeloid leukaemia and does not warrant inclusion into a routine test repertoire.

Orientador: Jean Leandro dos Santos / Banca: Gustavo Troiano Feliciano / Banca: Priscila Longhin Bosquesi de Oliveira / Resumo: A anemia falciforme (AF) é uma das doenças hematológicas genéticas mais prevalentes no mundo. Atualmente, as estratégias terapêuticas visando aumento dos níveis de hemoglobina fetal (HbF) contribuem significativamente na redução da morbidade e mortalidade associadas à doença. A compreensão dos mecanismos moleculares responsáveis pelo silenciamento gênico para produção de hemoglobina fetal pode permitir o desenvolvimento de fármacos indutores de HbF mais efetivos e seguros. Durante a eritropoiese, a expressão gênica de gama-globina é silenciada, em partes, pela ligação de um complexo protéico contendo entre outras proteínas a GATA-1, que se liga em sítios próximos aos promotores do gene de gama-globina impedindo o processo de transcrição. Especificamente, o recrutamento de GATA-1 é realizada pelo bromodomínio-3 (BRD-3). Neste trabalho, nossa hipótese foi a de que a inibição de BRD-3 possa constituir uma nova abordagem terapêutica capaz de promover o aumento dos níveis de HbF. Para tal, foi realizado um estudo prévio de triagem virtual com 2.2 milhões de moléculas, em que se identificaram alguns candidatos a ligantes do bromodomínio-3 (BRD-3). A partir destes, e aplicando a técnica do planejamento de fármacos baseado em fragmentos (FBDD), foi realizado um estudo de docagem molecular (docking) destes derivados, a fim de se explorar o padrão de substituição mais adequado para as interações com os resíduos de aminoácidos presentes no BRD-3. Os compostos finais apresentaram interaç... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sickle cell anemia (SCA) is the most common inherited hemoglobinopathy in the world. The therapies currently availables to increase the levels of fetal hemoglobin contribute significantly to reduce morbidity and mortality associated with the disease. An understanding of the molecular mechanisms for drug development in order to increase the fetal hemoglobina levels may allow the development of more effective and safe HbF-inducing drugs. During erythropoiesis, gamma-globin gene expression is partially inhibited by the binding of a protein complex containing GATA-1 among other proteins, which binds at sites close to the gamma-globin gene promoters by preventing the transcription. Specifically, recruitment of GATA-1 is performed by bromodomain-3 (BRD-3). In this work, our hypothesis was the BRD-3 inhibition may be a new therapeutic approach capable of promoting the increase of HbF levels. Preliminary study of virtual screening was carried out with 2.2 million molecules. It was identified some candidates for bromodromain-3 (BRD-3) ligands. Using Fragment Based Drug Design (FBDD), the fragments were optimized and a docking study was carried out, in order to explore the most adequate substitution pattern in order to improve the interactions present in BRD-31. The final compounds showed interactions with residues of asparagine-116, proline-58, glutamine-61 and tryptophan-57, and docking scores between -10.357 kcal/mol and -7.152 kcal/mol. It has been synthesized, identified and characterized nine compounds with yields between 17 and 86%. The compounds showed enzymatic inhibition range between 13 and 80% in BRD-3 (BD1). After 24 hours, compound IX was able to induce gamma globin expression in culture of K562 cells. These results suggest that compounds IX is a novel prototype, BRD-3 inhibitor, able to induce gamma-globin expression. / Mestre