The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called “seat-belt”, favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.

Agradeço em especial à minha orientadora e mestra Dra. Cibele Nunes Peroni, pela oportunidade, orientação, confiança e amizade tão importantes para a minha formação profissional e humana. Ao Dr. Paolo Bartolini, meu "co-orientador", pela amizade, incentivo e apoio em todas as fases deste projeto. Ao Dr. Carlos R. J. Soares e à Dra. Maria Teresa Ribela, pela amizade e incentivo. À Claudia Cecchi e à Eliza Higuti pela ajuda na realização dos experimentos e pelos ensinamentos. Aos amigos do Centro de Biotecnologia do IPEN pela ajuda e apoio: Arlete,

No abstract

Capítulo 1-Introdução geral e objetivos _____________________________ 04 1.1-Introdução Craniossinostoses___________________________________ 05 1.1.2-Craniossinostoses Sindrômicas _______________________________ 08 1.2-Aspectos clínicos das síndromes de craniossinostoses mais comuns _________________________________________________ 10 ABSTRACT Craniosynostosis, defined as the premature fusion of one or more cranial suture, are a very heterogeneous group of disorders, with an occurrence of about 1: 2000 live births. Except for rare syndromes, such as Apert, most of the cranioysnostotic conditions are characterized by wide clinical variability and genetic heterogeneity. Craniosynostosis can be classified into non syndromic forms-premature suture fusion is the only clinical alteration, and syndromic ones-premature cranial suture fusion associated with other anomalies. Apert, Pfeiffer, Crouzon and Saethre-Chotzen are the most common syndromic forms of craniosynostosis. The mutational mechanism is identified in approximately 58% of patients with Crouzon syndrome: FGFR2 mutations account for most of the cases (more than 80%) while mutations in TWIST1 and FGFR3 causes the remaining of them. Therefore, about 42% of the cases with clinical characteristics of Crouzon syndrome do not harbor mutations in any of these 3 genes and other mutational mechanisms might be responsible for the phenotype. Approximately 45% of Pfeiffer syndrome cases is not caused by mutations in the FGFR1 or FGFR2 genes, thus also suggesting genetic heterogeneity for this phenotype. On the other hand, all cases with Apert syndrome are caused by specific mutations in FGFR2. FGFR2 mutations show a partial genotype-phenotype correlation; however, severe cases, such as Pfeiffer types II and III are still very poorly characterized.

No abstract