Les Amélogenèses imparfaites constituent un groupe d’altération de l’émail dentaire d’origine génétique. Cette pathologie peut exister de manière isolée ou associée à d’autres symptômes dans le cadre de syndromes. Certains gènes impliqués sont déjà connus, cependant de nouvelles mutations et de nouveaux gènes restent à identifier. L’étude de familles informatives dans le cadre de ce projet de recherche sur le massif crânio-facial et bucco-dentaire, associée à des stratégies d’identification génétique telles que la sélection de gènes candidats, les zones d’homozygotie, le séquençage haut débit, ont permis d’obtenir des résultats probants. Des investigations futures passant par l’augmentation des cohortes, le développement des outils de séquençage de nouvelle génération, l’étude des modèles cellulaires et animaux permettront d'améliorer la compréhension de l’amélogenèse. / Amelogenesis imperfecta (AI) represents hereditary conditions affecting the quality and quantity of enamel. This disease can exist in isolation or in association with other symptoms in the form of syndromes. Several genes involved in AI are already known, however mutations in these genes are not sufficient to explain all cases of AI. This suggests that mutations in yet unidentified genes underlie AI. The study of informative families included in this research project on cranio-facial and oro-dental anomalies, by using genetic strategies such as candidate gene mutational analysis,homozygosity mapping and next generation sequencing, allowed the discovery of novel genes and mutations in AI. Future investigations based on the recruitment of new families, the development of new next generation sequencing tools and the establishment of cellular and animal models will improve our understanding of amelogenesis.

Les cellules épithéliales qui produisent l’émail, les améloblastes, sont séparées de l'émail au niveau de la zone de maturation par une membrane basale spécialisée (MBS) enrichie en laminine 332 (LM-332). Cette protéine hétérotrimérique (composée des chaînes α3, ß3 et γ2) assure l'intégrité structurelle des membranes basales (MB) et influence divers processus cellulaires épithéliaux tels que l'adhésion et la différenciation cellulaire. Des modèles de souris « knockout » (KO), où les gènes codant pour LM-332 ont été supprimés, meurent peu après la naissance. Néanmoins, ce phénotype létal peut être contourné en substituant chez la souris le gène produisant la chaîne γ2 de la laminine (LAMC2) par sa forme humaine, sous le contrôle de l’expression du promoteur-rtTA, de la cytokératine 14, inductible par la prise de doxycycline (Dox) - (Tet-on). Le but de ce projet est d’examiner si l’utilisation de cette protéine humaine chez la souris a un effet sur la structuration de la MBS ainsi que sur la maturation de l'émail. La phase de maturation de l’organe de l’émail chez la souris transgénique a été sévèrement altérée par rapport à une souris normale (WT). La MBS n’est plus visible, une matrice dystrophique s’est formée dans la couche d'émail dans la phase de maturation, et la présence d’une matrice résiduelle de l'émail est observée durant la phase tardive de maturation. Des micro-analyses tomographiques ont révélé une usure excessive des surfaces occlusales des molaires, un écroulement de l'émail sur les pointes des incisives et une hypominéralisation de l'émail. Cependant, aucune altération structurale due à cette recombinaison transgénique n’a été observée dans d'autres sites épithéliaux, tels que la peau, le palais et la langue. Ces résultats indiquent que, bien que ce modèle de souris humanisée soit capable de rétablir ses fonctions dans divers tissus épithéliaux, il est incapable de soutenir la structuration d'une MBS à l'interface entre les améloblastes et l’émail en maturation. Cet échec peut être lié à la composition spécifique de la MBS dans la phase de maturation et supporte l’hypothèse que la MBS est essentielle pour la maturation adéquate de l'émail. / The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-09-15T12:52:47Z No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:50:05Z (GMT) No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:50:10Z (GMT) No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Made available in DSpace on 2016-09-16T19:50:14Z (GMT). No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) Previous issue date: 2015-08-12 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Enamel formation is a two-step complex process by which proteins are first secreted to forming an extracellular matrix, followed by massive protein degradation simultaneously with the completion of mineralization. Excessive exposure to fluoride can disrupt this process and drive to a condition known as dental fluorosis. Recently it has been reported that genetic factors may influence the responses of mineralized tissues to fluoride, a phenomenon observed in A/J and 129P3/J mice strain. The present study aimed to map the protein profile of mouse enamel. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by two-dimensional electrophoresis and liquid chromatography, coupled to mass spectrometry. A total of 120 proteins were identified, from which 113 are well characterized, both experimentally and functionally. Seven proteins were classified as uncharacterized proteins. After functional enrichment, 9 proteins were significantly related to the terms "odontogenesis and tissue biomineralization”. Surprisingly, the COL1A1 and COL1A2 protein were found on secretory stage enamel of both strains. Protein interaction analysis showed interaction between the ENAM and other types of collagen. Another interesting finding was the possibility of uncharacterized sequence Q8BIS2 be an extracellular matrix protein involved on degradation of matrix proteins. These findings suggest that collagen is present on dental enamel and it seems to have a role in amelogensis. Furthermore, the existence of an new enzyme could be the key to elucidation of the mechanisms involved in the enamel biomineralization and the genetic susceptibility to dental fluorosis. / O desenvolvimento do esmalte dentário é um processo complexo no qual as proteínas da matriz do esmalte interagem para conduzir a deposição e o crescimento dos cristais de hidroxiapatita. Fatores como a exposição excessiva ao fluoreto podem atrapalhar esse processo, resultando em uma patologia conhecida como fluorose dentária. Recentemente tem sido relatado que fatores genéticos podem influenciar as respostas dos tecidos mineralizados ao fluoreto, fenômeno observado nas linhagens de camundongos A/J, tida como susceptível e 129P3/J, tida como resistente. Neste sentido, este estudo objetivou mapear as proteínas do esmalte de incisivos de camundongos A/J e 129P3/J, a fim de que se possa melhor entender os mecanismos moleculares envolvidos na patogênese da fluorose dentária. Para isso, amostras de matriz de esmalte foram obtidas de camundongos das linhagens A/J e 129P3/J e analisadas por eletroforese bidimensional e cromatografia liquida, associadas a espectrometria de massas. Foi identificada um total de 120 proteínas, das quais 113 são proteínas bem caracterizadas, tanto experimentalmente quanto funcionalmente, e 7 proteínas que foram classificadas como proteínas não caracterizadas. Após enriquecimento funcional utilizando ferramentas de bioinformática, verificou-se que 9 proteínas estavam significativamente relacionadas aos termos “odontogênese e biomineralização de tecidos”. Dentre essas, as proteínas COL1A1 e COL1A2 chamaram atenção, uma vez que o colágeno não é associado à amelogênese. As proteínas estavam presentes no esmalte em fase de secreção de ambas as linhagens, e, além disso, a análise de interação in silico mostrou a existência de interação física entre a enamelina e outros tipos de colágeno. Outro achado interessante foi a possibilidade da sequência não caracterizada Q8BIS2 ser uma proteína extracelular da matriz envolvida na degradação das proteínas de matriz. Em conclusão, os resultados levam a crer que o colágeno está presente no esmalte e é provável que tenha participação na amelogênese. Além disso, a existência de uma enzima ainda não caracterizada pode ser a chave para elucidação dos mecanismos envolvidos na biomineralização bem como na susceptibilidade genética à fluorose dentária. / 2008/03489-2 e 2009/53852-9

The proposed thesis concerns in the study of tribosphenic molar, the key apomorphy of mammalian clade, mainly in structure and development of its enamel coat. As the main model species served us European vespertilionid bat Myotis myotis. The aims of this thesis are: (1) to describe structure and microstructure of enamel in tribosphenic molars in detail; (2) to compare it with unicuspid teeth of the same dentition; (3) to describe mineralization process and enamel maturation in insectivorous dentition; (4) to describe late odontogenesis in molars of insectivorous dentition and its interlacing with enamel maturation, and how they come to occlusion.