Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.

Molecular defects in the p63 gene give rise to severe physiological abnormalities in patients with ectodermal dysplasia, however the mechanisms by which p63 mutations disrupt p63 function are unknown. In this study we examined four ΔNp63α mutants; Ectrodactyly-Ectodermal Dysplasia with Clefting (EEC) R204W, R304W and Ankyloblepharon-Ectodermal Dysplasia with Clefting (AEC) mutants, L514F and G530V, and characterized DNA binding, transcription factor activity, oligomerization with wild-type p63 and changes in protein stability/nuclear localization. We also investigated the putative OD-SAM interaction in p63 and p73. We demonstrated that both the EEC and AEC mutants cannot transcriptionally activate the PERP promoter and can hetero-oligomerize forming dominant negative complexes with wild-type p63. We show that both EEC mutants and AEC L514F mutants are more stable which is not due to aberrant degradation by the E3 ligase Itch. Finally, we discovered that a novel interaction between the p73 OD and SAM domain is absent in p63.

Thesis--University of Oklahoma. / Includes bibliographical references.

La dysplasie ectodermique hypohidrotique liée à l’X (DEX) est consécutive à la mutation du gène Eda. Ce projet de recherche avait pour but d’étudier les dysmorphoses cranio-faciales chez le mutant murin Tabby représentant l’équivalent phénotypique de la DEX. La forme des mandibules en vue latérale a été étudiée à partir d’approches quantitatives associant μ-CT, traitement d’images, analyses de Fourier elliptique et analyses métriques. Une première étude ex vivo menée sur 39 spécimens Tabby et 35 souris wild-type adultes a permis de mettre en évidence un hypo-développement mandibulaire chez Tabby. Deuxièmement, une étude longitudinale in vivo de la croissance mandibulaire mise en place sur une cohorte de 23 individus (12 WT et 11 Tabby) a montré que les individus Tabby présentaient cet hypodéveloppement dès le 1er mois et qu’il se maintenait à la fin de la première année de vie. Les défauts du gène Eda affectent ainsi le développement de la mandibule en plus des dérivés ectodermiques. / The X-linked hypohidrotic ectodermal dysplasia (XLHED) is the result of Eda gene defect. This research project studied the cranio-facial dysmorphoses in Tabby murin mutant which having a similar phenotype to the XLHED. A association of mutiple quantitive approachs (μ-CT, images processing, elliptical Fourier analyse and metric analyses) permitted to study the mandible’s shape in a lateral view. A first ex vivo study led on 39 specimens Tabby and 35 WT mice allowed to highlight a mandibular hypodevelopment in Tabby. Secondly, a longitudinal in vivo study of mandibular growth, based on 23 specimen (12WT and 11 Tabby), showed that Tabby presented this hypodevelopment from the 1st month and that it remained at the end of the first year of life. The Eda gene affects the development of mandible and ectodermal structures.

O sistema NADPH oxidase é um complexo enzimático gerador de superóxido. O NF-kB é um fator de transcrição envolvido no controle da expressão de diversos genes ligados à resposta inflamatória. Defeitos no eixo IL-12/23-IFN-g resultam em infecções recorrentes e à susceptibilidade mendeliana a micobacterioses, podendo diminuir a expressão do componente gp91-phox da NADPH oxidase. Estudamos qual é a relação direta do NF-kB e de defeitos no eixo IL-12/23-IFN-g na regulação dos genes CYBA, NCF1, NCF2 e NCF4 do sistema NADPH oxidase humano em células U937, células B EBV transformadas provenientes de pacientes com EDA-ID, DGC, ou de pacientes com defeitos no eixo IL-12/23-IFN-g. A expressão dos genes NCF1 e NCF2 foi diminuída em células com defeitos no eixo (IFNGR1 e INFGR2) e em células U937 IkB S32A/S36A. A expressão do gene NCF1 também foi diminuída em células EDA-ID S32I e em células EDA-ID NEMO/IKKg W420X. O NF-kB e os IFNGR1 e INFGR2 são necessários para a expressão dos genes NCF1 e NCF2 e para a ativação do sistema NADPH oxidase humano neste sistema modelo. / The NADPH oxidase system is an enzymatic complex that generates superoxide. The NF-kB is a transcriptional factor involved in the expression of several genes related to the inflammatory response. The IL-12/23-IFN-g axis defects lead to recurrent infections and to the mendelian susceptibility of mycobacterial disease (MSMD), and they can decrease the gp91-phox expression (a NADPH oxidase component). We studied the NF-kB and the IL-12/23-IFN-g axis defects consequences on the regulation of CYBA, NCF1, NCF2 and NCF4 genes of the human NADPH oxidase system in U937 cells, and in B EBV cells from patients with EDA-ID, DGC, or patients with IL-12/23-IFN-g axis defects. The NCF1 and NCF2 gene expression was decreased in IL-12/23-IFN-g axis defects cells (IFNGR1 and INFGR2) and in U937 IkB S32A/S36A cells. NCF1 gene expression was decreased in EDA-ID S32I and in EDA-ID NEMO/IKKg W420X cell lineages. The NF-kB and the IFNGR1 and INFGR2 are necessary for NCF1 and NCF2 gene expression and activation of the human NADPH oxidase in this model system.

La Dysplasie Ectodermique Hypohidrotique liée à l'X (DEX) est une maladie génétique liée à une mutation du gène EDA. Le phénotype exprimé par le modèle murin Tabby est l'équivalent de celui observé dans l'espèce humaine et présente des anomalies dentaires, cranio-faciales, vertébrales et des défauts de trabéculation osseuse. Dans ce contexte, une cartographie de ces anomalies chez le mutant Tabby était nécessaire et l'analyse de l’impact de la mutation Eda/Ta sur la croissance du squelette crânien et post-crânien a été étudiée. Un suivi longitudinal d'une cohorte d'individus murin Tabby (5 mâles hémizygotes EdaTa/Y, 6 femelles hétérozygotes EdaTa/+) et sauvages (n=12) a été réalisé à partir d’une succession d’acquisitions TDM pendant plus de 2 ans. L'observation des profils de croissance et de leurs paramètres a montré des anomalies de croissance du complexe crânio-facial, de la base du crâne (hypo-développement crânien) et un déficit de croissance relatif des os longs (fémur et humérus) chez les souris hémizygotes EdaTa/Y. Ces résultats mettent pour la première fois en évidence des anomalies de développement des os longs et confirment le rôle d’EDA-A dans la formation normale du squelette. Ces données constituent un pré-requis essentiel pour tester l’efficacité de tentatives de réversion phénotypique à partir de protéines recombinantes. / X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is a genetic disorder due to a mutation of the EDA gene. The phenotype expressed by Tabby mice, murine model of XLHED, is equivalent to that observed in humans including dental anomalies, craniofacial and vertebral trabecular bone defects. In this context, a mapping of these anomalies in Tabby mice was necessary and the impact of the EdaTa mutation on cranial and post -cranial skeletal growth was studied. A 2 years (112 weeks) μCT follow-up of Tabby mice (5 hemizygous males EdaTa/Y, 6 heterozygous females EdaTa/+) and Wild Type group (n = 12) hasbeen performed. The observation of growth patterns and parameters showed a relative cranial hypodevelopment, abnormal growth of the craniofacial complex and a relative hypo-development of appendicular skeleton (femur and humerus) in Tabby mice. These results allowed for the first time to highlight appendicular developmental abnormalities, confirming the role of EDA-A in the normal formation of the skeleton. While enriching the phenotypic picture of this syndrome, in a therapeuticperspective, all of these data are an essential prerequisite to test the effectiveness of attempts to phenotypic reversion from recombinant proteins.

O sistema NADPH oxidase é um complexo enzimático gerador de superóxido. O NF-kB é um fator de transcrição envolvido no controle da expressão de diversos genes ligados à resposta inflamatória. Defeitos no eixo IL-12/23-IFN-g resultam em infecções recorrentes e à susceptibilidade mendeliana a micobacterioses, podendo diminuir a expressão do componente gp91-phox da NADPH oxidase. Estudamos qual é a relação direta do NF-kB e de defeitos no eixo IL-12/23-IFN-g na regulação dos genes CYBA, NCF1, NCF2 e NCF4 do sistema NADPH oxidase humano em células U937, células B EBV transformadas provenientes de pacientes com EDA-ID, DGC, ou de pacientes com defeitos no eixo IL-12/23-IFN-g. A expressão dos genes NCF1 e NCF2 foi diminuída em células com defeitos no eixo (IFNGR1 e INFGR2) e em células U937 IkB S32A/S36A. A expressão do gene NCF1 também foi diminuída em células EDA-ID S32I e em células EDA-ID NEMO/IKKg W420X. O NF-kB e os IFNGR1 e INFGR2 são necessários para a expressão dos genes NCF1 e NCF2 e para a ativação do sistema NADPH oxidase humano neste sistema modelo. / The NADPH oxidase system is an enzymatic complex that generates superoxide. The NF-kB is a transcriptional factor involved in the expression of several genes related to the inflammatory response. The IL-12/23-IFN-g axis defects lead to recurrent infections and to the mendelian susceptibility of mycobacterial disease (MSMD), and they can decrease the gp91-phox expression (a NADPH oxidase component). We studied the NF-kB and the IL-12/23-IFN-g axis defects consequences on the regulation of CYBA, NCF1, NCF2 and NCF4 genes of the human NADPH oxidase system in U937 cells, and in B EBV cells from patients with EDA-ID, DGC, or patients with IL-12/23-IFN-g axis defects. The NCF1 and NCF2 gene expression was decreased in IL-12/23-IFN-g axis defects cells (IFNGR1 and INFGR2) and in U937 IkB S32A/S36A cells. NCF1 gene expression was decreased in EDA-ID S32I and in EDA-ID NEMO/IKKg W420X cell lineages. The NF-kB and the IFNGR1 and INFGR2 are necessary for NCF1 and NCF2 gene expression and activation of the human NADPH oxidase in this model system.

Doctor Scientiae (Odontology) - DSc(Odont) / Investigations of pathoses of the oral cavity encompass a relatively wide spectrum of diseases, abnormalities, tumours and tumour-like conditions affecting and occurring in the dental hard tissues and supportive structures, the bony skeleton of the face and the soft tissues of the. mouth. It involves a study of the normal - oral biology - and the abnormal - oral pathology. Oral pathology is a relatively new specialized field of dental science and practice. In South Africa, prior to the nineteen-fifties, research in oral pathology was primarily directed towards dental disease. Two people - Julius Staz of the University of the Witwatersrand and Tony Ockerse of the University of Pretoria - were the doyens in this field and made major contributions to dental science. Staz reported on the status of dental caries and tumorous malformations of teeth and Ockerse on the prevalence and severity of fluorosis in South Africa. During the fifties a second generation of dental surgeons, who were interested in soft tissue, bone and tumour pathology, emerged. They ,were Bertie Cohen, George Baikie, Mervyn Shear and John Lemmer who, at that time, were all from the University of the Witwatersrand. Bertie Cohen later joined the Royal College of Surgeons of England. Mervyn Shear led the field with his research on cysts of the oral cavity. The practice of oral pathology, moulded on anatomical pathology, was established in the early sixties and Mervyn Shear and the author, from the University of Pretoria, became known as oral pathologists. Research at that early stage comprised clinical and histological observations of oral lesions, diseases, tumours and tumour-like conditions. Observation techniques became more sophisticated during the sixties and seventies with the advent of histochemistry and electronmicroscopy. The next major development which blossomed in the seventies and early eighties was the application of epidemiological methods in the study of disease. Epidemiological principles enabled the correct recording of profiles of oral pathoses in the community. Much was learnt about the prevalence and distribution of oral conditions. The application and use of experimental models, especially laboratory animals, became popular in the eighties. Amongst others, a germfree animal unit was established in the Faculty of Dentistry of the University of Stellenbosch enabling workers to study the microbiological aetiology of dental and oral disease. Morphological observations of tumours and mucosal lesions were further enhanced during this period with the development of immunocytochemistry Experimental cell studies by means of cell culture techniques, commenced late in the eighties and was established in the early nineties. These models fostered molecular biology techniques which have become useful tools for the investigation of the aetiology of disease at a cellular and molecular level. At present molecular techniques are also popular in other spheres of oral pathology such as microbiological, immunological and oncological research. The author's first contact with oral pathology as a subject, forming an important and interesting part of dentistry, was the prescribed textbook "Oral and Dental Diseases", 2nd ed., 1951., by HH Stone of the University of Liverpool in the United Kingdom. Subsequently an enduring interest in the subject and research was cultivated by three teachers and colleagues, Ivor Kramer, Robert Bradlow and Mervyn Shear. Ivor Kramer, Professor of Oral Pathology in the Eastman Dental Institute of the University of London was a superb postgraduate teacher of oral pathology, and revelled in research. The Dean of the Institute, Professor Sir Robert Bradlow was a clinician and splendid diagnostician. He correlated the clinical and histopathological features of oral diseases. These two teachers set the course in oral pathology for the author during his postgraduate studies. In the sixties, after a spell at the University of Pretoria, the author joined Professor Mervyn Shear at the University of Witwatersrand. It was here that the author could further his skills of presenting lectures and research papers in an orderely manner and strengthen his love of research. The research carried out by the author reflects to a large extent the development of research in oral pathology in South Africa since 1960.. It includes studies of diseases and lesions of the oral mucosa, the dental hard tissues, tumours of the oral cavity and jaws and forensic odonto-stomatology. To date 139 articles have been published and accepted in scientific journals of which I was the first or co-author. The research presented here, however, comprises only those studies related to pathoses of the oral mucosa as it occurs in South Africa. Fifty-four papers and two abstracts are submitted. The papers are grouped into two divisions which include studies on (I) normal human oral and ectocervical mucosa and (II), those related to pathoses of the oral mucosa. The latter is subdivided into sections on: the profile of lesions of the oral mucosa in the community; cytological, clinical and morphological features of lesions of the oral mucosa; and studies on the aetiology of lesions of the oral mucosa. Each division and section is preceded by a declaration as to the contribution of the author or co-authors and a précis of the aims, objects and research findings. In the introduction of the précis statements are made explaining the aims of the study. These statements are not referenced because they appear in the respective articles.