Le contrôle combinatoire de l’ expression des gènes est une caractéristique importante du profil spatio-temporel de l'accumulation des flavonoïdes chez les plantes. Des résultats précédents avaient démontré chez Arabidopsis thaliana, que la régulation de l’accumulation des anthocyanes et des proanthocyanidines repose sur l'activité de facteurs de régulation de la transcription de type R2R3-MYB et bHLH qui forment des complexes ternaires ("MBW") avec la protéine TTG1 (WDR). L'objectif de la thèse était de caractériser les complexes MBW impliqués et leurs cibles, pour avoir une compréhension globale des mécanismes transcriptionnels qui contrôlent la biosynthèse des flavonoïdes.En utilisant différentes approches génétiques et moléculaires, nous avons montré que seuls les gènes « tardifs » (c’est à dire DFR, LDOX, BAN, TT19, TT12 et AHA10) sont des cibles directes des complexes MBW. Bien que le complexe de régulation impliquant les protéines TT2-TT8-TTG1 ait un rôle majeur dans la régulation de ces gènes structuraux dans la graine d’Arabidopsis, trois autres complexes contenant MYB5, GL3 ou EGL3 sont également impliqués de façon tissu-spécifique. Comme l’expression du gène TT8 joue un rôle clef dans ces régulations, une dissection fonctionnelle de son promoteur a été entreprise. Elle a montré la nature modulaire de ce promoteur avec deux domaines impliqués dans l’expression tissu-spécifique et un troisième dans la force du promoteur. Les résultats obtenus suggèrent également l’existence d’autres régulateurs qui restent à caractériser. Enfin, nous avons développé une nouvelle technique de caractérisation des interactions entre les facteurs de transcription et les promoteurs, basée sur l’expression transitoire dans des protoplastes de mousse (i.e. Physcomitrella patens). Nous avons ainsi pu identifier les éléments cis des promoteurs impliqués dans la régulation de l’expression de TT8 et de chacun des promoteurs cibles des complexes MBW.L’ensemble de ces travaux permet de fournir un modèle plus complet du réseau de régulations transcriptionnelles qui contrôle la biosynthèse des proanthocyanidines et des anthocyanes, ainsi que des outils et de nouvelles pistes pour poursuivre ces études chez Arabidopsis et d’autres plantes. / The combinatorial control of gene expression is a key feature of the spatio-temporal pattern of flavonoid accumulation in plants. Previous results have shown that the regulation of anthocyanins and proanthocyanidins (PAs or tannins) pigmentation relies on the transcriptional activity of R2R3-MYB and bHLH proteins that form “MBW” ternary complexes with TTG1 (WD-Repeats), in Arabidopsis thaliana. The purpose of the thesis was to figure out the nature and spatio-temporal activity of these MBW complexes and to identify their direct targets, which were essential steps toward a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. Using different molecular and genetic approaches this thesis has demonstrated that only late biosynthetic genes (namely DFR, LDOX, BAN, TT19, TT12 and AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex was shown to play the major role in regulating the expression of these structural genes in developing seeds, three additional MBW complexes that contain MYB5, GL3 or EGL3 are also involved, in a tissue-specific manner. Because the expression of TT8 is largely involved in these regulations, a functional dissection of its promoter was carried out. Two modules drive the tissue-specific activity of the TT8 promoter in PA- and anthocyanin-accumulating cells, and a third module is responsible for the strength of the promoter. Interestingly, this regulation involves at least six different MBW complexes. Our results also suggest that some putative new regulators remain to be discovered. Last, use of a newly developed fast and sensitive transient expression system that relies on protoplasts of the moss Physcomitrella patens has allowed the identification of both, specific cis-regulatory elements through which TT8 expression is regulated and the minimal promoter for each of the genes that are targeted by the MBW complexes.Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, we have provided a new and comprehensive view of the regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis, as well as new clues and tools for further investigation of this pathway in Arabidopsis and other plant species.

The Multiple Breath Washout (MBW) test is increasingly being recognised as a sensitive method of detecting early small airways lung disease. Indices of MBW include lung clearance index (LCI), Scond and Sacin. Factors that affect MBW variability have not been fully established. This thesis presents five studies which examine MBW repeatability in children with and without cystic fibrosis (CF) or asthma. MBW was performed using 0.2% sulphur hexafluoride and the modified Innocor (Innovision). Testing was performed at the Clinical Research Facility of the Royal Hospital for Sick Children in Edinburgh. (1) MBW and spirometry were performed in children with and without CF (n=20 in each group), initially while sitting and then 30 minutes after assuming a supine posture. LCI was found to significantly rise on lying supine in healthy children (p < 0.01) and children with CF (p=0.03). (2) Thirty two children with CF performed MBW and spirometry on four study visits, results were correlated with findings from high resolution chest computed tomography scans taken on the first visit. LCI showed the strongest correlation with extent and severity of bronchiectasis (r=0.66, p < 0.01 and r=0.69, p < 0.01 respectively). Variability of LCI was similar to FEV1 over the 4 visits. (3) MBW and spirometry of 66 healthy children were compared to 63 children with stable asthma; lung function of asthmatic children was related to symptoms and medication use. LCI was higher in the asthmatic group (6.7 vs 6.3, p < 0.01); within the asthmatic group LCI was significantly higher if asthma was less well controlled (p=0.02). (4) Children with and without asthma (n=21 in each group) performed MBW and spirometry before and after exercise and again after salbutamol, symptom data was collected from asthmatic children. Baseline LCI was abnormal in the asthmatic group who had severe exercise induced bronchospasm during testing. (5) Asthmatic children admitted to hospital due to exacerbation performed MBW and spirometry. Mean (SD) LCI was abnormally high at 8.5 (1.7) in the nine patients recruited and returned to normal 6.7 (0.6) in three patients who attended follow up. I have presented evidence that LCI is repeatable and sensitive to early disease in CF and asthma. I have described for the first time the effects of exercise and exacerbation on MBW indices in asthmatic children. MBW is potentially a very useful tool in paediatrics; standardisation of testing and equipment may enable clinical use.