Cette étude apporte un nouveau corpus à la description du paysage linguistique de Basse-Bretagne et participe de ce fait à une meilleure connaissance de la langue bretonne dans son ensemble. Les habitudes langagières constatées dans ce parler haut-vannetais seront décrites à partir d'exemples issus de langue parlée, et de langue chantée, collectés auprès de vingt-cinq locuteurs traditionnels originaires de Languidic, nés entre 1919 et 1950. Après avoir défini le terroir dialectal de ce breton, haut vannetais intérieur de transition, nous présentons son système vocalique, qui est un élément distinctif entre les parlers de ce sous-dialecte. Nous verrons quele timbre des voyelles peut être centralisé ou neutralisé selon leur quantité, qui est elle-même dépendante de celles des consonnes. Le système consonantique sera défini et exposé en tenant compte de la typologie du breton, étant une langue à mutations consonantiques. Le schéma accentuel, bien que principalement oxytonique, présente de nombreuses variations. Les mutations consonantiques des initiales s'organisent en trois principaux groupes, s'associant pour certains mutateurs, créant ainsi trois types de mutations hybrides, auxquels il faut ajouter trois mutations isolées. La morphologie et la syntaxe du parler sont exposées et tenant compte des différents usages, allant du registre familier quotidien, à la langue soutenue des chants, qui sont une des richesses de ce terroir, où les traditions orales ont su se maintenir jusqu'à nos jours. / This work presents a new study of work to the visual description of dialectology in the lower region of Brittany and participates in providing a better understanding of the Breton language in its entirety.The usages and customs of this micro-dialect from this part of Brittany known as haut-vannetais will be described from examples of spoken language and song. These are collected from twenty five traditional native speakers originally from Languidic, born between 1919 and 1950. After having defined the dialect area of this local speech, haut-vannetais in transition, we present its vocalic system, which in itself is a distinct element between different spoken sub dialects. We will see that the tone of vowels could be centralised or neutralised depending on their quantity. This also applies for the consonants. The consonantal system will be defined and exposed by considering the typology of Breton, with initial consonant mutations. The lexical stress, which is principally oxytonic presents numerous variations. There are three principal classes of mutation, three hybrid and three isolated mutations. The morphology and syntax of this sub dialect is exposed in consideration of different usages, ranging from familiar everyday language to received pronunciation used whilst singing, which give a richness to the region where the oral traditions have been retaineduntil today.

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MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Introduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.

Notre recherche intitulée « les cadres. Une population face aux mutations socioéconomiques dans un contexte postcolonial : le cas du Gabon (1970-2008) » retrace à travers les mutations sociales au Gabon et notamment par la colonisation, le «progrès » opérés sur les populations (durant des décennies de restructuration des strates sociales à la modernité intervenues dans cette partie de l’Afrique noire), l’institution scolaire et la rationalité étatique, une catégorie sociale porteuse d’une lisibilité de la société gabonaise. Cette étude tente de rendre manifeste les rapports de travail des cadres à travers les procédures de recrutement, la répartition des salaires, la mobilité sociale de cette catégorie socioprofessionnelle, leurs fonctions, les logiques de domination et de subordination d’un ordre social nouveau. Notre thèse rend compte de l’irruption de cette catégorie socioprofessionnelle dans le paysage politique et social au Gabon, de leur développement, de la manière dont les cadres se perçoivent, de la connaissance des relations qui sont à l’œuvre dans le travail, de l’influence de cette catégorie dans les rapports sociaux. Nous tentons de mettre en lumière une catégorie sociale non négligeable souvent délaissée au profit des classes ouvrières dont le positionnement et la trame qui se tissent, investissent différents champs sociaux. Par notre étude, nous essayons d’apporter un éclairage nouveau sur la sociologie du « cadre » au Gabon / My research entitled « the chief executives, a population face to socio-economic change in post-colony context: the case of Gabon (1970-2008) traces through the social changes in Gabon and in particular by colonization, the restructuring of social strata, modernity, the school and state rationality, a carrier of a social category readability of Gabonese society. This study makes clear labour relations executives through recruitment, social mobility, the logic of domination and subordination of one new social order. This thesis reports the emergence of leadership in the political and social landscape in Gabon, of development, of how managers perceive knowledge of the reports which expressed at work and in other social spheres. We also try to highlight a significant social group often neglected in favour of the working classes. In this study, we try to shed new light on the sociology of the chief executives of Gabon

Primary aldosteronism (PA) accounts for 5-10% of all hypertension. One of the major causes of PA is sporadic formation of aldosterone-producing adenomas (APAs). These benign tumours develop in the cortical region of adrenal glands and autonomously secrete excessive amounts of aldosterone. This hormone increases sodium retention and water reabsorption by the kidneys, leading to high blood pressure. Landmark discoveries of somatic mutations in APAs led to better understanding of molecular mechanisms causing autonomous aldosterone secretion. The first mutations were found in KCNJ5, followed by ATP1A1, ATP2B3 and CACNA1D, all encoding cation-channels or transporters. Several in vitro studies showed disruption of cellular ion-balance leading to the phenotype of hyper-aldosterone secretion from APAs. Following our lab's discovery of initial four somatic mutations by whole exome sequencing, over 30 single-base change mutations have been reported in the CACNA1D gene, which encodes the a1 subunit of an L-type Ca2+ channel (LTCC), CaV1.3. Initial and several subsequent mutations cause electrophysiological gain-of-function with increased activation and/or slowed inactivation of CaV1.3. Prior to the discovery of these mutations, L-type Ca2+ channels were not considered important in regulation of aldosterone production. In the first part of my thesis, I investigated two of the mutations and showed that the gain-of-function results in increased aldosterone secretion from an adrenocortical carcinoma cell line, H295R, when transiently transfected with the mutants. I also showed that CaV1.3 can play a role in physiological aldosterone secretion, finding that CYP11B2 expression is reduced by 50% in the adrenals of CaV1.3 knockout mice. The discovery of mutations in CACNA1D led to a drug discovery challenge award from a pharmaceutical company in which high-throughput screening of CaV1.3-expressing cells was undertaken against the company's 1.8M compound library. I identified the adrenal isoforms of the channel's alpha and beta subunits (CACNA1D and CACNB2), and helped development of the stable HEK293 cell line used for screening. This led to 3 tool compounds (A, B & C) that were selective antagonists for CaV1.3 over another family member of the ion channels in high-throughput electrophysiological experiments using IonWorks Barracuda and QPatch platforms. I showed compound B to effectively inhibit aldosterone secretion in both H295R and primary adrenal cells isolated from a normal adrenal. This finding is a significant step in developing compound B further into a CaV1.3-selective drug for treating PA patients without cardiovascular side effects as in the case of existing dihydropyridine class of Ca2+ channel blockers. The second part of my thesis focused on genotyping and whole exome sequencing of 59 APAs from 52 patients, in order to identify further genes underlying primary aldosteronism. Mutations in previously reported genes were identified in 34 of the APAs (57.6%). CACNA1D was the most commonly mutated gene (20.3%) in this cohort, but not KCNJ5 (16.9%) as previously reported. This variation in the frequencies observed is perhaps due to the different methods used for screening PA. For example, many of our patients were detected by renin measurement in resistant hypertension, and their APA identified by a unique PET-CT (using C11 metomidate), in place of adrenal vein sampling. In addition to this, novel somatic mutation was found in a gene not encoding an ion channel, however, this protein was previously linked to cell-cell adhesion and tumour suppression. The gene identified is CADM1, a cell adhesion molecule 1, and the mutation found leads to substitution of uncharged by negatively charged amino acid in the single transmembrane domain of this cell surface protein. The likely significance of this discovery was greatly enhanced when we ascertained that one of the 'private' somatic mutations found on whole exome sequencing of APAs in Munich was in fact a similar substitution in the adjacent amino acid of the membrane-spanning domain. High expression of CADM1 in zona glomerulosa (ZG) was found, the site of aldosterone synthesis in the adrenal cortex and in the APAs, as well as the aldosterone producing cell clusters (APCCs) within the ZG. In vitro experiments using H295R cells showed both mutations in CADM1 lead to 10-20 fold upregulation of CYP11B2 transcription, on qPCR, resulting in 2-4-fold increase of aldosterone secretion, compared to the wild-type CADM1. Despite the introduction of a negative charge into the transmembrane domain, both mutants could translocate to the cell surface. The evidence to date, points to the loss of cell-cell adhesion in the presence of mutant CADM1 as the cause of uncontrolled aldosterone synthesis. Silencing of CADM1 in H295R cells revealed downregulation of aldosterone synthesis and secretion. Transcriptome analysis by RNAseq, of H295R cells expressing wild-type or mutant CADM1 or silenced CADM1 showed a large number of differentially expressed genes. Mutant CADM1 upregulated genes involved in steroidogenesis and ACTH response pathways. A possible role of CADM1 was found to be in the regulation of inter-cell communication via gap junction protein, connexin-43 (Cx43). This was upregulated with higher expression on plasma membrane in the CADM1 silenced cells. TSG101, a protein involved in lysosomal degradation of Cx43 was downregulated in the absence of CADM1 and possibly the mechanism for increased Cx43 expression. Also, immunostaining of adrenal sections showed internalised para-nuclear staining localisation of Cx43 in the ZG, APAs and APCCs, regions with high CADM1 expression compared to membranous localisation of Cx43 in ZF. In contrast to the common and numerous mutations in CACNA1D, mutations in CADM1 are rare. Nonetheless, they may enhance our understanding of the functional significance of glomerular structure of the outer zone of adrenal cortex, where cell-cell adhesion and intercellular communication appear critical for the regulation of aldosterone secretion.

DNA methylation is a common epigenetic mark that affects gene regulation, genomic stability and chromatin structure. In mammals, methylation is mainly found in the CpG dinucleotides. The CpG methylation signals can be recognised by the Methyl-CpG-Binding Protein (MBP) family which includes MeCP2, MBD1, MBD2, MBD3, MBD4 and Kiaso. MeCP2 and MBD1-4 (except mammalian MBD3) recognise methyl-CpG via their MBD domain whereas Kaiso interprets methylation through its Zn finger DNA binding domain. The TRD domains of MeCP2, MBD1 and MBD2 have been reported to recruit transcriptional co-repressors to the methylated DNA. A thymine DNA glycosylase domain is located at the C-terminal region of MBD4. This study concerns the molecular details of the methyl-CpG recognition by the MBD domain of MeCP2. To achieve this, the MeCP2 MBD domain has been expressed, purified and co-crystallised with a 20 bp DNA fragment from the BDNF promoter. The DNA-protein cocrystal diffracted X-rays to a maximum resolution of 2.5Å using synchrotron sources. It belongs to space group C2 with unit cell dimensions: a = 79.71Å, b = 53.60Å, c = 65.73Å, and β = 132.1°. The X-ray structure of the MeCP2 MBD-DNA complex was solved using the SAD method. Structural analyses of the refined X-ray structure reveal that the methyl groups of the DNA make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. From a structure of the MBD domain in MBD1, established by NMR, the binding specificity of the MBD domain had been thought to depend on hydrophobic interactions between the cytosine methyl groups and a hydrophobic patch within the MBD domain. The findings of this study suggest that MeCP2 recognises the hydration pattern of the major groove of methylated DNA rather than cytosine methylation per se. The X-ray structure also identifies a unique role of T158 and R106, the sites of the two most frequent Rett missense mutations. Both residues stabilise the tandem Asx-ST motif at the C-terminal region of MBD domain. Disruption of this tandem motif destabilises the DNA-protein interaction. The BDNF sequence in this study contains an AT run which displays unique properties of AT tract DNA. Previously, mutation of the AT run has been reported to decrease MeCP2 binding specificity. This study however demonstrated that a significant reduction can only be observed when both AT runs close to the methyl-CpG have been mutated. The X-ray structure of the MeCP2 MBD-DNA complex in this study rationalises the effects of the most common Rett mutations and provides a general model for methylated DNA binding that is dependent on structured water molecules.

Background Somatic mutations accumulate in human cells throughout life. Some may have no adverse consequences, but some of them may lead to cancer. A cancer genome is typically unstable, and thus more mutations can accumulate in the DNA of cancer cells. An ongoing problem is to figure out which mutations are drivers - play a role in oncogenesis, and which are passengers - do not play a role. One way of addressing this question is through inspection of somatic mutations in DNA of cancer samples from a cohort of patients and detection of patterns that differentiate driver from passenger mutations. Results We propose QuaDMutEx an QuadMutNetEx, a method that incorporates three novel elements: a new gene set penalty that includes non-linear penalization of multiple mutations in putative sets of driver genes, an ability to adjust the method to handle slow- and fast-evolving tumors, and a computationally efficient method for finding gene sets that minimize the penalty, through a combination of heuristic Monte Carlo optimization and exact binary quadratic programming. QuaDMutNetEx is our proposed method that combines protein-protein interaction networks to the method elements of QuaDMutEx. In particular, QuaDMutEx incorporates three novel elements: a non-linear penalization of multiple mutations in putative sets of driver genes, an ability to adjust the method to handle slow- and fast-evolving tumors, and a computationally efficient method for finding gene sets that minimize the penalty. In the new method, we incorporated a new quadratic rewarding term that prefers gene solution set that is connected with respect to protein-protein interaction networks. Compared to existing methods, the proposed algorithm finds sets of putative driver genes that show higher coverage and lower excess coverage in eight sets of cancer samples coming from brain, ovarian, lung, and breast tumors. Conclusions Superior ability to improve on both coverage and excess coverage on different types of cancer shows that QuaDMutEx and QuaDMutNetEx are tools that should be part of a state-of-the-art toolbox in the driver gene discovery pipeline. It can detect genes harboring rare driver mutations that may be missed by existing methods.

Most human breast tumors have mutations in the growth factor/phosphatidylinositol 3’ kinase (PI3K) pathway. These can occur in genes coding for receptors, adaptor proteins, catalytic and regulatory subunits of PI3K, downstream kinases, or antagonistic tumor suppressors. While each genetic change results in elevated PI3K signaling, and all major breast cancer subtypes show pathway activation, the specific mutations involved in any one tumor may play an important role in defining tumor subtype, prognosis and sensitivity to therapy. Here, I describe mouse models of PI3K-induced breast cancer. First I generated mice that express Pik3ca cDNA under control of the ROSA26 promoter, in a Cre-dependent and therefore tissue specific way. I have generated four strains of knock-in mice: R26-Pik3cawt, R26-Pik3caE545K, R26-Pik3caH1047R, and R26-Pik3caE545K-H1047R, which can be induced to express wild type, helical domain, kinase domain and double mutant forms of mouse p110α, respectively. Mice expressing mutant Pi3kca develop mammary tumors, but the phenotypic spectrum for each mutation is unique. Indeed, many E545K mammary tumors are ii vascularized, whereas H1047R tumors are not. Using these models, I have compared downstream signaling properties of E545K and H1047R. The potential for improved breast cancer treatment lies in combination therapies that target more than one oncogenic pathway. To develop such treatments, we need good mouse models, and an understanding of the oncogenic network. To this end, my Pik3caH1047R model was mated to p53 and PTEN knockout mice, and to mice with active Notch1 signaling. In each case, genetic cooperation was observed and characterized. Oncogenic PI3K cooperated with p53-loss and active Notch1 to decrease survival and alter tumor phenotype in distinct ways. Loss of PTEN cooperated with oncogenic PI3K to alter tumor type, decrease average age at end point, and increase the number of tumors per mouse. Overall, I have shown that Pik3caE545K and Pik3caH1047R are sufficient to induce mammary tumors, and that tumor characteristics differ with these mutations, and with cooperating genetic changes.