Dissertations / Theses on the topic 'SoxB'

Chez les mammifères, le développement testiculaire des gonades XY est initié par les facteurs de transcription SRY/SOX9 qui promeuvent la différenciation des cellules de Sertoli. Chez l’embryon XX, la signalisation RSPO1/WNT/beta-catenin contrôle la différenciation des cellules de la granulosa et le développement ovarien. De fait, les souris XY n’exprimant pas Sox9 (KO) développent des ovaires et les souris XX n’exprimant pas Rspo1(KO) développent des ovotestis, constitués d’une partie testiculaire et une ovarienne. Leur formation est due à la différenciation précoce de cellules de granulosa et la reprogrammation d’une partie d’entre elles, en cellules de Sertoli. Chez les souris XX Rspo1 KO, SRY n’est pas nécessaire au développement testiculaire.De plus, les gonades des souris XX et XY présentant une double inactivation des gènes Rspo1 et Sox9 (Double Knockout/DKO) montrent une différenciation testiculaire partielle et complète respectivement, avec un développement d’ovotestis chez les individus XX DKO et un développement de testicules hypoplasiques chez les souris XY DKO. SOX9 et/ou SRY ne sont donc pas nécessaires à la différenciation testiculaire dans ce contexte, suggérant l’implication d’autres facteurs.L’objectif de ma thèse est de tester l’hypothèse selon laquelle SOX8, un facteur de transcription de la même famille que SOX9, pourrait induire le développement testiculaire chez les souris XX et XY DKO Rspo1 Sox9. Afin d’établir l’existence d’une compensation entre ces gènes SOX, nous avons analysé leur expression et le développement des gonades chez la souris DKO pour les gènes Rspo1 et Sox8 ou Sox9. Nous avons ensuite étudié les souris mutantes simultanément pour les gènes Rspo1, Sox8 et Sox9 (triple knockout/TKO). Notre hypothèse est qu’une perte d’expression des gènes Sox8 et Sox9 chez les souris TKO empêche la reprogrammation des cellules de la granulosa en Sertoli et par conséquent le développement testiculaire. Nous avons donc analysé la morphologie des gonades, les caractères sexuels secondaires, ainsi que l’organisation des gonades avec les différentes populations cellulaires qui les constituent par histologie et immuno-marquages à différents stades : à 17.5 jours de développement embryonnaire (E17.5) où la reprogrammation de cellules de granulosa en Sertoli commence dans la souris XX Rspo1 KO; chez les souris juvéniles au jour 10 (P10) où le développement somatique est achevé; et chez les souris adultes 40 jours après la naissance (P40).Nos résultats montrent que SOX8 et SOX9 sont exprimés de manière indépendante dans les gonades des souris XY and XX DKO Rspo1 Sox9 et DKO Rspo1 Sox8 à E17.5 et à P10. De plus, les souris XY et XX DKO Rspo1 Sox8 développent des testicules et des ovotestis indiquant que la perte d’un seul facteur SOX n’altère pas la formation des testicules, comme dans les souris XY et XX DKO Rspo1 Sox9. Cependant, chez les souris XX et XY TKO, la reprogrammation des granulosa en Sertoli à E17.5 et le développement testiculaire postnatal ne sont plus observés, démontrant que SOX8 peut compenser la perte de SOX9. De plus, les gonades des souris XY et XX TKO sont des ovaires atrophiques, indiquant que la différenciation ovarienne peut s’opérer.En résumé, nous avons analysé l’étiologie du développement physiopathologique des gonades chez les souris ayant une perte de fonction de RSPO1. Bien que SOX8 ne soit pas nécessaire à la différenciation testiculaire chez la souris, il peut promouvoir le développement testiculaire en l’absence de SRY et SOX9 en raison de sa redondance fonctionnelle avec SOX9. Chez l’Homme, dans les cas cliniques d’ambiguïtés sexuelles avec différenciation testiculaire, qui ne sont pas expliqués par le défaut d’expression de SRY ou SOX9, SOX8 pourrait ainsi être un facteur causatif
In humans and mice, testicular development in XY gonads involves SRY/SOX9 signaling to promote Sertoli cell differentiation and their formation as testis chords. For ovarian development in XX gonads, RSPO1/WNT/beta-catenin signaling is the main pathway for granulosa cell differentiation and their subsequent assembly into follicles. Indeed, XY Sox9 mutant mice develop ovaries, and XX Rspo1 mutant mice develop ovo-testes, a gonad containing a testicular and an ovarian part. In XX Rspo1 mutant mice, ovo-testicular development involves precocious differentiation of some granulosa cells and their and reprogramming as Sertoli cells. Thus, these single mutant studies demonstrated that SOX9 and RSPO1 are required for testicular and ovarian development respectively, and that SRY is dispensable for testicular development in XX Rspo1 mice. Interestingly, gonad development in XY and XX Rspo1 Sox9 double knockout (DKO) mice has challenged the requirement of SOX9 for testicular development. In XX Rspo1 single mutants, it was assumed that Sertoli cell differentiation was SOX9-dependent, but co-inactivation of Sox9 in DKO mice does not impair the ovo-testicular phenotype. For XY Sox9 single mutant mice developing ovaries, co-inactivation of Rspo1 in XY DKO mice rescues the sex reversal, though the testes are hypo-plastic. Thus, in XY and XX Rspo1 Sox9 DKO mice, SOX9 and/or SRY are dispensable for testicular differentiation, indicating that an alternate testis factor exists. For my research project, we hypothesized that a SOX9-related transcription factor, SOX8, acts redundantly for testicular development in XY and XX Rspo1 Sox9 DKO mice. Thus, to first establish redundancy among the SOX factors, we first analyzed their expression in Rspo1 mutant mice lacking Sox8 or Sox9, and then generated and analyzed gonad development in XY and XX Rspo1 Sox8 DKO mice. Then to test our hypothesis, we studied Rspo1 Sox8 Sox9 triple knockout (TKO) mice. We predicted that a loss of both Sox genes in TKO mice would prevent granulosa cell reprogramming as Sertoli cells and subsequent testicular development. To characterize gonad development and their effects in DKO and TKO mice, we performed analyses in embryonic day 17.5 (E17.5) mice, when granulosa-to-Sertoli cell reprogramming begins in XX Rspo1 single mutants; in juvenile post-natal day 10 (P10) mice, when gonad fate is set; and in young adult P40 mice. We examined a variety of parameters including gonad morphology and secondary sex characteristics, as well as gonad organization and cell population by histological and immunostaining analyses. We report that SOX8 and SOX9 are expressed independently in XY and XX Rspo1 Sox9 DKO and Rspo1 Sox8 DKO gonads in embryonic and juvenile mice. Next, XY and XX Rspo1 Sox8 DKO mice developed testes and ovo-testes, indicating that loss of one SOX factor does not impair testicular differentiation, as in XY and XX Rspo1 Sox9 DKO mice. In XY and XX Rspo1 Sox8 Sox9 TKO mice, granulosa-to-Sertoli cell reprogramming was impaired at E17.5 and post-natal gonads lacked testicular development. Thus, SOX8 can compensate for the loss of SOX9 in Rspo1 Sox9 DKO mice. In addition, gonads in XY and XX TKO mice developed as atrophied ovaries, indicating that ovarian fate is partially maintained.In total, we investigated the etiology of pathophysiological testicular development in RSPO1 loss-of-function mice. Remarkably, though SOX8 is dispensable for male sex determination in mice, it can promote testicular differentiation in the absence of SRY and SOX9 because of functional redundancy with SOX9. Thus, in human cases of sex reversal where testicular development cannot be explained by misexpression of SRY or SOX9, SOX8 could be a causative factor

SOX9 est un facteur de transcription, appartenant à la famille des protéines à domaine HMG, et connu pour réguler la transcription grâce à la liaison de ce domaine à l'ADN. Au laboratoire, il a été montré que SOX9 possédait des propriétés anti-oncogéniques, cependant, de façon paradoxale, SOX9 est surexprimé dans les tumeurs colorectales. Nous avons mis en évidence l'existence d'un nouveau variant d'épissage de SOX9, MiniSOX9, qui possède un effet dominant négatif vis-à-vis de l'activité transcriptionnelle de SOX9. MiniSOX9 est fortement exprimé dans les tumeurs en comparaison avec le tissu sain adjacent à la tumeur. Nous avons émis l'hypothèse que MiniSOX9 pourrait donc avoir, dans les tumeurs, un effet antagoniste à SOX9 et s'opposer à ses propriétés anti-oncogéniques. Grâce à la mise en place de modèles cellulaires tumoraux de surexpression de SOX9 et MiniSOX9, inductibles à la doxycycline, nous avons mis en évidence que SOX9 réduit la prolifération, la migration et l'invasion cellulaire. De manière surprenante, MiniSOX9 ne possède aucun effet sur la prolifération cellulaire, suggérant que les effets de SOX9 pourraient être dus à son activité transcriptionnelle. En revanche, SOX9 ainsi que MiniSOX9 réduisent la capacité clonale des cellules et leur capacité à former des tumorosphères. Dans ce cas, il serait probable que SOX9 et MiniSOX9 modulent l'activité de protéines partenaires
SOX9 is an HMG transcription factor known to regulate transcription by binding of its HMG domain to DNA. We previously demonstrated that SOX9 has anti-oncogenic-properties but SOX9 is overexpressed in colon tumors when compared to adjacent healthy tissu. This overexpression appears paradoxical, unless its anti-oncogenic activity cannot be exert. In this study, we report the discovery of MiniSOX9, a new SOX9 splice variant, which is highly expressed in colon tumors. MiniSOX9 acts as a SOX9 dominant negative isoform. Our hypothesis was that MiniSOX9 antagonizes the SOX9 anti-oncogenic activity in tumors.Using tumors cells lines inducible for SOX9 and MiniSOX9 overexpression, we showed that SOX9 reduces cell proliferation, migration and invasion. Surprisingly, MiniSOX9 has no effect on cell proliferation, suggesting that SOX9 effects could be du to his transcriptionnal activity. However, SOX9 and MiniSOX9 decrease cell clonal ability and tumorosphere formation. In this case, it is likely that SOX9 and MiniSOX9 modulate the activity of proteins partners

L'ovaire représente à la fois un organe cible et le principal organe producteur d'estrogènes et de progestérone qui maintiennent le développement des caractères sexuels féminins et une fonction de reproduction normale. Cette production hormonale est contrôlée par les gonadotropines FSH et LH produites dans l'hypophyse, responsables dans l'ovaire de la croissance folliculaire et de l'ovulation, respectivement. Mon travail de thèse a identifié la signalisation prostaglandine D2 (PGD2), comme un nouvel élément-clé dans la signalisation des gonadotropines, contribuant à l'activation de l'expression des récepteurs FshR et LhR et des enzymes de la stéroïdogenèse SCC et StAR. La PGD2, produite dans plusieurs tissus par deux enzymes de synthèse, les prostaglandines synthases H et L-PGDS, est impliquée dans de nombreuses fonctions physiologiques et pathologiques. Comme dans l'ovaire pathologique, nous avons montré que la PGD2 avait aussi un rôle anti-prolifératif dans la cellule de granulosa de l'ovaire normal. Le cancer de l'ovaire représente la 4ème cause de mortalité par cancer chez la femme. Les mécanismes moléculaires impliqués dans le développement de ces tumeurs sont encore peu connus, bien que l'implication des estrogènes et de la Prostaglandine E2 (PGE2) dans la progression des tumeurs ovariennes épithéliales soit bien établie. D'autre part, les ovaires des souris invalidées pour les gènes codant les récepteurs aux estrogènes ou l'aromatase, possèdent des structures tubulaires contenant des cellules de Leydig et des cellules de Sertoli re-différenciées exprimant le facteur de détermination sexuelle mâle SOX9, alors qu'il n'est pas exprimé dans l'ovaire sain. Mon travail a montré que les estrogènes inhibent la transcription des gènes Sox9 et L-Pgds dans les lignées ovariennes tumorales BG1 et COV434 et que cette régulation est la résultante d'une inhibition, via le récepteur ERa et d'une activation via le récepteur ERß. Ces résultats sont en accord avec les études sur les effets prolifératifs d'ERa et le rôle anti-prolifératif d'ERß et suggèrent donc un rôle anti-prolifératif de la PGD2 dans l'ovaire tumoral et une régulation négative directe ou indirecte de l'expression de Sox9 et des Pgds par les estrogènes
The prostaglandin D2 (PGD2) pathway is involved in numerous biological processes and while it has been identified as a partner of the embryonic sex determining male cascade, the roles it plays in ovarian function remain largely unknown. PGD2 is secreted by two prostaglandin D synthases (Pgds); the male-specific lipocalin (L)-Pgds and the hematopoietic (H)-Pgds. Here, we report the localization of H-Pgds mRNA in the granulosa cells from the primary to pre-ovulatory follicles. We used adult female mice treated with HQL-79, a specific inhibitor of H-Pgds enzymatic activity, to provide evidence of an interaction between H-Pgds-produced PGD2 signaling and FSH signaling. This leads to the activation of steroidogenic Scc and StAR gene expression through increased FshR and LhR receptor expression leading to progesterone secretion. We also identify a role whereby H-Pgds-produced PGD2 is involved in the regulation of follicular growth through inhibition of granulosa cell proliferation in the growing follicles. Indeed, we report an altered H-Pgds expression in human ovarian tumors alongside a partial or complete absence of H-Pgds protein in granulosa cell tumors, suggesting a potential association between decreased levels of H-Pgds expression and a tumoral phenotype. Together, these results show PGD2 signaling to be essential for FSH action within granulosa cells, thus identifying an important and unappreciated role for PGD2 signaling in controlling the balance of proliferation, differentiation and steroidogenic activity of these cells

La transició Epiteli-Mesènquima es dóna durant el desenvolupament embrionari i en els estadis tardans de la progressió tumoral permetent que es produeixi la metàstasi. Aquestes transicions necessiten una repressió de l'E-Cadherina i es pot reproduir en cèl·lules en cultiu amb l'expressió ectòpica de Snail1, un repressor de l'E-Cadherina. Durant la transició produïda per Snail es produeix la ràpida activació de gens mesenquimals com Fibronectina i LEF1. L'expressió forçada d'E-Cadherina fa disminuir els nivells de RNA de Fibronectina i LEF1, indicant que en l'activació d'aquests dos gens està implicat un cofactor sensible a l'E-Cadherina. En concordança, la transcripció de Fibronectina i LEF1 és depenent de -Catenina i NFB. La sobreexpressió d'E-Cadherina inhibeix l'activitat transcripcional d'aquests dos factors i disminueix la seva interacció amb el promotor de Fibronectina. De manera similar a la -Catenina, NFB es detecta associat a l'E-Cadherina i altres components dels contactes intercel·lulars. Quan es trenquen les unions adherents, com quan es sobreexpressa Snail, la interacció E-Cadherina-NFB disminueix i augmenta l'activitat transcripcional de NFB i-Catenina.
Epithelial to mesenchymal transitions takes place during embryo development and in the late stages of tumorigenesis allowing metastasis formation. These transitions require E-Cadherin downregulation and can be reproduced in cell culture by ectopic expression of Snail1, an E-Cadherin gene repressor. During Snail-induced transition a rapid upregulation of mesenchymal genes such as Fibronectin and LEF1 has been characterized. Forced expression of E-Cadherin strongly down-regulates Fibronectin and LEF1 RNA levels, indicating that an E-Cadherin sensitive cofactor is involved in the activation of these genes. Accordingly, transcription of Fibronectin and LEF1 was dependent on -Catenin and NFB. E-Cadherin over-expression downregulated the transcriptional activity of both factors and decreased their interaction to Fibronectin promoter. Similarly to -Catenin, NFB was detected associated to E-Cadherin and other cell adhesion components. Association of NFB to E-Cadherin required the integrity of this complex; conditions that disrupts adherens junctions, such as Snail over-expression, decreased E-Cadherin-NFB interaction and up-regulates NFB and -Catenin transcriptional activity. Therefore, -Catenin and NFB transcriptional activities are required for expression of the studied mesenchymal genes and these activities are inactivated by immobilizing -Catenin and NFB to functional E-Cadherin structures.

Comprendre la structure et l’organisation de la chromatine est une question fondamentale dans le domaine de la régulation de l’expression des gènes. La cristallographie par rayons-X et d’autres techniques biophysiques on permit de comprendre la structure du nucléosome avec une précision quasi atomique. Malgré de nombreuses études, les données structurelles au delà de la particule de cœur nucléosomale (NCP) demeurent imprécises. Au cours des dernières décennies plusieurs tentatives ont été faites pour montrer comment l’histone de liaison H1 interagit avec les particules nucléosomales pour les condenser en fibre de chromatine. Ces études ont mené à différents modèle décrivant la position de l’histone de liaison H1 sur la chromatine. De récentes avancées sur l’histone de liaison H1 suggèrent que le domaine globulaire de H1 (GH1) et la partie C-terminale interagit avec la dyade du nucléosome et les 2 bouts d’ADN de liaison (modèle à 3 contacts) qui sont contraintes de former une structure en tige. Cependant, la conformation et la position précise de l’histone de liaison H1 reste inconnues et la controverse à ce sujet persiste.Dans cette étude, nous avons déterminé la structure tridimensionnelle de nucléosomes contenant H1 par des techniques de cryo-microscopie électronique (cryo-EM) et de diffraction aux rayons-X dans des cristaux. Nous avons utilisé le chaperons d’histone, NAP1, pour déposer l’histone de liaison H1 sur les nucléosomes reconstitué à partir des histones de cœur recombinant et la séquence d’ADN positionnante 601 de 197 paires de bases (dite de Widom). Nos résultats de cryo-EM montrent que l’association de H1 compacte le nucléosome en réduisant la mobilité des ADNs et stabilisant ainsi les contacts entre les nucléotides précédant la sortie NCP et l’octamer d’histones. Nos résultats par diffusion de rayon-x dans des cristaux à une résolution de 7Ä montrent que la partie globulaire de H1 (GH1) est située sur la dyade et interagie simultanément avec les petits sillons de l’ADN à la dyade et les ADN de liaison à l’entrée et à la sortie du nucléosome. Les parties N- et C-terminales de H1 sont orientées vers l’extérieur du cœur du nucléosome à travers les différents ADN de liaison. Nous avons validé l’orientation de GH1 par des expériences de pontages ADN-proteine, après substitutions de cystéine par mutagénèse dirigée, empreinte par radicaux hydroxyles et « amarrage moléculaire ». Nos résultats révèlent l’effet de H1 sur la dynamique du nucléosome et apporte une vision détaillé de la conformation du « stem du nucléosome » lors de l’incorporation de H1.Nous avons également étudié l’association spécifique du facteur de transcription Sox6 à ces de reconnaissance consensus présent à l’intérieur du nucléosome, associé ou non avec l’histone de liaison H1 par une empreinte biphotonique avec laser UV. Nos résultats montrent que le domaine HMG de Sox6 se fixe spécifiquement sur son motif consensus situé profondément à l’intérieur du nucléosome à l’exception sur la dyade. Cette association n’est pas influencée par la « fermeture » des ADN de liaison avec l’histone H1 démontrant l’existence d’un autre façon de reconnaissance que le modèle de Widom basés sur fluctuations thermodynamiques des ADN de liaison. Le résultat que Sox6 est capable de surmonter la barrière nucléosomale (avec ou sans H1) suggère fortement que les facteurs de transcription de la famille Sox, de domaine de liaison de type HMG, jouent le rôle de facteurs « pionnier » dans la régulation de la transcription et en particulier dans l’initiation de la différentiation
Understanding the structural organization of chromatin is a fundamental issue in the field of gene regulation. X-ray crystallography and other biophysical techniques have enabled understanding of the nucleosome structure nearly at atomic precision. Despite numerous studies, the structural information beyond the nucleosome core particle (NCP) remains elusive. Over the last few decades several attempts have been made to reveal how the linker histone H1 interacts with the nucleosome particles and condenses them into a chromatin fiber. These studies have led to different models describing the position of linker histone H1 on chromatin. Recent advancements in linker histone H1 studies suggest that globular domain of histone H1 (GH1) interacts with the nucleosomal dyad and its C-terminal domain interacts with the linker DNA forming a stem like structure. However, the precise conformation of linker histone H1 and position of other domains still remains unknown.In this study, we resolved the three-dimensional structure of H1-containing nucleosomes by using cryo-electron microscopy (cryo-EM) and X-ray crystallography. We have used the chaperone NAP-1 to deposit linker histone H1 onto nucleosomes reconstituted from recombinant core histones and 197 base-pair of 601 strong nucleosome positioning DNA sequence. Our cryo-EM results showed that association of H1 gives a more compact appearance of the nucleosome as it restricts the mobility of the two linker DNAs keeping them in close proximity and thereby stabilizing contacts between the histone core and nucleotides preceding NCP exit. Our X-ray crystallography results at 7 Ä resolution reveal that the globular domain of histone H1 (GH1) is positioned onto the nucleosome pseudodyad and recognizes the nucleosome core and both linker arms by contacting the DNA backbone in the minor groove. The N- and C-terminal domains of H1 are oriented away from the nucleosome core towards different DNA linkers. We further validated the orientation of GH1 by cross-linking experiments followed after cysteine substitutions mutagenesis, hydroxyl radical footprinting and by molecular docking. Our results reveal the effect of H1 on nucleosome dynamics and also provide a detailed view of the nucleosome stem conformation upon H1 incorporation.We also studyed the nucleosome accessibility of transcription factor Sox6 and the impact of linker histone H1 incorporation to Sox6 binding on nucleosome by using UV laser biphotonic footprinting. Our results reveal that Sox6 HMG domain binds specifically to its consensus binding located deep inside of the nucleosomal DNA, but not at the nucleosomal dyad. Our in vitro footprinting results reveal that the “locking” of DNA linkers by incorporation of histone H1 on nucleosome does not show any impact on Sox6 HMG domain binding, evidencing an alternative to the Widom model based on thermal fluctuation “opening” of the nucleosome at the linkers.. The finding that Sox6 is able to overcome nucleosome (chromatosome) barrier in presence or absence of H1, strongly suggest that the HMG domain - based Sox family proteins it can act as a pioneer factor in transcription regulation, in particular in initiation of cell differentiation

Les cellules tumorales présentent des variations parfois importantes du taux de certaines protéines qui leur procurent un avantage sélectif de croissance par rapport aux cellules normales dont elles dérivent. Ces variations peuvent résulter d'altérations de l'ADN, premier support de l'information génétique, la transcription, la maturation et/ou la stabilité de l'ARN messager, ou encore la traduction et/ou la dégradation de la protéine. Les protéines kinases C (PKC) sont impliquées dans de nombreux processus cellulaires associés à la tumorigenèse, notamment le contrôle de la prolifération, de la différenciation et de l'apoptose. Or, d'importantes variations de leurs niveaux d'accumulation sont observées dans de nombreuses tumeurs humaines. Néanmoins, le lien causal entre les mécanismes régulant la transcription, la traduction ou encore la stabilité et la dégradation des PKC et ces variations est rarement établi. Nous avons pour notre part démontré que l'expression de PKCα est réprimée aussi bien in vitro qu'in vivo par le facteur de transcription SOX9 dans les cellules épithéliales intestinales. Cette répression ne nécessite pas l'interaction de SOX9 avec l'ADN via son domaine HMG mais est médiée par un nouveau mécanisme impliquant la région centrale de SOX9, très conservée entre les membres du groupe des SOXE (SOX8, 9, 10). Puisque SOX9 est une cible de la voie Wnt/APC dans les cellules épithéliales intestinales, nos résultats établissent un lien moléculaire entre voie Wnt/APC et PKCα et permettent d'expliquer pourquoi PKCα est diminuée dans les cancers colorectaux qui présentent pour 80% d'entre eux une activation constitutive de la voie Wnt/APC.

Les anomalies congénitales du rein et du tractus urinaire (CAKUT) sont l’une des malformations les plus fréquentes chez l’homme, et résultent d’un défaut du programme de dévelopement des organes. La famille de gènes Sox code pour 20 facteurs de transcription qui assurent des fonctions multiples et essentielles pendant l’organogenèse chez l’homme et la souris. Nous avons précedemment montré que Sox8 et Sox9 sont nécessaires au branchement de l’uretère, et la perte de ces gènes résulte en une agénésie rénale. L’objectif de ce projet de thèse était de caractériser le role des gènes Sox-C (Sox4/11/12) in vivo chez la souris. L’analyse des patrons d’expression a révélé que Sox4 , Sox11 et Sox12 sont co-exprimés dans les cellules progénitirices des néphrons, destinées à subir une transition mésenchyme epithelium (MET) pour former des vésicules qui s'allongent pour aboutir au néphron fonctionnel. L’analyse phénotypique a révélé une redondance fonctionnelle entre Sox4 et Sox11 pendant les processus de MET et de maturation des néphrons: les double mutants développent une hypodysplasie rénale dûe à une réduction dramatique du nombre et de la taille des néphrons. Le pool de progéniteurs de néphrons est intact chez ces mutants mais incapable de s’engager dans la nephrogenèse, probablement dû à un changement d’identité cellulaire. Par ailleurs, en l’absence de Sox11, des bourgeons uretéraux ectopiques se forment, conduisant à des reins duplex, phénotype présent dans une proportion de patients CAKUT. De manière importante, nous avons identifié une série de variants SOX11 dans une cohorte de patients CAKUT, suggérant l'implication de mutations SOX11 dans cette maladie chez l'homme
Congenital abnormalities of the kidney and the urinary tract (CAKUT) belong to the mostcommon birth defects in human and are caused by defects in the program governing organ development. The Sox gene family encodes 20 transcription factors that ensure multiple and essential functions during mouse and human organogenesis. We have previously shown that the homologous genes Sox8 and Sox9 are required for the branching process of the ureter and their loss results in renal agenesis. In this thesis project, we aimed to identify and characterize the role of the Sox-C genes (Sox4/11/12), in vivo using mouse models. Expression analysis revealed that Sox4, Sox11 and Sox12 are coexpressed in the self-renewing nephron precursors cells that are destined to undergo mesenchyme-to-eptihelial transition (MET) to form vesicles that elongate to give rise to the functional nephrons. Phenotypical analysis revealed a functional redundancy between Sox4 and Sox11 in MET and nephron maturation processes: double mutants display renal hypodysplasia, due to a dramatic reduction in the number and size of nephrons. The nephron precursor pool is intact in these mutants but unable to commit to nephrogenesis, probably because of a cell identity change. In addition, in the absence of Sox11, ectopic ureteric buds form, leading to duplex kidneys, a phenotype found in a proportion of CAKUT patients. Importantly, mutation analysis of a cohort suffering from CAKUT syndrome identified a series of SOX11 variants, thus suggesting an involvement of SOX11 mutations in this human disease

Terminally differentiated Schwann cells (SCs), the glial cells in the adult peripheral nerves, display a remarkable plasticity by adopting a de-differentiated phenotype following injury and becoming specialised to repair-type cells for promoting nerve regeneration. Adult SC plasticity is also hijacked by leprosy-causing Mycobacterium leprae during peripheral nerve infection, which make SCs susceptible to reprogramming and generation of progenitor/stem-like cells for bacterial advantage. Interestingly, de-differentiated SCs generated during nerve injury and infection reactivated stem cell transcription factor Sox2, which is essential for maintaining pluripotency in embryonic stem cells (ESCs). In this study we address what role Sox2 plays and how it is involved in adult SC plasticity. We identified that Sox2 binds to a network of gene targets in de-differentiated adult SCs across the mouse genome. This Sox2 target network is distinct from Sox2 target genes in core ESC pluripotency, and appears to be modulated by SC microenvironmental changes and pathological conditions, as nerve crush injury and infection-induced reprogramming expanded Sox2 binding to target genes. In vivo knockdown by shRNA of Sox2 in wild type adult nerves demonstrated reduction in SC de-differentiation. Mutant mice defective in natural nerve degeneration, de-differentiation and regeneration (Wallerian degeneration slow mice; Wlds) not only show impaired Sox2 binding to its target genes but also a delay in Sox2 and target gene expression after nerve crush injury. Together, these in vivo data reveal an impact of Sox2 and its target network on SC plasticity. Furthermore, altered expression of many of these target genes after Sox2 knockdown in wild type adult Schwann cells in vitro and in vivo as well as in injured Wlds nerves suggests a functional role of a Sox2 target network in nerve injury-repair processes. This includes Sox2 target genes such as Megf10, Btc, Atf3 and Nestin. By acting on these genes Sox2 may coordinate relevant gene functions ranging from phagocytosis/clearance, proliferation, transcription and cytoskeletal dynamics. Thus, this study proposes a novel concept of how reactivation of an embryonic stem cell regulator like Sox2 in adult tissues coordinates a gene network regulating Schwann cell plasticity and multiple biological functions facilitating the nerve injury-repair process. These findings may aid in developing strategies towards promoting nerve regeneration, or designing treatments for neuropathies in which deregulation of Schwann cell de-differentiation contributes to pathogenesis.

The work presented in this thesis is a study of human pancreas development. The principle goal of this work is to provide information that can be used in the development of treatments for Type 1 Diabetes and in pancreas regeneration methodologies. The transcription factor (TF) sex determining region Y homeobox gene 9 (SOX9) has been identified as a key factor in human pancreas development but its role has not been well characterized. The expression of SOX9 during early pancreas development was analyzed by immunostaining of fixed embryonic and fetal sections in the context of other developmentally important TFs. Modulators of SOX9 function, downstream targets and upstream regulatory pathways were investigated in human cell lines using coimmunoprecipitation, small interfering RNA (siRNA) knockdown, quantitative polymerase chain reaction (qPCR), luciferase assays and small molecule signaling pathway inhibitors. SOX9 was expressed in epithelial progenitors from initial human pancreas specification, but became excluded from the periphery of the epithelium and developing islet cells as differentiation proceeded. It was co-expressed with important endocrine and exocrine differentiation factors during the early stages of development. Some factors, such as Nirenberg and Kim 2, homeobox family member, drosophila, homolog of, 2 (NKX2.2) showed differing expression profile compared to murine development, while the widespread expression of endocrine factors before expression of the pro-endocrine gene neurogenin 3 (NGN3) suggested that these factors play an important role in initiating endocrine specification. Two transcription factors, GATA-binding protein 4 (GATA4) and neurogenic differentiation 1 (NEUROD1), were found to interact with SOX9 in potentially developmentally relevant complexes. This prompted the search for downstream targets of these transcriptional complexes by in silico analysis, which identified an array of novel potential downstream targets. Luciferase assay analysis of a subset of these genes showed SOX9 to activate a regulatory region of NGN3, and inhibit the regulatory regions of carboxy peptidase A6 (CPA6), v-ets avian erythroblastosis virus E26 oncogene homolog1 (ETS1) and SPONDIN1. An additional target of SOX9, osteopontin (OPN), was identified from a microarray of Sox9 knockout mouse pancreata. Investigation of SOX9 and OPN regulation by the Hedgehog signalling pathway (HH) identified both factors to be regulated by the pathway, suggesting SOX9 may act as a mediator of HH signalling. This is the first study to identify a range of SOX9 targets relevant to human pancreas development. While further characterization is required this work has provided essential clues to the function of SOX9, and provides a detailed framework of SOX9 expression and targets for future pancreatic studies to build upon.

Le maintien de l’homéostasie intestinale met en jeu un dialogue entre l’épithélium, le microbiote et le système immunitaire. Les CSI assurent le renouvellement et la régénération de l’intestin en cas de lésions mais elles peuvent être également à l’origine des tumeurs intestinales. Le facteur de transcription Sox9 est un candidat intéressant comme régulateur clé de l’homéostasie intestinale car il est exprimé dans les CSI, les cellules de Paneth et les cellules tuft. De plus, Sox9 est indispensable à la différenciation des cellules de Paneth puisque l’inactivation de Sox9 chez l’embryon (modèle murin Sox9LoxP/LoxP ; Villin-Cre) conduit à une absence de cellules de Paneth. Au cours de ma thèse, nous avons dans un premier temps déterminé la fonction de Sox9 dans l’épithélium intestinal adulte à l’aide du modèle murin inductible : Sox9LoxP/LoxP ; Villin-CreERT2. Ainsi nous avons démontré que la délétion de Sox9 dans les cellules de Paneth conduit à des altérations structurales et fonctionnelles de ces dernières, qui induisent une altération de la biodiversité d’espèces bactériennes (dysbiose). La dysbiose est « sentie » par les cellules tuft qui initient une réponse immunitaire de type 2. Cette étude a révélé le rôle clé de Sox9 dans les cellules de Paneth adultes pour réguler l’homéostasie intestinale, en prévenant l’établissement d’un microbiote pro-inflammatoire. Les cellules tuft, via leur fonction de « sensing », sont capables en réponse à une dysbiose de moduler l’immunité mucosale et participent ainsi à la formation d’un cercle vicieux délétère. De plus, nous nous sommes intéressés à la biologie des CSI, en intégrant la contribution des propriétés des cellules de Paneth qui participent à l’établissement de la niche. Nous avons étudié les propriétés des cellules souches dans un contexte sain ou au cours de l’initiation tumorale. L’ensemble de nos données indiquent qu’en contexte sain, Sox9 est requis pour la régulation du destin cellulaire des CSI, c’est à dire l’équilibre entre l’auto-renouvellement des CSI et leur différenciation cellulaire. Les mécanismes régulés par Sox9 mettent en jeu le métabolisme cellulaire, un acteur clé du destin des cellules souches. Nos travaux montrent également que le maintien d’une niche intacte est nécessaire au contrôle du devenir des CSI. La délétion de Sox9 altère l’intégrité mitochondriale et favorise la production de ROS mitochondriaux qui pourrait moduler le destin des CSI vers un état différencié. En parallèle, nous avons mis en évidence que l’invalidation de Sox9 après l’acquisition d’un événement initiateur tel que la perte de fonction du gène suppresseur de tumeur Apc, affecte de façon majeure le destin des CSC vers un état souche ainsi que leur métabolisme cellulaire. L’évaluation du rôle du facteur de transcription Sox9 dans le contrôle de l’homéostasie métabolique permettra de mieux comprendre les mécanismes de régulation de la biologie des CSI, et de proposer à terme de nouvelles stratégies thérapeutiques ciblant les CSC
The intestinal homeostasis maintenance involves a permanent crosstalk between the epithelium, the microbiota and the immune system. ISC are responsible for the intestine renewal and regeneration, but they can also cause intestinal tumors. The Sox9 transcription factor is an interesting candidate as a key regulator of intestinal homeostasis because of its specific expression in ISC, Paneth cells and tuft cells. In addition, Sox9 is essential for the differentiation of Paneth cells since the loss of Sox9 in the mouse embryo (model Sox9LoxP / LoxP, Villin-Cre) leads to the absence of Paneth cells. First, we analysed the function of Sox9 in the adult intestinal epithelium using the inducible mouse model: Sox9LoxP / LoxP; Villin-CreERT2. We demonstrated that the deletion of Sox9 in adult Paneth cells leads to structural and functional alterations of Paneth cells, which induce alterations of bacterial diversity (dysbiosis). Dysbiosis is "sensed" by tuft cells that initiate a type 2 immune response. This study revealed the key role of Sox9 in adult Paneth cells to regulate intestinal homeostasis, thus preventing the establishment of a proinflammatory microbiota. Tuft cells, via their sensing function, are able to modulate mucosal immunity in response to a dysbiosis and thus participate in the formation of a vicious circle. In addition, we studied the biology of ISC, by integrating the contribution of Paneth cells properties that participate in the establishment of the niche. We analysed the properties of stem cells in a healthy context or during tumor initiation. Our data indicate that in a healthy context, Sox9 is required for the regulation of ISC fate, namely the balance between ISC self-renewal and differentiation. The mechanisms regulated by Sox9 involve cellular metabolism, a key player in the stem cells fate. Our work shows that an intact niche maintenance is necessary to control ISC fate. The deletion of Sox9 alters mitochondrial integrity and promotes mitochondrial ROS production that could modulate the ISC fate toward a differentiated state. In parallel, we demonstrated that Sox9 deletion concomitant with the acquisition of an initiating event such as the loss of function of the tumor suppressor gene Apc, affects the CSC and their cellular metabolism. The evaluation of the role of the Sox9 transcription factor in the control of metabolic homeostasis will provide a better understanding of the regulatory mechanisms in ISC biology, and eventually new therapeutic strategies targeting CSC might be proposed

Problem Area: The American company Enron was the seventh largest company notated on the stock market when it went bankrupt due to extensive accounting frauds in December 2001. By manipulating the financial reports, the management of Enron was able to hide huge debts through transactions with external companies. Therefore, they were able to deceive the public by reporting huge profits while the company, in reality, was generating a negative result. As a result of the bankruptcy, 40 billion dollars disappeared and tens of thousands of employees lost their jobs and pension savings. After the Enron scandal, further accounting frauds were discovered in the US, such as WorldCom and Tyco. The WorldCom bankruptcy is, as of today, the largest bankruptcy in the world. Following the scandals and the accounting frauds in a number US companies, the US congress passed a law – the Sarbanes-Oxley Act of 2002 (SOX).

Purpose: The purpose of the study is to examine how companies in Sweden have managed to implement SOX Sec. 404 – for ensuring internal control. The researchers will examine how different Swedish companies have approached the issue and how they have chosen to implement SOX sec. 404 in their organisation. Have some companies been more effective in their implementation than others and why? Limitations: SOX cover several areas around internal control and reporting. The authors will mainly study Sec. 404, which treats internal control over financial reporting, and is the section that has demanded most effort to comply with. Methodology: The study was conducted by a qualitative research method by conducting personal interviews with respondents from three different companies in Sweden. All of the participating companies in the study are obliged to comply with SOX. Conclusion: The researchers in this study have identified differences in the approach towards SOX-compliance by the participating organisations. The lack of how to implement guidance and the different approaches taken by companies towards SOX compliance may suggest that there is no supreme implementation guide that is suitable for all companies in order to achieve compliance. Proposals for Further Research: As the implementation phase just recently was completed the authors would like to find out how companies are sustaining compliance in the future. Researchers could focus on sustainable compliance as compared to this study where sustainable compliance is merely limited to one section. In addition, it would be interesting to carry out an in-depth study on one company to evaluate their implementation on a deeper level. This would give the researcher a deeper understanding which is more difficult to achieve when conducting a comparative study.

Células-tronco embrionárias (CTE) são importantes para estudos de desenvolvimento embrionário, diferenciação e manipulação genética. Além disso, essas células podem ser utilizadas na terapia celular e organogênese in vitro. Na pesquisa sobre terapia celular a partir de CTE oriundas de embriões humanos, considerações éticas, morais e religiosas têm sido feitas por pesquisadores e leigos. Portanto, um modelo animal como o suíno (Sus scrofa) será bastante válido por transpor tais barreiras, visto que o suíno possui parâmetros fisiológicos semelhantes aos humanos. Apesar do alto potencial biomédico das CTE, existem dificuldades na manutenção da pluripotência in vitro dessas células em suínos. Portanto, estudos que visam elucidar os mecanismos de manutenção da pluripotência de CTE in vitro são necessários para viabilizar o cultivo dessas células. Os objetivos do presente estudo foram (1) isolar células-tronco embrionárias suínas a partir de blastocistos produzidos in vitro e in vivo; (2) comparar dois sistemas de cultivo in vitro das massas celulares internas (MCI) isoladas, MEF ou Matrigel e (3) identificar e comparar a expressão dos fatores de transcrição Nanog, Sox2 e FoxD3 em CTE e blastocistos suínos produzidos in vitro e in vivo. Assim, blastocistos suínos foram produzidos in vitro a partir da maturação e fecundação in vitro de oócitos de ovários obtidos em matadouro. Os embriões foram cultivados in vitro por 7 dias, até atingirem o estágio de blastocisto. Blastocistos suínos também foram produzidos in vivo, através de superovulação seguida de inseminação artificial de marrãs com 150 dias de idade. Para a colheita dos embriões, foi realizada lavagem dos cornos uterinos post-mortem cinco dias após a ovulação. Tanto blastocistos produzidos in vitro quanto os produzidos in vivo foram submetidos à imunocirurgia para isolamento da MCI. Brevemente, a zona pelúcida foi digerida com solução de pronase e os embriões incubados com soro de coelho anti-suíno para remoção das células do trofoectoderma e soro complemento de cobaia. A MCI resultante foi cultivada em meio para células-tronco (GMEM acrescido de 15% SFB, 0,1 mM ß-mercaptoetanol, 1% aminoácidos não essenciais e 4 ng/mL de bFGF) sobre monocamada de fibroblastos fetais murinos (MEF) inativados por radiação ou sobre Matrigel. Não foi observada diferença entre os dois sistemas de cultivo in vitro (MEF e Matrigel) na adesão das MCI isoladas. Também não foi verificada diferença entre os grupos de blastocistos, produzidos in vitro e in vitro, nas taxas de adesão das MCI cultivadas. Contudo, nenhuma colônia de CTE suínas foi obtida. A análise da expressão gênica em blastocistos produzidos in vitro e in vitro demonstrou que os genes Nanog e Sox2 são menos expressos em blastocistos produzidos in vitro. Contudo, a expressão do gene FoxD3, demonstrada pela primeira vez em suínos no presente trabalho, se mostrou semelhante entre os dois grupos de embriões. Visto que nenhuma linhagem de CTE legítima foi isolada em suínos até o momento, sugere-se que esta espécie possua requerimentos diferentes dos já conhecidos para as espécies murina e humana. Portanto, novos estudos são necessários para o estabelecimento de protocolos mais efetivos para o isolamento de CTE de suínos.
Embryonic stem cells (ESC) represent a useful tool to study embryonic development, cell differentiation and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral and religious considerations by scientists and laymen alike. Therefore, an animal model like the pig (Sus scrofa) is valuable by overcoming such hurdles, since this species holds physiologic parameters similar to humans. In spite of the high biomedical potential of ESC, many difficulties have been faced to maintain these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro and in vitro produced swine blastocysts, (2) to compare two in vitro culture conditions to maintain isolated inner cell masses (ICM), MEF or Matrigel and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2 and FoxD3 at ESC and in vitro and in vitro produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vitro produced blastocysts were obtained by superovulation followed by artificial insemination of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing five days after ovulation. in vitro and in vitroproduced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. The resultant ICM was cultured in stem cells media (GMEM added by 15% SFB, 0.1 mM ß-mercaptoethanol, 1% non essential amino acids and 4 ng/mL of bFGF) over monolayer of irradiated murine fetal fibroblasts (MEF) or Matrigel. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro and in vitro produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis of in vitro and in vitro produced blastocysts showed that Nanog and Sox2 are less expressed in in vitro produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Since no ESC lineage was obtained in swine until now, we believe this species have different requirements compared to murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC.

Lors de la formation du blastocyste, l'embryon de souris est constitué d'un épithélium externe, le trophectoderme (TE), et d'une masse cellulaire interne (MCI). L’épiblaste (EPI) et l’endoderme primitif (EPr) se spécifient au sein de la MCI sous un patron de « sel et poivre » caractérisé par l’expression complémentaire de NANOG, marqueur de l’EPI et de GATA6, marqueur de l’EPr. Nanog est nécessaire pour l’acquisition d’une identité EPI et Gata6 induit le devenir en EPr. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr et la perturbation de son activité impacte directement sur le ratio EPr/EPI dans la MCI. Je recherche des facteurs qui serait exprimés de manière hétérogène avant la spécification des cellules internes et pourraient faire pencher la balance vers un destin ou l’autre. Pour cela, j’ai disséqué l’évolution des cellules de la MCI au sein des embryons Nanog-/- et Gata6-/-. Ces embryons forment correctement le TE et la MCI qui ne se spécifie ni en EPI ni en EPr. En effet, les cellules internes des embryons Nanog-/- ; Gata6-/- restent bloquées autour du stade E3.25. De manière étonnante, dans les cellules de la MCI, le facteur de transcription SOX2 est présent et ce, de manière hétérogène. De plus, grâce à des traitements inhibiteurs de la voie FGF/MAPK, je montre que cette voie n’est pas responsable de l’hétérogénéité d’expression de SOX2. Ainsi, l’expression hétérogène de SOX2 dans les cellules internes des embryons est donc indépendante de Nanog, de Gata6 et de la voie FGF/MAPK
During mouse blastocyst formation, the embryo consists of an outer epithelium, the trophectoderm (TE), and the inner cell mass (ICM). The epiblast (EPI) and the primitive endoderm (PrE) are specified within the MCI in a "salt and pepper" pattern characterized by the complementary expression of NANOG, marker of EPI and gata6, marker of PrE. Nanog is mandatory to acquire an EPI identity and Gata6 induces the PrE identity. FGF /MAPK pathway plays a critical role in the acquisition of a PrE identity and disruption of its activity directly impacts the PrE/Epi ratio within the ICM. I’m looking for factors that would be expressed heterogeneously before the specification of internal cells and might tilt the balance towards one fate or the other. For this, I dissected the evolution of ICM cells within Nanog-/- ; Gata6-/- embryos. These embryos form properly the TE and MCI that specifies neither EPI nor PrE. Indeed, the internal cells of Nanog-/- ; Gata6-/- embryos remain stuck around the stage of E3.25. Surprisingly, in the MCI cells, the transcription factor SOX2 is present and this, heterogeneously. Moreover, using inhibitors treatments of the FGF/MAPK pathway, I show that this pathway is not responsible for the heterogeneity of expression of SOX2. Thus, the heterogeneous expression of SOX2 in the inner cells of the embryos is therefore independent of Nanog, Gata6 and the FGF/MAPK pathway

Le carcinome basocellulaire (CBC) est le cancer le plus fréquent chez l’homme et est à lui seulresponsable de plus de cas que l’incidence cumulée de tous les autres cancers réunis. Des travauxrécents qui se sont intéressés aux étapes précoces de la tumorigenèse ont fourni un supportprécieux pour l’analyse du développement tumoral précoce. Ils ont mis en évidence le rôlecentral de gènes impliqués dans les progéniteurs embryonnaires des cellules souches du folliculepileux. Dans ce travail, nous avons étudié le rôle du facteur de transcription Sox9, un gène connupour être crucial dans la spécification de ces progéniteurs et dans les cellules souches du folliculepileux. Nous avons montré que Sox9 est requis pour la progression des lésions paranéoplasiquesvers les CBC et qu’il était également requis pour la maintenance des cellules cancéreuses. Enutilisant des méthodes d’analyse génomique de pointe, nous avons montré que Sox9 agit tantcomme un activateur que comme un répresseur de la transcription. Nous avons montré qu’ilpromeut directement le renouvellement cellulaire, l’invasion ainsi que la quiescence des cellulescancéreuses.Dans la seconde partie de ce travail, nous avons découvert le mécanisme d’action de l’inhibiteurd’Hedgehog Vismodegib sur les cellules cancéreuses. En utilisant des modèles génétiques desouris, nous avons montré que l’inhibiteur empêche la reprogrammation embryonnaire qui anormalement lieu durant la formation du CBC et qu’il promeut la différentiation des cellulescancéreuses vers une identité interfolliculaire, d’infundibulum ou sébacée suivant l’origine de cescellules. Nous avons également observé qu’une population caractérisée par l’expression du gèneLgr5 ainsi que par une activation de la voie de signalisation Wnt est plus résistante au Vismodegibchez la souris et nous avons trouvé des indices suggérant qu’il pourrait en être de même chezl’humain. Enfin, nous avons pu montrer que l’inhibition combinée des voies de signalisation Wntet Hedgehog annihile cette résistance et résulte en la disparition complète des CBCs.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

SOX9 est un facteur de transcription à domaine HMG. Il est impliqué dans de multiples processus biologiques au cours du développement et de la vie adulte. En particulier, SOX9 joue un rôle important dans l'homéostasie de l'épithélium intestinal. Nous avons montré que SOX9, cible positive de la voie de signalisation oncogénique Wnt/(beta)-caténine, réprime l'expression de PKC(alpha). Cette répression implique un nouveau mécanisme d'action qui ne nécessite ni la fixation du domaine HMG à l'ADN ni le domaine de transactivation de SOX9. Nous avons également identifié MiniSOX9, un nouveau variant d'épissage de SOX9, résultant de la rétention du second intron. MiniSOX9 est fortement exprimé dans les tumeurs coliques. Il agit en tant que dominant négatif de SOX9, inhibiteur de l'expression du suppresseur de tumeurs PKC(alpha) et activateur de la voie de signalisation oncogénique Wnt/(beta)-caténine. Nos données suggèrent ainsi un rôle primordial de MiniSOX9 dans la tumorigenèse intestinale. Enfin, notre étude protéomique des partenaires de SOX9 et de MiniSOX9 permet d'ouvrir de nouvelles perspectives quant aux rôles de ces deux protéines dans l'homéostasie et la tumorigenèse intestinale
SOX9 is an HMG transcription factor involved in numerous biological processes during development and adult life. It plays an important role especially in the intestinal epithelium homeostasis. In the present study, we demonstrate that SOX9, a positive target of the oncogenic signaling pathway Wnt/(beta)-catenin, represses PKC(alpha) expression. This repression involves a new mechanism of action requiring neither HMG domain binding to DNA nor the transactivation domain of SOX9. We also report the discovery of MiniSOX9, a new SOX9 splice variant, resulting from the second intron retention. MiniSOX9 is highly expressed in colon tumors. It acts as a SOX9 dominant negative, as a repressor of the expression of the tumor suppressor PKC(alpha), and as an activator of the oncogenic signaling pathway Wnt/(beta)-catenin. Our data suggest a crucial role of MiniSOX9 in intestinal tumorigenesis. Finally, a proteomic analysis allowed us to identify potential new SOX9 and MiniSOX9 partners which will be useful to decipher the roles of these two proteins in intestinal homeostasis and tumorigenesis

Inledning: De senaste årens redovisningsskandaler, där bland annat Enron och Worldcom var inblandade, har lett till att förtroendet för den finansiella rapporteringen har minskat eftersom rapporteringen av bolagens ekonomiska ställning inte har varit sanningsenlig. Debatten kring redovisningsskandalerna har främst handlat om att den finansiella rapporteringen inte har varit tillförlitlig på grund av bristande intern kontroll hos bolagen. För att vinna tillbaka marknadens förtroende införde George W Bush en ny lagstiftning som kom att kallas Sarbanes Oxley Act. Alla bolag som är registrerade på amerikanska börsen måste följa lagen, vilket även påverkar svenska bolag. Sarbanes Oxley Act är en omfattande lag som består av ett antal sektioner. Den del av lagen som sägs innebära störst förändringar för bolagens arbete är SOX Sektion 404, Management assesment of internal control, det vill säga ledningens utvärdering av intern kontroll.Syfte: Syftet med rapporten är att genom en fallstudie skapa kunskap om Sarbanes Oxley Act Sektion 404 påverkan på ett företags externa rapportering och om implementeringen av denna lag ökar tillförlitligheten och minimerar risken för felaktig rapportering och bedrägerier i ett företag.Metod: I denna del beskriver vi de metoder som vi valt att använda oss av i vår uppsats samt vad de innebär. Vi tillämpar följande metoder; hermeneutisk ansats, kvalitativ metod, abduktiv metod och som undersökningsmetod har vi använt oss av en fallstudie. Vi beskriver även vår datainsamlingsprocess samt hur vi förhåller oss källkritiskt till den insamlade datan.Analys och slutsatser: Vi har kommit fram till att det har krävts en del förändringar hos det företag vi har valt att studera, Parker Hannifin AB, för att leva upp till kraven som ställs i SOX Sektion 404. SOX har påverkat tillförlitligheten i den finansiella rapporteringen genom bland annat införande av nya IT – system, tydligare ansvarsfördelning och fler behörighetsbegränsningar i IT – systemen i syfte att bland annat minska risken för obehörig åtkomst till data samt eliminera risken för felaktiga transaktioner. Vi har i undersökningen även kommit fram till att Parker i samband med implementeringen av SOX har fått tillämpa en ny modell för intern kontroll för tillförlitlig finansiell rapportering. Modellen heter CAVR som skall säkerställa att kraven för intern kontroll för finansiell rapportering uppfylls.
Uppsatsnivå: C

Sox9 est un facteur de transcription exprimé au cours du développement de l'intestin et son expression est maintenue à l'âge adulte dans trois populations cellulaires : les cellules souches, les cellules de Paneth, et les cellules tuft. L'inactivation de Sox9 dans l'épithélium intestinal embryonnaire entraîne, chez l'adulte, une hyperplasie des cryptes ainsi que l'absence de cellules de Paneth. Ce projet de thèse vise à déterminer le rôle de Sox9 dans les cellules de Paneth (dont la fonction est altérée chez les patients atteints de la maladie de Crohn), à identifier les mécanismes par lesquels Sox9 régule la prolifération et à proposer des cibles de Sox9 dans les cellules tuft. À l'aide de modèles murins d'inactivation de Sox9 au niveau de l'épithélium intestinal adulte, nous avons montré que la perte de ce facteur conduit à une augmentation de la prolifération dans les cryptes, confirmant ainsi que Sox9 régule négativement ce processus. Nos résultats indiquent que Sox9 est essentiel au maintien de l'identité des cellules de Paneth et nous proposons qu'il assure cette fonction en réprimant des gènes requis pour la différenciation des cellules de Goblet : Muc2 et Klf4. La perte de Sox9 dans les cellules de Paneth s'accompagne d'une réduction importante des molécules antimicrobiennes, ce qui entraîne une dysbiose intestinale. Dans un environnement spécifique (en présence du « mouse norovirus »), les souris déficientes en Sox9 présentent une perméabilité intestinale augmentée et une susceptibilité à l'inflammation accrue. Les dysfonctionnements des réponses antimicrobiennes et immunitaires dans notre modèle sont comparables à ceux observés chez les patients atteints de la maladie de Crohn, suggérant une implication potentielle de Sox9 dans cette pathologie. De plus, ces altérations pourraient expliquer l'augmentation de l'apparition des tumeurs observée chez les souris dont l'épithélium intestinal est déficient en Sox9, dans le contexte d'une mutation du gène suppresseur de tumeur Apc. Enfin, nous avons identifié des gènes potentiellement régulés par Sox9 qui pourraient expliquer son rôle dans le contrôle de la prolifération. Ces découvertes seront importantes pour mieux comprendre le processus du renouvellement de l'épithélium intestinal et identifier précisément le rôle de Sox9 dans le maintien de l'homéostasie et au cours du processus de la tumorigenèse intestinale
Sox9 is a transcription factor expressed during the intestinal development and its expression is maintained throughout adult age in at least three populations of cells: stem cells, Paneth cells and tuft cells. Sox9 inactivation in the embryonic intestinal epithelium leads to crypts hyperplasia and to the loss of the Paneth cell lineage. The aim of this project is to determine Sox9 function in the adult intestinal epithelium, especially its role in Paneth cells (which function is altered in patients affected by inflammatory diseases such as Crohn disease), to identify how Sox9 controls proliferation and to propose molecular targets of Sox9 in tuft cells. Using mice models to inactivate Sox9 in adult intestinal epithelium, we could show that Sox9 is required to limit proliferation in the crypts, thus validating the hypothesis that Sox9 regulates negatively proliferation. Our results indicate that Sox9 is essential to maintain Paneth cells identity and we proposed that it ensures this function by repressing genes specific for Goblet cells differentiation: Muc2 and Klf4. Loss of Sox9 in Paneth cells is associated with a reduction of antimicrobial molecules which causes intestinal dysbiosis. In a specific environment (in presence of the « mouse norovirus »), Sox9-deficient mice have a defective intestinal permeability and are more susceptible to inflammation. The dysfunctions of the mucosal defences and of immunity responses in our model resemble those observed in Crohn patients, thus suggesting a potential implication of Sox9 in this pathology. In addition, these alterations could be responsible for the increased susceptibility of our deficient model to develop tumors in the context of a mutation of the tumor suppressor gene Apc. We started to unravel potential molecular targets of Sox9 that are involved in the control of proliferation, that will be important to better understand Sox9 function in the intestinal epithelium self-renewal and to identify precisely Sox9 function to maintain homeostasis and during intestinal tumorigenesis

Medulloblastoma (MB) is the most common malignant brain tumor affecting children. The transcription factors MYCN and SOX9 are associated with initiation, maintenance and recurrence of MB and are also connected to more aggressive tumors. In this study, a ChIP was performed to isolate DNA from genes that are transcriptionally regulated by these proteins. Identification of these target genes will reveal new potential drug targets and help us better understand the functions of MYCN and SOX9. The ChIP was not fully optimized during this project and the target genes were not sent for sequencing and identified. To study the connection between SOX9 and recurrence, cells with different levels of SOX9 were treated with drugs, after which cell viability was measured. No significant difference in resistance could be measured. Change in expression level of MYCN, SOX9 and other relevant genes after drug treatment was also studied. The results show an increase in SOX9 and HES1, suggesting that these genes are involved in tumor recurrence.

Le corépresseur nucléaire NCOR1 est un répresseur transcriptionnel qui régule l’expression génique en s’associant à des récepteurs nucléaires ou à des facteurs de transcription comme AP-1 et NF-B. Ce projet de recherche visait à déterminer comment NCOR1 régule la prolifération et la réponse inflammatoire dans les cellules épithéliales intestinales (CEIs). Nous avons utilisé deux modèles murins de délétion du gène Ncor1 (Ncor1ID et Ncor1Exon11) ainsi que la Cre-recombinase sous le contrôle du promoteur du gène Vil1 (12.4KbVilCre) afin d’invalider le gène Ncor1 au niveau des CEIs (Ncor1IDΔCEI et Ncor1Exon11ΔCEI). Nous avons induit une réponse inflammatoire dans les CEIs en utilisant de l’IL-1 et du LPS. Nous avons observé que l’expression de NCOR1 est augmentée dans les CEIs lors de la réponse inflammatoire. Nous avons également traité les souris invalidées pour le gène Ncor1 avec du DSS afin d’induire une colite chimique expérimentale. Nous avons observé que les animaux Ncor1IDΔCEI et Ncor1Exon11ΔCEI sont plus susceptibles que les témoins. Nous avons analysé l’expression génique chez les animaux Ncor1IDΔCEI et Ncor1Exon11ΔCEI. L’analyse des gènes modulés dans les animaux Ncor1IDΔCEI a révélé que l’expression de la Retnlb est augmentée chez ces animaux, ce qui suggère un dérèglement dans la microflore. Dans les animaux Ncor1Exon11ΔCEI, nous avons noté que l’expression du gène Ido1, un puissant immunosuppresseur, est augmentée et permettrait possiblement à ces animaux de se maintenir dans un état homéostatique en absence de stress. Lorsque nous avons diminué l’expression de NCOR1 dans les CEIs à l’aide de shARN (shNCOR1_655), nous avons observé que celles-ci arrêtent de proliférer et sont sénescentes. De plus, nous avons remarqué une induction de molécules inflammatoires associées au SASP dans ces cellules. Nous avons analysé le transcriptome des cellules shNCOR1_655. Nous avons identifié le facteur de pluripotence SOX2 comme étant induits lorsque l’expression de NCOR1 est diminuée. Finalement, nous avons utilisé la technologie SILAC et la spectrométrie de masse afin de déterminer la composition du complexe de répression de NCOR1 dans les CEIs. Nous avons identifié de nouveaux partenaires d’interaction potentiels de NCOR1.

The nBmp2 protein was first identified in a DNA affinity chromatography/mass spectrometry screen designed to detect proteins that interact with a cartilage-specific enhancer element (called D/E) from the type XI collagen gene Col11a2. The transcription factor SOX9, a protein from the Sox (SRY-related HMG box) family, binds to and activates gene expression from this enhancer. nBmp2 has no transcriptional activity of its own on this enhancer, but when co-transfected with SOX9 it increases SOX9's activation of D/E nearly 2-fold. SOX9 also activates cartilage-specific enhancer elements from the Col2a1, Col27a1, and Col9a1 genes. The purpose of this project was to determine 1) whether nBmp2 similarly effects SOX9-dependent expression from these enhancers, and 2) whether it does so by binding (either directly or indirectly) to the Col2a1, Col27a1, and Col9a1 enhancers. The work described in this thesis has shown that nBmp2 increases luciferase levels produced from three enhancer/reporter plasmids, but it does so without binding directly to the enhancers. This work has opened up a new area of exploration into the function of the novel protein nBmp2 to examine its potential effects on a variety of different gene regulatory processes.

Les duplications de gènes et de génome sont considérées comme des moteurs de l’évolution des génomes eucaryotes. Trois duplications de génome complet (ou polyploïdisations) sont survenues au cours de l’évolution des vertébrés, dont deux à la base des vertébrés, et une troisième chez l’ancêtre commun des poissons téléostéens. La diversité morphologique, anatomique et écologique des espèces qui partagent un ancêtre commun polyploïde chez les chordés suggère un rôle des duplications de génome dans la diversification des espèces. En particulier, les duplications de génome semblent avoir facilité l’émergence du plan d’organisation des vertébrés, et être à l’origine de la radiation évolutive survenue chez les poissons téléostéens. Cependant, la portée évolutive des duplications de génome, et notamment les deux hypothèses majeures formulées ci-Avant, restent des questions ouvertes et en grande partie non résolues. Le groupe des téléostéens, qui compte plus de la moitié des espèces vertébrés existantes et partage un ancêtre commun polyploïde, constitue un modèle pertinent pour évaluer la contribution des duplications de génome dans l’expansion des familles multigéniques chez les vertébrés, pour comprendre les mécanismes évolutifs qui façonnent l’évolution des familles de gènes, et finalement tester les hypothèses moléculaires qui peuvent relier duplication de génome et biodiversité. Ainsi, nous avons étudié l’impact de la duplication de génome survenue à la base des téléostéens sur l’évolution de la famille multigénique sox, essentielle pour le développement et l’homéostasie des vertébrés. Notre analyse du contenu et de l’organisation des gènes sox dans 15 génomes de vertébrés, dont 10 téléostéens, révèle une importante expansion de l’ensemble de la famille des gènes sox dans ce vaste groupe de vertébrés, et démontre que cette expansion est essentiellement due à la duplication de génome survenue à la base des téléostéens. Les gènes sox dupliqués par duplication de génome semblent avoir été perdus par non-Fonctionnalisation dans certaines lignées, et préservés en deux copies par sous-Fonctionnalisation et/ou néo-Fonctionnalisation dans certaines autres lignées. Notre étude indique en effet une divergence lignée-Spécifique des patrons d’expression entre les gènes sox dupliqués chez différentes espèces de téléostéens. Ainsi, l’expansion du répertoire des gènes sox à la base des téléostéens semble avoir été suivi d’une évolution lignée-Spécifique du contenu et des fonctions de la famille des gènes sox chez les poissons téléostéens. Cette étude supporte l’hypothèse d’un rôle des duplications de génome dans l’enrichissement et la diversification subséquente des répertoires de gènes du développement tels que les gènes sox, et son rôle potentiel dans la diversification des espèces vertébrés
Gene and genome duplications are major engines of eukaryotic genome evolution. Three rounds of whole genome duplication (WGD) have occurred during vertebrate evolution, two rounds at the base of the vertebrate lineage, and a third round in the common ancestor of the teleostean fish (the so-Called teleost-Specific WGD). In chordates, species that share a polyploid ancestor are characterized by a huge morphological, anatomical and ecological diversity suggesting a role of WGDs in species diversification. For instance, it is considered that these drastic genomic events provided the raw material for the emergence of the vertebrate body plan, and facilitated speciation processes during the teleost radiation. However, how WGD is related to phenotypic diversification or to major evolutionary transitions are fundamental questions that remain largely unsolved. Teleostean fish constitute more than half of all extant vertebrates and share a polyploid ancestor. Thus, they provide a relevant model to study the importance of WGDs in gene families expansion, to understand evolutionary mechanisms that drive the evolution of these families and, finally, to test molecular hypotheses that might relate WGD and biodiversity. In this project, we studied the impact of the teleost-Specific WGD on the evolution of the sox gene family which are involved in development and homeostasis in vertebrates. Our analysis of the content and the genomic organization of the sox genes in 15 vertebrate genomes, including 10 teleosts, reveals an important expansion of this family in the teleost lineage, and demonstrates that this expansion is mainly due to the teleost-Specific WGD. The duplicated sox genes seem to have been lost by non-Functionalization in certain lineages, and preserved in two copies in others by neo-Functionalization and/or sub-Functionalization. Indeed, this study indicates lineage-Specific divergence in expression patterns between duplicated sox genes in different teleostean species. Hence, the sox family expansion that occurred in the last common ancestor of teleostean fish seems to have been followed by a lineage-Specific evolution of the content and functions of the sox family in this group. Our study supports the hypothesis for a role of WGDs in the enrichment and diversification of developmental genes repertories and its potential role in species diversification in vertebrates

Group B Sox genes play a critical developmental role in both vertebrates and insects. Within the model species Drosophila melanogaster, two SoxB genes, Dichaete and SoxNeuro, have been shown to act as ‘master regulators’ in the early development of the central nervous system. SoxB genes have only been characterised in a handful of arthropod species thus far, with most work to date focusing on drosophilids. The purpose of this investigation was twofold. First, I set out to resolve the phylogenetic origins of arthropod SoxB genes, as mutually exclusive models explaining their emergence are still contested. I have identified and annotated the SoxB of several invertebrate taxa. In total, my investigation includes 24 different metazoan taxa, and represents the largest investigation of arthropod SoxB phylogeny to date. In light of this research, I have proposed a new model of SoxB evolution which resolves the conflicting elements of the two primary competing models. Second, to study the evolution of SoxB in terms of functional conservation/divergence, I selected the emerging model organism Tribolium castaneum to draw a comparative analysis with Drosophila melanogaster. I first began by characterising the spatiotemporal expression patterns of SoxNeuro mRNA in early Tribolium embryos using whole mount in situ hybridisation, and examined published Dichaete expression patterns in the context of central nervous system development in T. castaneum. Using these data, I draw a comparison to the expression profiles of Dichaete and SoxNeuro orthologues in Drosophila melanogaster and other species. I have found that both Dichaete and SoxNeuro expression patterns in the developing central nervous system are remarkably well-conserved across species. I also attempted to characterise genome-wide binding for both Dichaete and SoxNeuro proteins in Tribolium in what would have represented the first genomic investigation of its kind in this emerging species. Using a tethered DNA adenine methyltransferase (Dam) enzyme for both SoxNeuro and Dichaete, I hoped to characterise the genomic loci with which each protein interacts within the beetle genome (a technique known as DamID). Unfortunately, these last set of experiments have proved unsuccessful, despite several attempts which have made use of different promoters, different DNA enrichment methodologies, and tackling unforeseen DNA contamination issues. Nevertheless, the troubleshooting experiments that I have carried out will pave the way for further genomic experiments in Tribolium, easing the establishment of genomic research in this emerging organism.

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro
Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

Les activitats industrials com són les industries papereres, farmacèutiques, minera, de processat d’aliments, etc. generen aigües residuals amb un alt contingut de sulfat. El sulfat com a tal no resulta altament perjudicial per a la salut, però si s’aboca en rius o sistemes de clavegueram, els microorganismes coneguts com bactèries reductores de sulfat (sulfate reducing bacteria, SRB) el poden transformar en sulfur d’hidrogen. El sulfur d’hidrogen és un compost que fa mala olor, és corrosiu i s’ha demostrat tòxic inclús a baixes concentracions. Per aquests motius el tractament d’efluents rics en sulfat és indispensable. A més a més, la recuperació de sofre elemental d’aquests efluents per poder ser reutilitzat com a fertilitzant o matèria primera a la indústria és una oportunitat de recuperació de recursos en el marc de l’economia circular. Els sistemes bioelectroquímics (bioelectrochemical systems, BES) són una tecnologia innovadora basada en l’habilitat d’alguns bacteris d’intercanviar electrons amb un elèctrode sòlid. Últimament, l’estudi dels BES s’ha focalitzat en el tractament d’aigües residuals i en la recuperació de productes gràcies a l’activitat dels microorganismes que colonitzen els elèctrodes. En aquesta tesi s’ha estudiat l’ús de BES per al tractament i recuperació de compostos de sofre, concretament, el tractament d’aquestes aigües residuals amb sulfat. El sistema permet la reducció de sulfat en un biocàtode mentre en l’ànode succeeix l’electròlisi d’aigua per generar el flux d’electrons necessari. Els microorganismes que colonitzen la superfície del càtode utilitzen l’hidrogen generat a partir dels electrons per transformar el sulfat en sulfur d’hidrogen. No obstant això, els resultats obtinguts han demostrat que gràcies a l’electròlisi de l’aigua que té lloc a l’ànode es produeix un flux d’oxigen cap al càtode que permet el creixement dels microorganismes capaços de produir sofre a partir del sulfur d’hidrogen, anomenats bacteris oxidants de sulfur (sulfide oxidising bacteria, SOB). Per tal de millorar l’eliminació de sulfat i la producció de sofre es va estudiar com el pH del compartiment del càtode i el potencial de càtode podien influir en el procés. Es va observar que el pH neutre (pH = 7) era més beneficiós ja que un pH àcid (pH = 5.5) podria inhibir l’activitat de les SRB i un pH bàsic (pH = 8.5) requeria de més energia per aconseguir resultats similars a causa de la limitació en la producció d’hidrogen a un pH elevat. En quant al potencial del càtode, es va poder observar que a menors potencials, major eliminació de sulfat, però a partir d’un potencial de -1.0 V vs. SHE, el sistema no podia augmentar la velocitat d’eliminació. A més a més, també s’ha estudiat el tractament d’aigua residual real procedent d’un sistema de dessulfuració de gasos de combustió. S’ha observat que amb l’aigua real l’eliminació de sulfat es reduïa, però en canvi la producció de sofre elemental augmentava. Finalment, com que el flux d’oxigen de l’ànode al càtode no es podia controlar amb els sistemes anteriors, s’han dissenyat dues noves configuracions per poder millorar la producció de sofre elemental. La primera ha consistit en l’addició d’una cel·la electroquímica per tal d’oxidar el sulfur d’hidrogen en l’ànode permetent el control del potencial i així poder-ne controlar la producció. La segona configuració ha consistit en l’addició d’una cel·la de combustible amb un càtode exposat a l’aire aprofitant la capacitat del sulfur d’hidrogen a ser oxidat en un ànode espontàniament i així produir energia en comptes de requerir-la en el procés d’oxidació.
Las actividades industriales tales como las industrias papeleras, farmacéuticas, minera, de procesado de alimentos, etc. generan aguas residuales con un alto contenido en sulfato. El sulfato como tal no resulta muy perjudicial para la salud, pero si se vierte en ríos o sistemas de alcantarillado, los microorganismos conocidos como bacterias reductoras de sulfato (sulfate reducing bacteria, SRB) lo pueden transformar en sulfuro de hidrógeno. El sulfuro de hidrógeno es un compuesto que huele mal, es corrosivo y se ha demostrado tóxico incluso a bajas concentraciones. Por estos motivos, el tratamiento de efluentes ricos en sulfato es indispensable. Además, la recuperación de azufre elemental de estos efluentes para poder ser reutilizado como fertilizante o materia prima en la industria es una oportunidad de recuperación de recursos en el marco de la economía circular. Los sistemas bioelectroquímicos (bioelectrochemical systems, BES) son una tecnología innovadora basada en la habilidad de algunas bacterias de intercambiar electrones con un electrodo sólido. Últimamente, el estudio de los BES se ha focalizado en el tratamiento de aguas residuales y en la recuperación de productos gracias a la actividad de los microorganismos que colonizan los electrodos. En esta tesis se ha estudiado el uso de BES para el tratamiento y recuperación de compuestos de azufre, concretamente, el tratamiento de estas aguas residuales con sulfato. El sistema permite la reducción de sulfato en un biocátodo mientras en el ánodo se produce la electrólisis del agua para generar el flujo de electrones necesario. Los microorganismos que colonizan la superficie del cátodo utilizan el hidrógeno generado a partir de los electrones para transformar el sulfato en sulfuro de hidrógeno. Sin embargo, los resultados obtenidos han demostrado que gracias a la electrólisis del agua que tiene lugar en el ánodo se produce un flujo de oxígeno hacia el cátodo que permite el crecimiento de microorganismos capaces de producir azufre a partir del sulfuro de hidrógeno, llamados bacterias oxidantes de sulfuro (sulfide oxidizing baceria, SOB). Para mejorar la eliminación de sulfato y la producción de azufre se estudió como el pH del compartimento del cátodo y el potencial de cátodo podían influir en el proceso. Se observó que el pH neutro (pH = 7) era más beneficioso ya que un pH ácido (pH = 5.5) podría inhibir la actividad de las SRB y un pH básico (pH = 8.5) requería más energía para conseguir resultados similares debido a la limitación en la producción de hidrógeno a un pH elevado. En cuanto al potencial del cátodo, se pudo observar que a menores potenciales, mayor eliminación de sulfato, pero a partir de un potencial de -1.0 V vs. SHE, el sistema no podía aumentar la velocidad de eliminación. Además, también se ha estudiado el tratamiento de agua residual real procedente de un sistema de desulfuración de gases de combustión. Se ha observado que con el agua real la eliminación de sulfato se reducía, pero en cambio la producción de azufre elemental aumentaba. Finalmente, dado que el flujo de oxígeno del ánodo al cátodo no se podía controlar con los sistemas anteriores, se han diseñado dos configuraciones nuevas para mejorar la producción de azufre elemental. La primera ha consistido en la adición de una celda electroquímica para oxidar el sulfuro de hidrógeno en el ánodo permitiendo el control del potencial y así poder controlar la producción. La segunda configuración ha consistido en la adición de una celda de combustible con un cátodo expuesto al aire aprovechando la capacidad del sulfuro de hidrógeno a ser oxidado en un ánodo espontáneamente y así producir energía en vez de requerirla en el proceso de oxidación.
Industrial activities such as paper, pharmaceutical, mining, food processing, etc. generate wastewater with high sulfate content. Sulfate as such is not very harmful to health, but if it is poured into rivers or sewage systems, the microorganisms known as sulfate reducing bacteria (SRB) can transform it into hydrogen sulfide. Hydrogen sulfide is a compound with bad odour, is corrosive and has been shown toxic at low concentrations. For these reasons, the treatment of sulfate-rich effluents is essential. In addition, the recovery of elemental sulfur from these effluents in order to be reused as fertilizer or raw material in the industry is an opportunity to recover resources in the framework of the circular economy. Bioelectrochemical systems (BES) are a novel technology based on the ability of some bacteria to exchange electrons with a solid electrode. Lastly, the study of the BES has focused on the treatment of wastewater and the recovery of products thanks to the activity of the microorganisms that colonize the electrodes. In this thesis, the use of BES for the treatment and recovery of sulfur compounds was studied, specifically, the treatment of these wastewaters with sulfate in a biocathode. The system allows the reduction of sulfate at a biocatode while at the anode electrolysis of water occurs to generate the necessary electron flow. The microorganisms that colonize the surface of the cathode use the hydrogen produced from the electrons to transform the sulfate into hydrogen sulfide. However, the results obtained showed that thanks to the water electrolysis that takes place at the anode an oxygen flow to the cathode is generated, allowing the growth of microorganisms capable of producing sulfur from hydrogen sulfide, called sulfide oxidizing bacteria (SOB). The influence of pH of the cathode compartment and the cathode potential was studied in order to improve sulfate removal and sulfur production. It was observed that neutral pH (pH = 7) was more beneficial since an acidic pH (pH = 5.5) could inhibit the activity of the SRB and a basic pH (pH = 8.5) required more energy to achieve similar results due to the limitation in the production of hydrogen at a high pH. Regarding the potential of the cathode, it could be observed that lower potentials led to greater sulfate removal rate, but from a potential of -1.0 V vs. SHE, the system could not increase the removal rate. In addition, the impact of real wastewater coming from a flue gas desulphurization system in the system was also studied. It was observed that with real water the sulfate removal decreased, however, the production of elemental sulfur increased. Finally, since the oxygen flow from the anode to the cathode could not be controlled with the previous systems, two new configurations were designed to improve the production of elemental sulfur. The first one consisted in the addition of an electrochemical cell to oxidize the hydrogen sulfide at the anode, allowing the control of the potential and thus controlling the production. The second configuration consisted in the addition of a fuel cell with a cathode exposed to the air taking advantage of the capacity of the hydrogen sulfide to be oxidized at an anode spontaneously and thus produce energy instead of requiring it in the oxidation process.

Heart valve structures open and close during the cardiac cycle to provide unidirectional blood flow through the heart, critical for efficient cardiovascular function. Valve dysfunction results in either incomplete opening or incomplete closure of the valve. Both types of valve dysfunction decrease efficiency of blood flow, increasing the load on the myocardium and leading to secondary heart disease such as pathological hypertrophy and heart failure. There are currently no effective treatments to prevent or slow the progression of valve disease, and there are no pharmacological treatments for advanced valve disease. Although most valve disease is associated with aging, increasing evidence suggests that valve disease often has origins in development. Congenital valvuloseptal defects affect many newborns, ranging from life-threatening malformations requiring immediate repair to more subtle, often undiagnosed defects that increase susceptibility to valve disease later in life. Therefore, an improved understanding of the mechanisms of heart valve formation and maintenance of adult valves may serve as an important step in improving valve disease treatment options. In this work, the mechanisms of normal valve development and the role of Sox9 in developing and mature valves are further studied. The temporal and spatial expression of extracellular matrix genes and proteins are examined throughout normal murine valve development. Sox9 function in the processes of valve development and valve maintenance is examined using mouse models of conditional Sox9 loss-of-function. Heart valve phenotypes in mice with reduced Sox9 function are examined throughout development and in adult mice with resultant calcific valve disease. The possible causative mechanisms of calcific valve disease in mice with reduced Sox9 function are further investigated by identification of novel possible targets of Sox9 transcriptional regulation. Together these studies improve our understanding of heart valve development, characterize a model of heart valve calcification with genetic etiology, and identify and characterize novel targets of Sox9.

Despite recent advances in oncology and extensive research efforts, gliomas remain essentially incurable. Glioblastoma multiforme (GBM, WHO grade IV) is the most common glioma and may arise de novo or progress from a lower-grade lesion. GBM is characterized by invasive growth, aberrant angiogenesis and necrosis. The heterogeneity of GBM is further complicated by the contribution of the inflammation that is facilitated by immune cells that reside in and infiltrate this immuno-privileged organ. One of the cells types present in the tumor microenvironment are mast cells (MC) that accumulate in the tumor in a grade-dependent manner. GBM cells secrete a plethora of cytokines acting as chemoattractants in MC recruitment and to a lesser degree induce MC proliferation in situ. Expression of one of the cytokines secreted by GBM cells - macrophage migration inhibitory factor (MIF) - correlates with MC accumulation in vivo. GBM cells invade the surrounding parenchyma making complete resection impossible. Here, migration was studied with the focus on RAP1 and its negative regulator RAP1GAP. Activation of RAP1 signaling by lentiviral silencing of RAP1GAP lead to decrease in cell migration and a shift in expression of SOX2 and GFAP, presumably enhancing stem cell phenotype. MicroRNAs are small non-coding RNAs known to regulate the mRNA network. miR-21 is highly overexpressed in the majority of cancers including GBM. Its expression is strictly regulated during embryonic development of the brain. SOX2 is co-regulated with miR-21 demarcating a cell population with neural/glial progenitor/stem cell properties. In an experimental mouse model, expression of miR-21 can be sustained by forced expression of PDGF-BB leading to gliomagenesis. GBM cells seem to be addicted to oncogenic properties of miR-21 as its knockdown leads to extensive apoptosis. This observation combined with the fact that miR-21 is absent in the normal adult mammalian brain suggest miR-21 to be an excellent therapeutic target. Effects of conventional therapy (surgery combined with radiochemotherapy) on prolonging patient survival have reached a plateau. New effective personalized therapeutic modalities need to be designed and implemented. Targeting the tumor microenvironment as well as cell intrinsic properties like invasive potential, stemness and onco-miR addiction studied in this thesis will hopefully lead to efficient disruption of GBM’s aberrant ecosystem.

The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.

Pluripotency is defined as the capacity to differentiate into cells from each of the three primary germ layers, the ectoderm, mesoderm and endoderm. This is a property of cells located in the inner cell mass (ICM) of preimplantation blastocysts and in the epiblast layer of postimplantation, presomite embryos. Preimplantation and postimplantation pluripotency can be captured indefinitely in cultured embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively. Preimplantation pluripotency in ES cells is regulated by a network of genes centred on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a mutually-reinforcing circuit and cooperatively stimulate transcription of downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in the postimplantation epiblast. Functional studies established a role for Oct4 and Nanog in the specification of ICM cell identity, and a role for Oct4 in the maintenance of postimplantation pluripotency. Although the role of Sox2 in preimplantation ICM cells is unclear, it is critical for the establishment of egg cylinder following implantation and indispensable for ES cell pluripotency. However, despite the presence of Sox2 in postimplantation pluripotent cells the role of Sox2 in postimplantation pluripotency is unknown. In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog are expressed in opposing gradients in the gastrulation-stage postimplantation epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly suggests that coexpression of Sox2 and Oct4 is important for establishing postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively correlated with NanogHIGH cells. This reciprocal relationship emerged during the transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively influences Sox2 expression post-transcriptionally. The negative influence of Nanog on Sox2 protein level was confirmed using doxycycline-inducible Nanog overexpressing EpiSCs. This negative relationship indicates that the regulation of Sox2 expression is different in postimplantation pluripotency and that Nanog may negatively regulate Sox2 on the protein level in the posterior epiblast. Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this was further investigated by microarray transcription profiling using cells in which a fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2- tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated cell identity and Sox2-tdTomatoLOW cells are associated with non-neural differentiation. Interestingly the global profile of ES cells and EpiSCs that share similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different roles in different pluripotent states. ES cells with enforced Sox2 expression were unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on the maintenance of EpiSC pluripotency. This is likely due to the presence of redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in ES cells. Taken together, these results suggest the hypothesis that postimplantation pluripotency is maintained by multiple Sox factors, while Nanog negatively regulates Sox2 post-transcriptionally to repress neural specification in the posterior epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible negative feedback loop to balance the proneural and pluripotent properties of Sox2 in postimplantation pluripotency.

Nous avons montré que SOX9 était exprimé in vitro et in vivo par les mélanocytes et jouait un rôle clé dans la mélanogenèse induite par les radiations ultraviolettes de type B (UVB). Activé par l’AMP cyclique puis de la protéine kinase A (PKA), SOX9 va augmenter l’expression des enzymes de la mélanogenèse en agissant directement sur le facteur de transcription MITF, augmentant in fine la production de mélanine au sein des mélanosomes. Par ailleurs, nous avons également montré qu’Agouti Signaling Protein (ASP), connue pour inhiber la mélanogenèse, était capable de diminuer l’expression de SOX9 dans les mélanocytes. Cette action permet d’expliquer, du moins en partie, le mécanisme d’action encore méconnu d’ASP. Dans un second temps nous avons étudié l’expression et le rôle de SOX9 dans les cellules de mélanome. Nous avons montré que l’expression de SOX9 était faible ou nulle dans la grande majorité des biopsies de mélanomes que nous avons analysées. SOX9 inhibe la prolifération des cellules de mélanomes en activant directement et indirectement le promoteur de p21. Par ailleurs, nous avons montré que SOX9 pouvait restaurer la sensibilité des cellules de mélanome à l’acide rétinoïque en diminuant l’expression de PRAME. La surexpression de SOX9 réduit sensiblement la tumorigenicité des cellules de mélanomes in vivo chez la souris et ex vivo sur des modèles de peaux reconstruites humaines. Nous avons enfin montré que des agents capables d’augmenter ou d’activer SOX9, tels que la prostaglandine D2 (PGD2), reproduisaient son action sur la prolifération des mélanomes et permettaient également de restaurer la sensibilité à l’acide rétinoïque
We showed that SOX9 is expressed in vitro and in vivo by melanocytes and plays a key role in ultraviolet B (UVB) induced melanogenesis. After the activation by cyclic AMP and protein kinase A (PKA), SOX9 increases the expression of melanogenic enzymes by acting directly on the transcription factor MITF, leading to an increased production of melanin within melanosomes. We also showed that Agouti Signalling Protein (ASP), known to inhibit melanogenesis, was able to decrease the expression of SOX9 in melanocytes. This action could explain, at least partially, the poorly understood mechanism of action of ASP. Then, we studied the expression and the role of SOX9 inhibits the proliferation of melanoma cells by acting and indirectly on p21 promoter. In the same time, SOX9 restores the sensitivity of melanoma cells to retinoic acid by decreasing the expression of PRAME. The over expression of SOX9 strongly reduces the tumorigenicity of melanoma cells in vivo in mice and ex vivo in reconstructed skin models. Finally, chemical agents, capable of increasing or activating SOX9, such as prostaglandin D2 (PGD2), reproduce its action on proliferation of melanomas and also restore the sensitivity to retinoic acid

京都大学
新制・課程博士
博士(医学)
甲第23374号
医博第4743号
新制||医||1051(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 藤田 恭之, 教授 椛島 健治, 教授 斎藤 通紀
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM

Made available in DSpace on 2016-03-15T19:32:36Z (GMT). No. of bitstreams: 1 Anderson Herold.pdf: 812968 bytes, checksum: 0752767c7ec3a71a980b4bb6a7463ca0 (MD5) Previous issue date: 2013-03-06
Banco Itaú S.A.
The purpose of this dissertation was to investigate the contribution of compliance of Sarbanes-Oxley Act (SOX) as innovation in financial institutions, and also identify the main factors that affect the innovation (SOX) adoption process. There is a vast study on efficiency, Information Technology (IT) and also about innovation, however, all works on these themes aimed at exploring them as possible source of competitive advantage. It is noteworthy that before anything could be done, one should ensure the appropriate use of innovation, be it IT or other area, that will bring improvements to users and to the organization at large. In this study we decided for the financial sector as it is one of the sectors most audited and controlled, at the same time one of the most innovative sectors that constantly offer new type of services and facilities to its customers. For the development of the research we employed qualitative and quantitative nature techniques. Qualitative analysis was performed through the content analysis in order to identify how the adoption of an innovation affected the performance of the organization. In the quantitative aspect, it is characterized as descriptive, where questionnaires were responded by users of SOX in order to ascertain how they perceived the characteristics of an innovation in the use of compliance and how it impacts the performance of their services and processes. The multivariate factor analysis was employed. Overall, results of the study indicated that the process of innovation, that is SOX compliance in the financial sector depends on a number of factors among which we highlight the statement of adoption of innovation, relative advantage obtained with the adoption, the individuals and the institutional image portrayed with adoption, usability, experimentation innovation in decision making and voluntary usage in the organization. These interrelated factors could effectively contribute to innovation adoption in the financial sector and consequently trigger improvements in control processes across the organization.
A proposta desta dissertação foi estudar a contribuição de compliance da Sarbanes-Oxley Act (SOX) como inovação em instituições financeiras, onde são identificados os principais fatores que afetam o processo de adoção da inovação (SOX). Existe um material vasto de pesquisas sobre eficiência, sobre Tecnologia da Informação (TI) e também sobre inovação. Todos os trabalhos preocupados em apresentar esses temas como possível fonte de vantagem competitiva. Antes de qualquer coisa deve-se garantir o uso adequado da inovação, seja ela de TI ou de outra área, pois assim será possível trazer melhorias aos usuários e para a organização. Neste estudo optou-se o setor financeiro, pois e um dos setores mais auditados e controlados e ao mesmo tempo um dos setores mais inovadores que constantemente disponibilizam novos tipos de serviços e facilidades aos seus clientes. Para o desenvolvimento da pesquisa empregou-se técnicas de natureza Qualitativa e Quantitativa. A análise qualitativa foi efetuada por intermédio da analise de conteúdo para poder identificar de que forma a adoção de uma inovação pode afetar o desempenho da organização. Na parte quantitativa, caracterizada como descritiva, foram aplicados questionários fechados junto a usuários da SOX, com o propósito de verificar de que maneira os usuários percebem as características de uso em uma inovação de compliance e como ela impacta na realização dos serviços e processos. Na analise quantitativa empregou-se técnicas estatísticas multivariadas como a Analise Fatorial. Os resultados do estudo indicaram que o processo de adoção de inovações de compliance no setor financeiro depende de um conjunto de fatores entre os quais se destacam a demonstração do resultado do uso da inovação, vantagem relativa que se obtém com a adoção da inovação, a imagem, a visibilidade que a inovação proporciona aos indivíduos e a instituição, a usabilidade, a experimentação da inovação no processo de decisão e o uso voluntario pelos usuários da inovação. Estes fatores interligados podem contribuir efetivamente na adoção de inovações no setor financeiro e consequentemente desencadear melhorias nos processos e controles em toda a organização.

The thesis deals with the Sarbanes -- Oxley Act of 2002 (SOX). Reasons leading to its acceptance and the consequences are analysed. The costs and benefits of SOX implementation are compared from the individual and global view. The process of implementation is explained on the example of purchasing and account payable.

Les anomalies du développement sexuel recouvrent un spectre phénotypique large. Les hommes XX présentent dans la majorité des cas un développement testiculaire normal, lié à la présence SRY sur un des chromosomes X. Dans 10% des cas, aucune cause n’est retrouvée. Chez les femmes, l’origine de l’insuffisance ovarienne prématurée (IOP) n’est identifiée que dans 20% des cas. L’objectif de cette thèse a été d’identifier de nouveaux mécanismes moléculaires impliqués dans le développement gonadique, testiculaire et ovarien, ainsi que dans son fonctionnement. L’étude d’une cohorte de patients DSD 46,XX SRY négatifs a mis en évidence une duplication de la région RevSex dans un désert génique en amont de SOX9 chez quatre patients. Ceci a permis de redéfinir la région minimale impliquée dans l'activation de l'expression de SOX9 à une taille maximale de 41.9 kb et de proposer un mécanisme permettant cette expression chez des hommes XX. Le séquençage d’exome chez dix patients de la cohorte n’a pas mis en évidence de mutations dans des gènes d’intérêts. L’ensemble de ces résultats pose la question du rôle des régions régulatrices dans la survenue des DSD.L’analyse d’une cohorte de patientes ayant une IOP a permis d'identifier des délétions incluant le gène CPEB1. Des études précédentes chez la souris ont montré son implication dans le développement folliculaire. Le séquençage du gène CPEB1 dans la cohorte n'a pas mis en évidence de mutation pathogène. Ce travail a permis de montrer que la délétion impliquant le gène CPEB1est une cause rare mais récurrente d'IOP et concerne environ 1% des patientes. Une microdélétion contenant le gène CASP3 un gène de la voie des caspases impliquée dans la régulation du pool folliculaire a également été identifiée chez une patiente. L'ensemble de ces résultats montre l'intérêt de l'étude génétique des patients présentant une anomalie du développement de la gonade ou de son fonctionnement par des techniques d'étude globale du génome
Disorders of Sex Development (DSD) can be identified in new-born and during infancy but also in adults because of infertility. Most 46,XX testicular DSD have a normal testicular development due to the presence of the SRY gene at the tip of one of their X chromosome. However, the genetic causes of 46,XX-SRY negative testicular DSD remain poorly defined. In women, disorders of gonadal development can be responsible for primary ovarian insufficiency (POI) and genetic causes are identify in only 20% of cases. The aim of this thesis was to identify molecular mechanisms involved in gonadal development and in its functioning. The cohort study of 46,XX testicular DSD identified four patients with a duplication in the previously reported RevSex region located about 550 kb upstream of SOX9. One duplication allowed us to refine the minimal region associated with 46,XX-SRY negative DSD to a 40.7–41.9 kb element. Exome sequencing of 10 patients from the cohort did not show any mutation in genes implicated in DSD or in new candidate genes. These results raise questions about the role of the regulatory sequences in the onset of DSD.The cohort study of POI patients identified three patients carrying a microdeletion including CPEB1 a good candidate gene for POI as study in mice showed the implication of CPEB1 in follicular development. Sequencing CPEB1 gene did not identified any mutation. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. This microdeletion is rare but recurrent and was identified in about 1% of patients with POI. Another microdeletion containing CASP3 gene that belongs to the caspase family, which is implicated in the regulation of the follicular pool, was identified in a patient. Further studies are needed to confirm the role of CASP3 in POI. These results demonstrate the importance of genetic study of patients presenting with DSD or POI using whole genome techniques

Syfte:

Vårt samhälle går igenom en stor förändring genom att industrisamhället alltmer ersätts med dagens informationssamhälle. Denna förändring lämnar ingen människa oberörd utan alla berörs på något sätt, endera privat eller i arbetslivet. Syftet med denna uppsats är att jag vill skapa en uppfattning hur IT utvecklingen har förändrat företagens sätt att bedriva handel och hur det i sin tur har påverkat revisionen och revisorernas metoder och arbetssätt.

Metod:

Då arbetet var tänkt att undersöka om denna IT utveckling har förändrat revisionen eller revisorns arbetssätt, har jag valt att undersöka detta utifrån en kvalitativ metod i den empiriska undersökningen för att på detta sätt få så uttömmande svar som möjligt. Mitt syfte med de respondenter som jag valde, var att se om de skiljde något i deras uppfattning beträffande metoder och arbetssätt i och med IT utvecklingen. Jag valde därför respondenter med snarlika förutsättningar. De arbetade alla inom stora revisionsbyråer med liknande arbetsuppgifter och med kunder inom både små och stora företag. Eftersom målsättningen inte varit att göra en kvantitativ undersökning gjordes valet av respondenter inte slumpmässigt.

Resultat & slutsats:

I min undersökning har jag kommit fram till att företagens förändrade handelssätt och IT utvecklingen varit en stor bidragande orsak till revisionens och revisorns förändring av arbetssätt. Men det är inte bara IT utvecklingen som har skapat dessa förändringar. Revision och revisorer påverkas mycket av lagar och regler, så dessa förändringar har även skapats på grund av att dessa har förändrats. Den största förändringen som framkom med revisionen var att pappersdokumenten harförsvunnit alltmer och ersatts med elektroniska dokument som idag inte skrivs ut som det gjordes tidigare, utan idag sparas dokumenten på olika optiska medium. En IT revision är ingen fristående revision utan den ingår i den traditionella revisionen. Det som granskas vid en IT revision är, att företagens operativsystem, nätverk och databaserfungerar på ett tillfredsställande sätt och det som är viktigt att granska är intern kontroll och IT säkerhet. För att granska detta så använder sig revisorn allt mer av olika datoriserade revisionsverktyg och standarder. Dagens affärssystem integreras med ehandel produktionsstyrning och resultatet är att systemen blir alltmer sammansatta. Så för att granska de komplexa systemen så har efterfrågan på särskilt utbildade IT revisorer ökat i branschen. I både teori och praktik så framkom det under arbetets gång,att om IT revision utförs av revisorer med en gedigen kunskap av IT system så ansågs det att det kommer att skapa en mer trovärdig revision och att revisionen kommer att genomföras på ett säkrare och mer effektivt sätt, vilket i sin tur gynnar hela samhället.

Förslag till fortsatt forskning:

Ett skäl som framkom under arbetets gång till att marknaden efterfrågar en mer gedigen IT kompetens förutom IT systemens komplexitet var den lag som infördes i USA 2002 (Sarbanes Oxley Act). Denna lag infördes efter de uppmärksammade redovisningsskandalerna i USA och innebär hårdare redovisningskrav framförallt på revisorers oberoende och högre krav på den interna kontrollen. Lagen påverkar även Svenska företag som är registrerade på den amerikanska börsen fr.o.m. den 15 Juli 2005. I både teori och praktik så framkom det att troligtvis kan denna lag innebära att de Svenska företagen som är noterade på den amerikanska börsen kan komma att lyda under två regelverk. Det skulle vara intressant att undersöka hur denna lag kommer att påverka revisorernas arbetssätt.

Uppsatsens bidrag:

Internet uppfanns redan på 1960-talet och mycket har hänt sedan dess. Från början så fanns det inget vinstintresse med Internet, utan det var framförallt studenter från USA och runt om i världen som använde Internet eller ARPAnet som det kallades för att maximera den lilla dataprestanda som fanns på den tiden. Men i slutet av 1990-talet så hade sättet utvecklats hur man på nätet både kunde distribuera information och betjäna människor genom att utföra beställningar, läsa eller sända information dygnet runt. Detta har gjort att Internet har utvecklats till en stor och vinstdrivande marknadsplats. Som en följd av denna samhällsutveckling och den snabba IT utveckling som har skett, blir IT teknologin en allt viktigare framgångsfaktor i dagens affärsprocesser. För att företag ska kunna vara framgångsrika och överleva på dagens globala och konkurrensutsatta marknad bör de hänga med i denna utveckling. För att klara Internet och e-handel så måste företagen investera i olika former av affärssystem.Ett affärssystem är en programvara som används för att styra olika processer t.ex. inom försäljning, e-handel eller ekonomi. Dessa system utvecklas till att blir alltmer komplexa, detta leder i sin tur till att det ställs allt högre krav på kompetensen både inom företaget samt på revisorerna som ska granska dessa IT system.

Aim:

Our community is going through a huge change because nowadays information community is replacing the industrial community. This change leaves no human untouched, every one is affected somehow either private or in operational life.The aim with this study has been to create an understanding for my self and the reader how IT development has change the audit and auditors methods and their way of work.

Method:

Since the aim with this study was to investigate and to get such a full picture of the subject as possible if the IT development has change the audit and the auditors methods and way of work have I chosen to investigate this subject based on quality method in the practical search. My arrangement with the respondents that I choused was to see if there opinion differs from each other if IT development has change there methods and way of work. I choose auditor in similar situations. They all worked in big accounting firms with similar tasks and their customers were both from small and big firms. The plan with this study wasn’t to make a quantity investigation, therefore the respondents wasn’t chosen by random.

Result & Conclusions:

In this study I have skilled that the IT development has strongly contributed to change the working situation for the audits and the way they work. But it’s not only the IT development that has contributed to these changes. Laws and rules have also have a large affect on the audit and Auditors. The biggest change I discovered in the auditors way of work was that documentation on paper is less common nowadays. Today documentation is done on optical media and it’s not printed out like it used to do. An IT audit is not an isolated audit; it is a part of the traditional audit. The things that are highlighted in an IT audit are that the operating systems, networks and databases all work in a god manner. Another things that are important to look into will be matter such as internal control and security regarding IT. To review these areas use the auditors more often nowadays-special data auditor tools. In today’s business systems ecommerce is integrated with processing controls, which results in more complex systems. To audit this complex systems the market demands for specially educated IT auditors have increased. In both theory and practice it came to light during the work process, that if the IT audit is preformed by auditors with a genuine knowledge of the systems that they are auditing it will accomplish an audit with high credibility. And the audit will be preformed safer and more efficient, which is a benefit for all of society.

Suggestions for future research:

One of the reasons that emerge during the research to why the market demands a genuine IT knowledge, other than the complexity of the systems, is the law how become active in USA 2002, the Sarbanes Oxley Act. This law was activated after the audit scandals in USA and involve higher demands when it comes to the auditor’s independence and internal control. This law also affects Swedish company’s who is registered on the US stock exchange from 15 July 2005, witch means that they have to take consideration to two different laws. It would be interesting to study how this law will affect the auditors work procedures.

Contributions of the thesis:

Internet was invented in the sixties and much has happened since then. In the beginning profit was not the motivation with Internet, the users was students in the U.S. and around the world, and their target was to maximize the little computer recourses that was available in those days. At the end of the ninetieth methods has been developed how to use the Internet for distributing information and offer customer services such as ordering, reading and send information around the clock. This has created Internet to expand into a big and profitable marketplace. As a direct consequence of the fast IT growth and the development in the society, the IT technologies are playing a bigger part as a way to success in today’s business processes. If a company wants to survive and make progress in today’s global and highly competitive state of affairs they must keep up with this development. To be able to work with Internet and e-business, must the companies invest in different kinds of information-systems. An information-system is software that is used to control different types of processes for example selling, e-business or finance. These systems are constantly under development and getting more and more complex, for this reason demands on competence of the people who handle these systems are constantly getting higher, both inside the company and on the auditor who will verify these IT systems.

Glioma is the most frequent primary tumor of the central nervous system. By using the RCAS/tv-a mouse glioma model, we have studied mechanisms controlling glioma development and the effect of cell of origin on these processes. SOX5 was identified as a brain tumor locus in a retroviral insertional mutagenesis screen of PDGF-B induced mouse gliomas. Here we found that SOX5 could suppress PDGFB-induced glioma development particularly in Ink4a-/- mice. Analysis of putative PDGF-B signaling pathways revealed that the underlying mechanism could involve the activation of AKT and p27, which caused an acute cellular senescence. When cultured in a highly selective serum free medium, glioma-initiating cells could be isolated from mouse GBMs and their self-renewal and proliferation was independent on exogenous EGF and FGF2. Addition of serum into the medium induced aberrant differentiation that was reversible. Specific depletion of viral PDGF-B demonstrated that PDGF-B was necessary for stemness and tumorigenicity of GICs by preventing them to differentiate. Subsequently, by applying the same culture conditions, GICs of APC, NSC and OPC origins were isolated from mouse GBMs. GICs derived from NSCs exhibited higher self-renewal, faster proliferation and more potent tumorigenicity than those of APC or OPC origin. Furthermore, addition of 5% serum significantly inhibited the proliferation of APC- and OPC-derived GICs, but did not in NSC-derived GICs. Transcriptome analysis revealed that GICs of the same cell of origin displayed distinct expression profiles. In the last study, we showed that OPCs could serve as the origin for astrocytic glioma. Results from immunostainings revealed that these tumors might belong to a different molecular subtype than the oligodendroglial tumors induced in OPCs. We also found differences in tumorigenic potential between OPCs in neonatal and adult mice, which suggest that developmental age of the cell of origin is important for its susceptibility to oncogenic transformation.

Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.

SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.

Pax3 has numerous integral functions in embryonic tissue morphogenesis while knowledge of its complex function in cells of adult tissue continues to unfold. Across a variety of adult tissue lineages, the role of Pax3 is principally linked to maintenance of the tissue’s resident stem and progenitor cell population. In adult peripheral nerves, Pax3 is reported to be expressed in nonmyelinating Schwann cells, however, little is known about the purpose of this expression. Based on the evidence of its role in other adult tissue stem and progenitor cell maintenance, it was hypothesised that the cells in adult peripheral nerve that express Pax3 may be Schwann glioblasts. Here, methods have been developed for visualisation of Pax3 expressant cells in normal 60 day old mouse peripheral nerve. Visualisation allowed morphological, anatomical and phenotypic distinctions to be made between these Pax3 expressing cells and Remak bundle nonmyelinating Schwann cells. The distinctions described herein, together with the finding that Pax3 expressing cells co-express stem cell marker Sox2, provides compelling support for the suggestion that a progenitor Schwann cell population may be present in adult mouse peripheral nerve.

In the first essay using a sample of 3437 U.S. companies over the period 1992-2014, I demonstrate that international business activities of newly listed firms influence their corporate policies. Specifically, firms earning foreign pre-tax income at an early phase of their growth and development have higher investment and a higher propensity to acquire. I show that cash holdings are lower for firms involved in foreign activities, supportive of Duchin’s (2010) coinsurance theory. Further, CEOs of globally diversified firms have less pay-for-performance sensitivity than those of purely domestic firms. The second essay examines the impact of the Sarbanes-Oxley Act (SOX, 2002) on excess valuation calculated with the “chop shop” approach, which is typically used to measure the diversification discount. The results indicate a significant drop in excess valuation after SOX for both pure-play and multi-segment companies. Additional investigation of the calculation methodology and a difference-in-differences model show no distinction in this impact between un-accelerated and accelerated companies. There is no evidence to support that the Sarbanes-Oxley Act leads firms to diversify or focus. I run several robustness tests by including 2003 observations, creating a 2000-2006 subsample, excluding geographic segments. Finally, when in a firm's life would it fit for it to become involved in global strategies? What are the important influences on the decisions of young and mature firms to go international? I answer these questions in the third essay by examining the determinants that affect the choices of born-globals (BGs) and born-again globals (BaGs) to expand worldwide. My study is based on pre-existent theories of diversification, and I place specific emphasis on the conceivable role of peer influence and the motivation or desire for growth. I further study the entrenchment, the idiosyncratic risk, and the innovation caliber hypothesis. My results document that innovation efficiency strongly enhances BG’s propensity to global diversify. On the other hand, peer pressure, CEO ownership and idiosyncratic risk level significantly influence BGs not to globalize. In contrast, BaGs are positively influenced by their industry peers, showing how competition works in the financial markets for youthful versus mature companies.

I examine firm governance characteristics for a sample of companies disclosing material weaknesses under section 404 of SOX to examine what factors impact the likelihood that a company will disclose those material weaknesses prior to their first section 404 report (under section 302 reporting requirements). I find companies that were audited by industry leading auditors, that have higher quality audit committees, that have shorter auditor/client relationships, that recently restated their financial statements or have been the subject of an SEC AAER, or that have experienced poor financial performance are more likely to discover and disclose weaknesses in their controls under section 302. I find moderate evidence of a positive relationship between company's that have a CFO with financial accounting background and disclosure prior to the SOX 404 report and a negative relationship between a company's institutional ownership concentration and the probability that they disclose weaknesses in their controls prior to the SOX 404 report. In sensitivity tests, I find a positive relationship between a company's institutional ownership concentration and the probability that they disclose significant deficiencies in their controls prior to the SOX 404 report suggesting systematic misclassification of control problems as significant deficiencies rather than material weaknesses in high institutional ownership concentration settings.