Os genes Hox padronizam o corpo dos vertebrados durante o desenvolvimento embrionário, e a compreensão de sua evolução pode elucidar mecanismos genéticos subjacentes à evolução morfológica. A evolução molecular dos genes Hox imprime assinaturas em regiões regulatórias, as quais potencialmente afetam sua expressão gênica, como os elementos cis-regulatórios (CREs) que ladeiam o cluster D de Hox e seus RNAs não-codificantes (ncRNAs). Essa Tese de Doutorado enfoca a evolução regulatória de genes HoxD envolvidos no estabelecimento dos eixos corpóreos axial ântero-posterior (AP) e apendiculares em linhagens de aminiotas que exibem características morfológicas homoplásticas peculiares: os squamatas serpentiformes (Capítulos I e II) e os marsupiais Diprotodontia (Capítulo III). No Capítulo I investigou-se, em serpentes e anfisbênias, se assinaturas regulatórias envolvidas no estabelecimento das morfologias serpentiformes foram impressas na Sequência Conservada B (Conserved Sequence B, CsB), um CRE centromérico de Hoxd10-13. Usando lagartos e outros tetrápodes como referência para a morfologia serpentiforme, regiões conservadas de CsB foram sequenciadas em 38 espécies de Squamata, cujos TFBS foram preditos e comparados. Ambas linhagens serpentiformes exibem assinaturas regulatórias divergentes e convergentes ausentes em lagartos; a convergência localizou-se em um segmento de CsB que concentra perda nas linhagens serpentiformes de diversos TFBS com funções no desenvolvimento de membros e a aquisição de um sítio de ligação para PBX1. Essa assinatura convergente impressa durante evoluções independentes da morfologia serpentiforme pode estar relacionada à elongação corpórea e à perda dos membros, evidenciando um papel do CsB no desenvolvimento do eixo AP. No Capítulo II, foi investigado se um CRE telomérico (CNS65) e um centromérico (Island I) de Hoxd, os quais regulam respectivamente regiões proximais e distais dos membros tetrápodes em desenvolvimento, retêm suas capacidades regulatórias em Serpentes. Expressões de gene repórter desses CREs de serpentes foram realizadas em camundongo transgênico, revelando deficiência de suas atividades regulatórias nos brotos de membro. A comparação dos TFBS preditos nesses elementos entre serpentes e outros tetrápodes revelou que TFBS relacionados ao desenvolvimento dos membros foram perdidos nas sequências das serpentes. Ainda, essa comparação indicou um elemento em CNS65 potencialmente envolvido especificamente na regulação da formação de estilopódio/zeugopódio, e três elementos na Island I exclusivamente reguladores do desenvolvimento autopodial. A perda de membros em x serpentes aparentemente imprimiu assinaturas nesses CREs de Hoxd que possivelmente contribuíram para sua degeneração funcional, putativamente indicando módulos específicos de regulação nos membros. No Capítulo III, ncRNAs do cluster D de Hox foram estudados no contexto da evolução morfológica do autopódio posterior e heterocronia entre o desenvolvimento de membros anteriores e posteriores em Macropus eugenii. Os ncRNAs mapeados sobre o cluster D de Hox foram selecionados a partir de transcritoma de membros de embriões de M. eugenii nos dias 23 (d23) e 25 (d25) de gravidez, e sua conservação, perfis transcricionais e padrões de expressão foram explorados. A comparação com sequências ortólogas de outros mamíferos revelou cinco ncRNAs conservados em mamíferos, e três aparentemente exclusivos dos marsupiais. Os perfis transcricionais de genes HOXD10-13 e dos ncRNAs do cluster D de Hox foram predominantemente equivalentes. Os padrões de expressão de XLOC46 foi similar aos dos genes HOXD terminais de camundongo e M. eugenii, enquanto que XLOC52 e XLOC53 apresentaram expressão idêntica à desses genes em M. eugenii, exceto pela baixa expressão de XLOC53 no d25. Os ncRNAs intergênicos/intrônicos aos genes HOXD9-12 possivelmente regulam a expressão de genes HOXD terminais em mamíferos, enquanto que XLOC52 e XLOC53 constituem bons candidatos para investigação relacionada à evolução dos membros de marsupiais. Esta Tese demonstra como estudos de assinaturas regulatórias na evolução de genes do desenvolvimento contribuem para o entendimento das histórias evolutivas de divergência entre linhagens e d / Hox genes pattern the vertebrate body during embryonic development, and understanding their evolution may unravel genetic mechanisms subjacent to morphological evolution. Molecular evolution of Hox genes entails signatures in regulatory regions that potentially affect gene expression, such as the cis-regulatory elements (CREs) that surround the HoxD cluster and its noncoding RNAs (ncRNAs). In this PhD Thesis, I have explored regulatory evolution of HoxD genes engaged in the development of appendicular and anterior-posterior body (AP) axes in amniotic lineages that exhibit homoplastic morphological peculiarities: snakelike squamates (Chapters I and II) and diprotodontid marsupials (Chapter III). In Chapter I, I investigated in snakes and amphisbaenians, whether equivalent regulatory signatures were registered in the Conserved Sequence B (CsB), a centromeric Hoxd10-13 CRE, during evolution of snakelike morphologies. Using lizards and other tetrapods to represent the lacertiform morphology, conserved regions within CsB were sequenced from 38 squamate species, and transcription factor binding sites (TFBS) were predicted and compared among groups. Both snakelike lineages carry divergent and convergent regulatory signatures not identified in lizards; the convergence located in one CsB segment comprised loss of limb-related TFBS and gain of a binding site for PBX1. This convergent regulatory signature registered along two independent processes of snakelike evolution may relate to body elongation and limb loss, and evidences a role of CsB for AP axis development. In Chapter II, I investigated whether a telomeric (CNS65) and a centromeric (Island I) Hoxd enhancer that regulate gene expression respectively at proximal and distal regions of developing limbs retain their regulatory capacities in Serpentes. Gene reporter expression of these CREs from snakes were performed in transgenic mice and revealed that their regulatory activities were abrogated in limb buds. Comparison of predicted TFBS in these elements between snakes and limbed tetrapods revealed limb-related TFBS apparently lost in snakes, and pointed to one potential stilopodium/zeugopodium-specific element in CNS65 and three likely autopodium-specific elements in Island I. Limb loss in snakes registered signatures in Hoxd CREs that possibly contributed for their functional impairment, putatively indicating limb-specific modules. Finally, in the chapter III, I studied ncRNAs from HoxD cluster in the context of hindlimb morphological evolution and heterochrony between fore and hindlimb development in the tammar wallaby Macropus eugenii. The ncRNAs mapped to HoxD cluster were selected from transcriptome performed using tammar embryo limbs at days 23 (d23) and 25 (d25) of viii pregnancy, and their conservation, transcriptional profiles and expression patterns were explored. Comparison with orthologous sequences in other mammals revealed five ncRNAs conserved among mammals, and three transcripts apparently exclusive to marsupials. Transcriptional profiles of HOXD10-13 and HoxD ncRNAs were mostly equivalent. XLOC46 expression patterns resembled those of mouse and tammar terminal HOXD genes, whereas XLOC52 and XLOC53 showed identical expression patterns to those genes of tammar, except for XLOC53 low expression at d25. The ncRNAs intergenic/intronic to HOXD9-12 may regulate expression of terminal HOXD genes in mammals, and XLOC52 and XLOC53 are suitable for investigation regarding limb evolution in marsupial. This PhD Thesis demonstrates how studies of evolutionary footprints in regulatory elements of developmental genes contribute for elucidating specific processes during lineages divergence as well as functional aspects of these genes during development.

No description available.

Einleitung: Die Osteoarthritis ist für einen Großteil der Lahmheiten beim Pferd verantwortlich. Durch die starken Belastungen der equinen Gelenke schreitet die Osteoarthritis unweigerlich fort und setzt diese Tiere einem hohen Leidensdruck aus. Bisherige Therapien reichen nicht aus, um in osteoarthritischen Gelenken die physiologische Integrität des Knorpels wiederherzustellen. Humane und caprine nasale Knorpelzellen zeigten in präklinischen und klinischen Studien ein hohes Regenerationspotential und eine hohe Integrität nach autologer Implantation in Defekte des Gelenkknorpels. Dies wurde auf ihr „Hox-Gen-negatives“ Expressionsprofil zurückgeführt, das sich nach der Implantation dem des artikulären Knorpels anpasste. Ziel der Studie: Das Hox-Genexpressionsprofil nasaler Chondrozyten sollte mit denen artikulärer Chondrozyten in der Zellkultur verglichen werden, um den nasalen Knorpel auch beim Pferd als mögliche autologe Knorpelquelle zu identifizieren. Tiere, Material und Methoden: Knorpelgewebe wurde von 7 verstorbenen Pferden aus dem Nasenseptum und einem vorderen sowie hinterem Fesselgelenk entnommen, Chondrozyten isoliert und bis zur vierten Passage zweidimensional kultiviert. Während der Kultivierung wurden die Chondrozyten alle 3 bis 4 Tage mittelseines eigens erstellten Beurteilungsbogens evaluiert. Zellen aus der ersten (T1) und der dritten (T2) Subkultivierung wurden lysiert, die RNA extrahiert und in cDNA umgeschrieben. Es folgte eine qPCR, um die Expressionslevel von drei Hox-Genen (A3, D1, D8) und zwei knorpeltypischen Genen (SOX9, Kollagen II) an den drei verschiedenen Lokalisationen während T1 und T2 zu bestimmen. Die Quantifizierung der relativen Genexpression erfolgte anschließend mit der ΔΔCT-Methode unter Verwendung von RPL32 und GAPDH als Housekeeping-Gene. Zur statistischen Auswertung wurden die Multiple Lineare Regression, eine einfaktorielle Varianzanalyse (ANOVA) und ein zweiseitiger t-Test herangezogen. Die Signifikanzniveaus aller statistischen Tests wurden auf α= 0,05 festgesetzt. Ergebnisse: Die Hox-Genexpressionen unterschieden sich in Bezug auf die drei Lokalisation und die Messzeitpunkte nicht signifikant voneinander. Die nasalen Chondrozyten wiesen während der ersten Subkultivierung gegenüber den artikulären Chondrozyten signifikant höhere Kollagen-II-Expressionen auf. Eine „Hox-Gen-negative“ Expression konnte für die Tierart Pferd nicht bestätigt werden. Die vorliegende Arbeit zeigt, dass Pferde wahrscheinlich ein speziesspezifisches Hox- Gen-Expressionsmuster aufweisen und dass die equinen Hox-Genexpressionsprofile statistisch signifikanten individuellen Einflüssen unterliegen. Schlussfolgerung: Das equine Hox-Genexpressionsprofil unterliegt statistisch signifikanten individuellen Einflüssen, die bei einer potenziellen Zelltherapie zu beachten sind. Nasale Chondrozyten eignen sich beim Pferd aufgrund ihrer genetischen Ähnlichkeit, bezogen auf die Expressionen der untersuchten Hox-Gene, zu artikulären Chondrozyten und ihrer hohen Bereitschaft zur Kollagen-II-Bildung wahrscheinlich als potenzielle Quelle für die autologe chondrozytäre Implantation (ACI).:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75 / Introduction: Osteoarthritis is responsible for most of the lameness in horses. Due to severe stress on equine joints, osteoarthritis inevitably progresses and results in a high degree of suffering. Current therapeutic options are not sufficient to restore the physiological integrity of the cartilage in osteoarthritic joints. Human and caprine nasal chondrocytes demonstrated high regenerative potential and integrity after autologous implantation into articular cartilage defects in preclinical and clinical studies. This was attributed to their “Hox gene negative” expression profile, matching the profile of articular cartilage after implantation. Aim of the study: The Hox gene expression profile of nasal chondrocytes was compared with those of articular chondrocytes in a cell culture to address nasal cartilage as a possible autologous source also in horses. Animals, Material and Methods: Cartilage was harvested from the nasal septum, one anterior and one posterior fetlock joint of deceased 7 horses, chondrocytes were isolated and cultured two-dimensionally until the fourth passage. During cultivation, chondrocytes were evaluated every 3 to 4 days using a specially designed assessment sheet. Cells were harvested during the first (T1) and third (T2) subcultivation. RNA was extracted and transcribed into cDNA. Subsequently, qPCR was performed to determine the expression levels of three Hox genes (A3, D1, D8) and two tissue-identifying genes (SOX9, collagen II) of the three locations after T1 and T2. Quantification of relative gene expression was performed with the ΔΔCT method using RPL32 and GAPDH as housekeeping genes. Multiple linear regression, one-way analysis of variance (ANOVA), and two-tailed t-test were used for statistical analysis. The significance levels of all statistical tests were set at α= 0.05. Results: Hox gene expressions were not significantly different in terms of localization and measurement time points. Nasal chondrocytes exhibited significantly higher collagen II expression than articular chondrocytes during the first subcultivation. 'Hox gene negative' expression could not be confirmed in horses. This study demonstrates that equine Hox gene expression pattern is likely species-specific and that equine Hox gene expression profiles are subject to statistically significant individual influences. Conclusion: The equine Hox gene expression profile is subject to statistically significant individual influences that should be considered in potential cell therapy. Nasal chondrocytes are probably suitable as a potential source for autologous chondrocyte implantation (ACI) in horses due to their genetic similarity, in terms of the expressions of the examined Hox genes, to articular chondrocytes and their high propensity for collagen II formation.:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

One of the major goals of developmental biology is to understand how specialized groups of cells arise from an initially unspecified cell population. The vertebrate hindbrain is transiently segmented along its anterior-posterior axis into lineage-restricted compartments called rhombomeres, making it an excellent model in which to study the genetic mechanisms of axial patterning. Hox homeodomain transcription factors (TF), in close partnership with the Pbx and Meis families of TALE-class homeodomain proteins, impart unique molecular identities to the hindbrain rhombomeres, thereby specifying functionally specialized neurons within each segment. The broad goals of this thesis are to clarify the roles of Meis1 and Tshz3b TFs in Hox-dependent hindbrain patterning, and to examine the Hox-independent roles of Pbx and Meis proteins in axial patterning of the visual system. While it is clear that Hox-Pbx-Meis complexes regulate hindbrain segmentation, the contributions of individual Meis proteins are not well understood. I have shown that Meis1-depleted embryos exhibit neuronal patterning defects, even though the hindbrain retains its segmental organization. This suggests that Meis1 is making important contributions to neuronal development downstream of rhombomeric specification. A zinc-finger TF called Teashirt (Tsh) cooperates with Hox-Pbx-Meis complexes to establish segmental identity in Drosophila, but this role not been tested in vertebrates. I found that overexpression of tshz3b produces segmentation defects reminiscent of Hox-Pbx-Meis loss of function phenotype, likely by acting as a transcriptional repressor. Thus, Tshz3b may be a negative regulator of Hox- dependent hindbrain patterning. Like the hindbrain, visual system function requires that positional information be correctly specified in the retina and midbrain. I found that zebrafish Pbx and Engrailed homeodomain TFs are biochemical DNA binding partners, and that this interaction is required to maintain the midbrain as a lineage- restricted compartment. Additionally, I show that Meis1 specifies positional information in both the retina and midbrain, thereby helping to organize the axonal connections between the eye and brain. Taken together, this thesis clarifies our understanding of Hox-dependent hindbrain patterning, and makes the claim that Pbx and Meis perform a general axial patterning function in anterior neural tissues such as the hindbrain, midbrain and retina. / Molecular Biology and Genetics

No description available.

La protéine de fusion E2A-PBX1 induit une leucémie lymphoblastique aigüe des cellules B pédiatrique chez l’humain. E2A-PBX1 possède de puissantes propriétés de trans-activation et peut se lier à l’ADN ainsi qu’aux protéines homéotiques (HOX) via des domaines conservés dans sa portion PBX1, ce qui suggère qu’une dérégulation des gènes cibles de HOX/PBX1 contribue à la leucémogénèse. Précédemment, Bijl et al. (2008) ont démontré que certains gènes Hox collaborent de manière oncogénique avec E2A-PBX1, et que ces interactions sont cellules-spécifiques et varient en fonction du gène Hox impliqué. Une mutagénèse d’insertion provirale suggère et supporte la collaboration des gènes Hoxa et E2A-PBX1 lors de la leucémogénèse des cellules B. La présence de ces interactions dans les cellules B et leur implication dans l’induction des B-ALL est pertinente pour la compréhension de la maladie humaine, et reste encore mal comprise. Notre étude démontre qu’Hoxa9 confère un avantage prolifératif aux cellules B E2A-PBX1. Des expériences de transplantation à l’aide de cellules B E2A-PBX1/Hoxa9 positives isolées de chimères de moelle osseuse démontrent qu’Hoxa9 collabore avec E2A-PBX1 en contribuant à la transformation oncogénique des cellules, et qu’Hoxa9 seul n’induit aucune transformation. Une analyse par Q-RT-PCR nous a permis de démontrer une forte inhibition de gènes spécifiques aux cellules B dans les leucémies co-exprimant Hoxa9 et E2A-PBX1, en plus d’une activation de Flt3, suggérant une inhibition de la différenciation des cellules B accompagnée d’une augmentation de la prolifération. De plus, la surexpression de Hoxa9 dans des cellules leucémiques de souris transgéniques E2A-PBX1, confère aussi un avantage prolifératif aux cellules in vitro, qui semblent être influencé par une augmentation de l’expression de Flt3 et Pdgfδ. En conclusion, nous démontrons pour la première fois à l’aide d’un modèle murin qu’Hoxa9 collabore avec E2A-PBX1 lors de la transformation oncogénique des cellules B et que la signalisation via Flt3 est impliquée, ce qui est potentiellement pertinent pour la maladie humaine. / The fusion protein E2A-PBX1 induces pediatric B cell leukemia in human. It has strong transactivating properties and can bind to DNA and homeobox (HOX) proteins through conserved domains in the PBX1 portion, suggesting that deregulation of HOX/PBX target genes contribute to leukemogenesis. Previously, we reported oncogenic interactions between Hox genes and E2A-PBX1, which are dependent on cell type as well as on the particular Hox member. A proviral insertional mutagenesis screen provided support for collaboration between Hoxa genes and E2A-PBX1 in B cell leukemogenesis. Whether these interactions occur in B cells and lead to B-ALL, relevant for human disease is still not clear. Here we report that Hoxa9 confers a proliferative advantage to E2A-PBX1 B cells. Transplantation experiments with E2APBX1/Hoxa9 positive B cells isolated from bone marrow (BM) chimeras showed that Hoxa9 interacts with E2A-PBX1 contributing to the oncogenic transformation of B cells, but is unable to transform B cells alone. Q-RT-PCR analysis demonstrated a strong repression of B cell specific genes in leukemias co-overexpressing Hoxa9 and E2A-PBX1 in addition to Flt3 activation, indicating inhibition of B cell differentiation in combination with enhanced proliferation. Overexpression of Hoxa9 in E2A-PBX1 mouse leukemic B cells also resulted in a growth advantage in vitro, likely mediated by the enhanced expression of Flt3 and Pdgfδ. In conclusion we show for the first time that Hoxa9 collaborates with E2A-PBX1 in the oncogenic transformation of B cells in a mouse model that involves Flt3 signaling, which is potentially relevant to human disease.

Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...

Les leucémies aiguës myéloïdes (LAM) sont des proliférations malignes de progéniteurs bloqués lors de la différenciation myéloïde. Le caryotype des blastes leucémiques identifie 3 groupes pronostiques distincts. Parmi les LAM à risque cytogénétique favorable, figurent les leucémies aiguës promyélocytaires (LAP), et celles avec inv(16) ou t(8;21). Les micro-ARNs sont des acteurs clef de l’hématopoïèse et sont aussi impliqués dans la leucémogénèse des LAM. Ils sont très stables dans le sérum et sont utilisés comme biomarqueurs dans les cancers. Le but de cette thèse était d’évaluer si une caractérisation pangénomique des micro-ARNs sériques permettait de distinguer ces 3 types de LAM entre elles ainsi que les LAM avec caryotype normal (NK-AML); d’identifier des micro-ARNs circulants fortement surexprimés dans les NK-AML par rapport à des sujets sains, pour une utilisation ultérieure comme marqueurs de maladie résiduelle; et de mieux préciser le pronostic des NK-AML. Ainsi, nous avons identifié une signature sérique spécifique des LAP liée à une dérégulation des micro-ARNs localisés dans la région DLK1-DIO3 soumise à l’empreinte, en 14q32. Ces micro-ARNs, dont l’origine était le blaste leucémique, étaient corrélés aux facteurs pronostiques connus des LAP. Par ailleurs, deux micro-ARNs, miR-10a-3p et miR-196b-5p, distinguant les NK-AML des LAM avec inv(16) ou t(8 ;21) ont été montré surexprimés dans les NK-LAM avec une mutation de NPM1 et/ou de FLT3-ITD. Enfin l’expression de ces deux micro-ARNs est corrélée à la dérégulation transcriptionnelle et à la méthylation de l’ADN affectant les gènes HOX et TALE. En conclusion, cette étude des micro-ARNs sériques ouvre un nouveau champ d’exploration à visée pronostique dans les LAM. / Acute myeloid leukaemia (AML) is a malignant proliferation of progenitors blocked during myeloid differentiation. The karyotype of the leukemic blasts identified three distinct prognostic groups. Among the favourable risk cytogenetics AML include acute promyelocytic leukaemia (APL), and those with inv (16) or t (8; 21). Micro-RNAs are key players in hematopoiesis and are also involved in leukemogenesis of AML. They are very stable in serum and used as biomarkers in cancers. The aim of this thesis was to evaluate whether a genome-wide characterization of serum micro-RNAs possible to distinguish these three types of AML them as well as AML with normal karyotype (NK-AML); identify micro-RNAs circulating highly overexpressed in NK-AML compared to healthy subjects, for later use as markers of residual disease; and to better define the prognosis of NK-AML. Thus, we have identified a specific serum signing of LAP related to a deregulation of micro-RNAs located in the DLK1-DIO3 under the imprinting, in 14q32. These micro-RNAs whose origin was the leukemic blasts were correlated with known prognosis factors of APL. In addition, two micro-RNAs, miR-10a-3p and miR-196b-5p, distinguishing the NK-AML with inv (16)-AML or t (8; 21)-AML have been shown overexpressed in NK-AML with mutation NPM1 and / or FLT3-ITD. Finally the expression of these two micro-RNAs correlates withtranscriptional deregulation and DNA methylation affectingTALE and HOX genes. In conclusion, this study of serum microRNAs opens a new field of exploration to assess the prognosis in AML.