Spinor Bose-Einstein Condensates and Topological Defects

This thesis concentrates on the topological defects of spin-1 and spin-2 Bose-Einstein condensates, the ground states of spin-3 condensates, and the inert states of spinor condensates with arbitrary spin. Our work is based on the description of a spinor condensate of spin-S atoms in terms of a state vector of a spin-S particle. The results of the homotopy theory are used to study the existence and structure of the topological defects in spinor condensates. We construct examples of defects, study their energetics, and examine how their stability is affected by the presence of an external magnetic field. The ground states of spin-3 condensates are calculated using analytical and numerical means. Special emphasis is put on the ground states of a chromium condensate, whose dependence on the magnetic dipole-dipole interaction is studied. A simple geometrical method for the calculation of inert states of spinor condensates is presented. This method is used to find candidates for the ground states of spin-S condensates.

Linear and nonlinear terrain deformation maps from a reduced set of interferometric SAR images

In this paper, an advanced technique for the generation of deformation maps using synthetic aperture radar (SAR) data is presented. The algorithm estimates the linear and nonlinear components of the displacement, the error of the digital elevation model (DEM) used to cancel the topographic terms, and the atmospheric artifacts from a reduced set of low spatial resolution interferograms. The pixel candidates are selected from those presenting a good coherence level in the whole set of interferograms and the resulting nonuniform mesh tessellated with the Delauney triangulation to establish connections among them. The linear component of movement and DEM error are estimated adjusting a linear model to the data only on the connections. Later on, this information, once unwrapped to retrieve the absolute values, is used to calculate the nonlinear component of movement and atmospheric artifacts with alternate filtering techniques in both the temporal and spatial domains. The method presents high flexibility with respect to the required number of images and the baselines length. However, better results are obtained with large datasets of short baseline interferograms. The technique has been tested with European Remote Sensing SAR data from an area of Catalonia (Spain) and validated with on-field precise leveling measurements.

Validation of a model to predict adverse outcomes in patients with pulmonary embolism.

AIMS: To validate a model for quantifying the prognosis of patients with pulmonary embolism (PE). The model was previously derived from 10 534 US patients. METHODS AND RESULTS: We validated the model in 367 patients prospectively diagnosed with PE at 117 European emergency departments. We used baseline data for the model's 11 prognostic variables to stratify patients into five risk classes (I-V). We compared 90-day mortality within each risk class and the area under the receiver operating characteristic curve between the validation and the original derivation samples. We also assessed the rate of recurrent venous thrombo-embolism and major bleeding within each risk class. Mortality was 0% in Risk Class I, 1.0% in Class II, 3.1% in Class III, 10.4% in Class IV, and 24.4% in Class V and did not differ between the validation and the original derivation samples. The area under the curve was larger in the validation sample (0.87 vs. 0.78, P=0.01). No patients in Classes I and II developed recurrent thrombo-embolism or major bleeding. CONCLUSION: The model accurately stratifies patients with PE into categories of increasing risk of mortality and other relevant complications. Patients in Risk Classes I and II are at low risk of adverse outcomes and are potential candidates for outpatient treatment.

The role of Receptor Activator of NFKB Ligand (RANKL) in mammary gland development

Abstract : The female reproductive hormones estrogen, progesterone and prolactin control postnatal breast development and are important to breast carcinogenesis. The mechanisms by which they elicit proliferation and morphogenesis remain poorly understood. Using the mouse as a model to study the molecular mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we found the Receptor Activator of NFκB Ligand, a Tumor Necrosis Factor family member, to be strongly induced by progesterone. Recent publications suggested that hormone dependant RANKURANK signals are involved in the terminal differentiation of mammary gland alveolar buds into lobulo-alveolar structures competent for lactation. I show that in the absence of epithelial RANKL a distinct earlier stage of mammary gland development, side branch formation, is blocked. RANKL acts as a major mediator downstream of progesterone; it is required for progesterone-induced paracrine proliferation and completely rescues the mutant phenotype when ectopically expressed in progesterone receptor (PR) KO mammary epithelia. RANKL is not required for cell autonomous division of estrogen receptor alpha (ERa) /PR positive cells. Cyclin D1, previously implicated as a mediator of RANKL, is not affected by ablation of RANKL and is not required for RANKL-induced paracrine proliferation but for the cell autonomous proliferation. Gene expression arrays to find specific RANKL downstream targets have identified Id4, ElfS and one secreted metalloprotease (Adamtsl8) as potential candidates validated by Q-RT-PCR. Interestingly, Id4 and Adamtsl8 are expressed by the myoepithelial cells. Their expression additionally coincides with RANKL mRNA expression at mid pregnancy, possibly implying a functional contribution of both genes to RANKL mediated sidebranch formation. ElfS in contrast, is found to be strongly expressed by the end of pregnancy supporting recent findings of a prolactin mediated regulation. As for RANKL, this gene was in particular induced in luminal cells. Taken together, I report that progesterone is the major proliferative stimulus in the adult mammary gland eliciting proliferation of ERaJPR positive cells by a cell autonomous, cyclin D1-dependent and a paracrine RANKL-dependent mechanism. My work moreover suggests, that RANKL acts as a major orchestrator affecting different downstream mediators, through which progesterone exerts its effects concomitantly on different cellular compartments. Résumé : Les hormones sexuelles telles que l'oestrogène, la progestérone et la prolactine contrôlent le développement postnatal du sein et sont impliquées dans la cazcinogenèse. Les mécanismes par lesquels elles induisent la prolifération et la morphogénèse demeurent incompris. En utilisant la souris comme modèle, J'ai trouvé que le ligand activateur du récepteur de NFκB, une protéine de la famille du facteur de nécrose des tumeurs, peut être fortement induit par la progestérone. Les publications récentes ont suggéré que cette protéine est nécessaire à la fin de la grossesse, quand les cellules sécrétrices du lait apparaissent. Par des techniques de transplantation d'épithélium, je montre contrairement aux études précédentes, qu'en l'absence de RANKL dans l'épithélium une partie distincte du développement mammaire, la formation de branches latérales, est bloquée. La progestérone agit de manière pazacrine par l'intermédiaire de 12ANKL pour induire la prolifération tandis que la mort cellulaire n'est pas affectée. De plus, l'injection d'une protéine recombinante RANKL dans une souris mutante pour le récepteur à la progestérone induit la prolifération des cellules épithéliales en l'absence de grossesse ; la surexpression de RANKL dans ces mêmes mutants mène à une réversion complète du phénotype. Mes expériences démontrent que la progestérone induit deux types distincts de prolifération. Un premier type direct dans laquelle les cellules positives au récepteur à la progestérone prolifèrent. Cette division cellulaire est alors indépendante de RANKL mais dépendante de la cycline D1. Le second type de prolifération est induit par un mécanisme pazacrine et dépend de RANKL mais pas de la cycline D1. Ici, les cellules négatives au récepteur à la progestérone prolifèrent. Pour détecter des gènes cibles de la voie de signalisation du RANKL, un profil d'expression des gènes a été généré. Les facteurs de transcription Id4, EIf5 et une métalloprotéase sécrétée (Adamtsl8) ont été identifiés en tant que cibles potentielles. D'autres analyses de validation démontrent qu'Id4, Adamtsl8, RANKL mais pas E1f5 sont fortement exprimés au cours de la grossesse, coïncidant avec la formation de branchements latéraux induit par progestérone. EIf5 s'est avéré être exprimé vers la fin de la grossesse appuyant des résultats récents proposant une régulation par la prolactine. Le système canalaire mammaire se compose de couches cellulaires: une couche interne de cellules luminales et une externe de cellules myoépithéliale. Les expériences génétiques d'expression ont révélé que RANKL. et E1f5 sont exprimés dans la partie luminale tandis qu'Id4 et Adamtsl8 sont dans les cellules myoépithéliales. En conclusion, je prouve que la progestérone est le stimulus principal induisant la prolifération dans la glande mammaire d'adulte. Deux mécanismes de prolifération sont impliqués: l'un direct dépendant de la cycline Dl et l'autre paracrine dépendant de RANKI.. Mon travail suggère par ailleurs que RANKL agit en tant que médiateur important, par lequel la progestérone exerce ses effets sur différents compartiments cellulaires tels que la coordination de la prolifération des cellules épithéliales avec la réorganisation de la matrice extracellulaire et de la membrane basale exigées pour la morphogénèse du système canalaire latéral.

A methodology to estimate the potential to move inpatient to one day surgery.

BACKGROUND: The proportion of surgery performed as a day case varies greatly between countries. Low rates suggest a large growth potential in many countries. Measuring the potential development of one day surgery should be grounded on a comprehensive list of eligible procedures, based on a priori criteria, independent of local practices. We propose an algorithmic method, using only routinely available hospital data to identify surgical hospitalizations that could have been performed as one day treatment. METHODS: Moving inpatient surgery to one day surgery was considered feasible if at least one surgical intervention was eligible for one day surgery and if none of the following criteria were present: intervention or affection requiring an inpatient stay, patient transferred or died, and length of stay greater than four days. The eligibility of a procedure to be treated as a day case was mainly established on three a priori criteria: surgical access (endoscopic or not), the invasiveness of the procedure and the size of the operated organ. Few overrides of these criteria occurred when procedures were associated with risk of immediate complications, slow physiological recovery or pain treatment requiring hospital infrastructure. The algorithm was applied to a random sample of one million inpatient US stays and more than 600 thousand Swiss inpatient stays, in the year 2002. RESULTS: The validity of our method was demonstrated by the few discrepancies between the a priori criteria based list of eligible procedures, and a state list used for reimbursement purposes, the low proportion of hospitalizations eligible for one day care found in the US sample (4.9 versus 19.4% in the Swiss sample), and the distribution of the elective procedures found eligible in Swiss hospitals, well supported by the literature. There were large variations of the proportion of candidates for one day surgery among elective surgical hospitalizations between Swiss hospitals (3 to 45.3%). CONCLUSION: The proposed approach allows the monitoring of the proportion of inpatient stay candidates for one day surgery. It could be used for infrastructure planning, resources negotiation and the surveillance of appropriate resource utilization.

Heart transplantation: current practice and outlook to the future.

QUESTIONS UNDER STUDY: The field of heart transplantation has seen substantial progress in the last 40 years. The breakthroughs in long-term survival were followed by a period of stagnation in the last decade. This review summarises current recommendations for the identification of candidates for heart transplantation and their immunological and non-immunological postoperative follow-up. RESULTS: The progress made in the treatment of patients with advanced heart failure has considerably changed the profile of candidates for heart transplantation. Patients are older, and the load of co-morbidities is more important requiring careful evaluation for candidacy. Long-standing research in the field of immunosuppression made available various drugs, which decrease the risk of acute allograft rejection and prolong survival after heart transplantation. Powerful new molecules are entering early phase clinical studies, suggesting further improvement in the near future. As a consequence, treatment of non-immunological co-morbidity after heart transplantation will gain in importance, however, the base of evidence guiding current recommendations is poor. CONCLUSIONS: The substantial progress in heart failure treatment and immunosuppression after heart transplantation has changed the profile of heart transplant recipients. The arrival of new molecules will provide additional alternatives for immunosuppressive treatment while studies have to address non-immunological treatment in order to improve long-term survival after heart transplantation.

Phylogenetic position of Fit insect toxin producing pseudomonads

Fit produced by Pseudomonas fluorescens CHA0 is a novel insect toxin in root colonizing pseudomonads, of which a homologue is described in Photorhabdus species.However, occurrence and abundance of insect pathogenicity in plant-associated pseudomonads is still unclear. An extensive screening outside the P. fluorescens complex identified strains of Pseudomonas chlororaphis as further Fit toxin producing candidates. Sequences of five different P. chlororaphis strains generated in this study were used to reconstruct the evolutionary history of the Fit toxin gene and to analyse its mode of evolution. We found that P. chlororaphis is closely associated with a small subgroup of 2,4-diacetylphloroglucinol and pyoluteorin- producing pseudomonads, both when analyzing four housekeeping genes and the nucleotide sequences for the Fit toxin gene. Additionally, we identified purifying selection to be the predominant mode of Fit toxin evolution.

Identification of in vitro HSC fate regulators by differential lipid raft clustering.

Most hematopoietic stem cells (HSC) in the bone marrow reside in a quiescent state and occasionally enter the cell cycle upon cytokine-induced activation. Although the mechanisms regulating HSC quiescence and activation remain poorly defined, recent studies have revealed a role of lipid raft clustering (LRC) in HSC activation. Here, we tested the hypothesis that changes in lipid raft distribution could serve as an indicator of the quiescent and activated state of HSCs in response to putative niche signals. A semi-automated image analysis tool was developed to map the presence or absence of lipid raft clusters in live HSCs cultured for just one hour in serum-free medium supplemented with stem cell factor (SCF). By screening the ability of 19 protein candidates to alter lipid raft dynamics, we identified six factors that induced either a marked decrease (Wnt5a, Wnt3a and Osteopontin) or increase (IL3, IL6 and VEGF) in LRC. Cell cycle kinetics of single HSCs exposed to these factors revealed a correlation of LRC dynamics and proliferation kinetics: factors that decreased LRC slowed down cell cycle kinetics, while factors that increased LRC led to faster and more synchronous cycling. The possibility of identifying, by LRC analysis at very early time points, whether a stem cell is activated and possibly committed upon exposure to a signaling cue of interest could open up new avenues for large-scale screening efforts.

Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome.

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.

Sex ratio and Wolbachia infection in the ant Formica exsecta.

Sex allocation data in social Hymenoptera provide some of the best tests of kin selection, parent-offspring conflict and sex ratio theories. However, these studies critically depend on controlling for confounding ecological factors and on identifying all parties that potentially manipulate colony sex ratio. It has been suggested that maternally inherited parasites may influence sex allocation in social Hymenoptera. If the parasites can influence sex allocation, infected colonies are predicted to invest more resources in females than non-infected colonies, because the parasites are transmitted through females but not males. Prime candidates for such sex ratio manipulation are Wolbachia, because these cytoplasmically transmitted bacteria have been shown to affect the sex ratio of host arthropods by cytoplasmic incompatibility, parthenogenesis, male-killing and feminization. In this study, we tested whether Wolbachia infection is associated with colony sex ratio in two populations of the ant Formica exsecta that have been the subject of extensive sex ratio studies. In these populations colonies specialize in the production of one sex or the other. We found that almost all F. exsecta colonies in both populations are infected with Wolbachia. However, in neither population did we find a significant association in the predicted direction between the prevalence of Wolbachia and colony sex ratio. In particular, colonies with a higher proportion of infected workers did not produce more females. Hence, we conclude that Wolbachia does not seem to alter the sex ratio of its hosts as a means to increase transmission rate in these two populations of ants.

Pienen kansainvälistyvän ohjelmistoyrityksen kumppaninvalintakriteerit

Tutkielman tavoite: Tämän tutkimuksen tavoitteena oli luoda pienelle ohjelmistoyritykselle kansainvälisen kumppanin valintaa helpottavat arviointikriteerit. Tutkimuksessa selvitettiin ohjelmistoyritysten kansainvälistymistä, sekä kuinka kansainvälistymisen apuna käytettyjen kumppanien valinta tapahtuu ja millaisia kriteerejä kumppaneille tulee asettaa. Tutkimusmetodologia ja tutkimusmenetelmät: Informaation keräämiseksi käytettiin laadullisia tutkimusmenetelmiä. Tutkimus toteutettiin case- study tutkimuksena. Tietoja kerättiinkirjallisuuteen ja aikaisempiin tutkimuksiin perehtymällä, sekä teemahaastattelujen avulla. Haastattelumateriaali analysoitiin tutkimuksen aiheesta nousseiden teemojen avulla. Tulokset ja päätelmät: Tutkimuksella voitiin osoittaa että kumppaninvalintakriteerit ovat vahvasti kontekstisidonnaisia. Lisäksi tutkimuksessa luotiin case - yritykselle kumppanin-valintakriteeri- malli, jota yritys voi käyttää konkreettisena työkaluna kumppanin valinnassaan.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rheinhuprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and ()-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and ()-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and ()-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rheinhuprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and ()-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and ()-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and ()-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Replicating adenoviruses targeting tumours with constitutive activation of the Wnt signalling pathway

Abstract Activation of the Wnt pathway through mutation of the adenomatous polyposis coli and 13-catenin genes is a hallmark of colon cancer. These mutations lead to constitutive activation of transcription from promoters containing binding sites for Tcf/LEF transcription factors. Tumour-selective replicating oncolytic viruses are promising agents for cancer therapy. They can in principle spread throughout a tumour mass until all the cancerous cells are killed, and clinical trials have shown that they are safe except at very high doses. Adenoviruses are interesting candidates for virotherapy because their biology is well understood and their small genome can be rapidly mutated. Adenoviruses with Tcf binding sites in the E2 early promoter replicate selectively in cells with an active Wnt pathway. Although these viruses can replicate in a broad panel of colon cancer cell lines, some colorectal cancer cells are only semi-permissive for Tcf-virus replication. The aim of my thesis was to increase the safety and the efficacy of Tcf-viruses for colon cancer virotherapy. I replaced the endogenous ElA viral promoter by four Tcf binding sites and showed that transcription from the mutant promoter was specifically activated by the Wnt pathway. A virus with Tcf binding sites in the ElA and E4 promoters was more selective for the Wnt pathway than the former Tcf-E2 viruses. Moreover, insertion of Tcf binding sites into all early promoters further increased viral selectivity, but reduced viral activity. I showed that Tcf-dependent transcription was inhibited by the interaction between ElA and p300, but deletion of the p300-binding site of ElA generally led to viral attenuation. In the semi-permissive cell lines, replication of Tcf-viruses remained lower than that of the wild-type virus. The E2 promoter was the most sensitive to the cell type, but I was unable to improve its activity by targeted mutagenesis. To increase the toxicity of the Tcf-E1A/E4 virus, I decided to express a suicide gene, yeast cytosine deaminase (yCD), late during infection. This enzyme converts the prodrug 5-FC to the cytotoxic agent 5-FU. yCD was expressed in a DNA replication-dependent manner and increased viral toxicity in presence of 5-FC. Tcf-ElA and yCD adenoviruses are potentially useful vectors for the treatment of liver metastases from colorectal tumours. Résumé Dans la quasi-totalité des cancers du côlon, la voie Wnt est activée par des mutations dans les gènes codant pour APC ou pour la (3-caténine. Ces mutations activent de façon constitutive la transcription de promoteurs contenant des sites de liaison pour les facteurs de transcription Tcf/LEF. Les virus réplicatifs spécifiques aux tumeurs sont des agents prometteurs pour la thérapie cancéreuse. En principe, ces vecteurs peuvent se propager dans une masse tumorale jusqu'à destruction de toutes les cellules cancéreuses, et des études cliniques ont démontré que de tels vecteurs n'étaient pas toxiques, sauf à de très hautes doses. Les adénovirus sont des candidats intéressants pour la thérapie virale car leur biologie est bien définie et leur petit génome peut être rapidement modifié. Des adénovirus comportant des sites de liaison à Tcf dans leur promoteur précoce E2 se répliquent sélectivement dans les cellules qui possèdent une voie Wnt active. Ces virus sont capables de se répliquer dans un grand nombre de cellules cancéreuses du côlon, bien que certaines de ces cellules ne soient que semi-permissives pour la réplication des virus Tcf. Le but de ma thèse était d'augmenter la sécurité et l'efficacité des virus Tcf. Le promoteur viral endogène ElA a été remplacé par quatre sites de liaison à Tcf, ce qui a rendu son activation dépendante de la voie Wnt. Un virus comportant des sites de liaison pour Tcf dans les promoteurs ElA et E4 était plus sélectif pour la voie Wnt que les précédents virus Tcf-E2, et un virus comportant des sites Tcf dans tous les promoteurs précoces était encore plus sélectif, mais moins actif. J'ai montré que l'interaction entre ElA et p300 inhibait la transcription dépendante de Tcf, mais la délétion du domaine concerné dans ElA a eu pour effet d'atténuer les virus. Dans les cellules semi-permissives, la réplication des virus Tcf était toujours plus basse que celle du virus sauvage. J'ai identifié le promoteur E2 comme étant le plus sensible au type cellulaire, mais n'ai pas pu augmenter son activité par mutagenèse. Pour augmenter la toxicité du virus Tcf-E1A/E4, j'ai décidé d'exprimer un gène suicide, la cytosine déaminase (yCD), pendant la phase tardive de l'infection. Cette enzyme transforme la procirogue 5-FC en l'agent cytotoxique 5-FU. yCD était exprimée après réplication de l'ADN viral et augmentait la toxicité virale en présence de 5-FC. Les virus Tcf-ElA et yCD sont des vecteurs potentiellement utiles pour le traitement des métastases hépatiques de cancers colorectaux.

Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.