Supplementary material to figures contained on DVD only available with manuscript. / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences, 2005. / Bibliography: p. 221-244. / Introduction -- Materials and methods -- Isolation of the hex1 gene from Trichoderma reesei and Ophiostoma floccosum -- Expression of DsRed under the cbh1 promoter and the hex1 promoter with random integration -- Modified expression vectors containing a fusion to a portion of hex1 gene sequence -- Expression of DsRed from the hex1 locus and the phenotypic characteristics of a hex1 deletion mutant -- Summary and concluding discussion. / For Trichoderma reesei to be developed as an effiecient producer of a large variety of proteins, the expression system requires diversification. In particular, the choice of promoters available needs to be broadened to include promoters which are active in conditions other than those conducive to induction of cellulase expression. Using proteomics, the HEX1 protein was identified as an abundant protein of the cell envelope of T. reesei when grown on a range of carbon sources, suggesting that a strong constitutive promoter drives the expression of this physiologically important protein. This thesis is an exploration into the hex1 gene promoter and the role of hex1 in the maintenance of mycelium integrity in T. reesei with consideration for the application of this gene in the further development of filamentous fungi as protein expression systems. -- The single copy hex1 gene and flanking regions were isolated from T. reesei and another biotechnologically important fungus, Ophiostoma floccosum. The fluorescent reporter protein DsRed1-E5 was expressed under the T. reesei hex1 promoter and promoter activity was monitored by fluorescence CLSM and RNA analysis. During the rapid growth phase of a culture, the hex1 promoter was active in a range of carbon sources and three transcipt types with alternative tsp and splicing sites were discovered for the hex1 gene. The distribution of fluorescence throughout the mycelium suggested spatial regulation of the hex1 promoter as well as temporal regulation. The promoter was continually active in the absence of a functional hex1 gene product suggesting that the hex1 promoter is regulated in part, by negative feedback from the endogenous gene product. Interruption of the hex1 gene produced hyphae that leaked excessive volumes of cytoplasm when physically damaged which may be advantageous for the externalisation of selected protein products. The results indicate that the regulation of the hex1 hene promoter is complex and that the hex1 gene is integral to the maintenance of the integrity of the fungal mycelium. / Mode of access: World Wide Web. / xv, 244 p. ill

Submitted by FELIPE AUGUSTO DARRIBA BATTAGLIN null (darriba@bol.com.br) on 2016-08-17T23:44:40Z No. of bitstreams: 1 DIS_MEST2016_BATTAGLIN FELIPE.pdf: 6951544 bytes, checksum: 2d13cd64aaec5226e6031c18795aaca7 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-19T18:47:28Z (GMT) No. of bitstreams: 1 battaglin_fad_me_soro.pdf: 6951544 bytes, checksum: 2d13cd64aaec5226e6031c18795aaca7 (MD5) / Made available in DSpace on 2016-08-19T18:47:28Z (GMT). No. of bitstreams: 1 battaglin_fad_me_soro.pdf: 6951544 bytes, checksum: 2d13cd64aaec5226e6031c18795aaca7 (MD5) Previous issue date: 2016-07-06 / Filmes de alumina foram depositados a partir de uma nova metodologia de deposição a plasma, utilizando o pó de acetilacetonato de alumínio (AAA) como precursor. Em trabalho prévio do grupo, foi demonstrada a viabilidade do sputtering do AAA em plasmas de argônio para deposição de filmes finos. Os bons resultados obtidos estimularam o desenvolvimento do presente trabalho, visando o aperfeiçoamento da metodologia de deposição. Para isso, primeiramente foram investigados os efeitos da alteração da composição química da atmosfera do plasma, por meio da incorporação de diferentes proporções de oxigênio (O2%) ao argônio, tornando o processo um sputtering reativo. As deposições foram realizadas espalhando-se o pó do AAA no eletrodo inferior de um sistema de plasma acoplado capacitivamente. Argônio, oxigênio ou a mistura de ambos foram admitidos até a pressão de 11,0 Pa. O plasma foi gerado pela aplicação de sinal de radiofrequência (13,56 MHz, 150 W) ao eletrodo contendo o pó, mantendo-se o eletrodo superior, também utilizado como porta-amostras, aterrado. O tempo de deposição foi de 90 minutos. Investigou-se o efeito da O2%, variada de 0 a 100%, nas propriedades dos filmes. Na etapa subsequente, filmes foram depositados por sputtering reativo utilizando-se a condição considerada ótima na última etapa do trabalho (O2% = 25%) e mantendo-se as condições de pressão, potência e tempo de tratamento constantes. Todavia, ao invés de aterrar o porta-amostras, pulsos retangulares negativos (600 V, 2 kHz, 1-100% de ciclo de trabalho) foram aplicados, promovendo bombardeamento iônico durante a deposição por sputtering reativo. O efeito do ciclo de trabalho dos pulsos nas propriedades dos filmes foi avaliado. Na última etapa do trabalho, filmes foram depositados pelo sputtering reativo a partir de atmosferas contendo 25% de O2 e 75% de Ar e em condições mais energéticas que as utilizadas nos ciclos anteriores. Para tal um primeiro conjunto de amostras foi preparado mediante aquecimento resistivo do porta-amostras (410ºC) em plasma de menor pressão (4,0 Pa) que a anteriormente utilizada. O tempo de deposição foi de 28 minutos. Um segundo conjunto de amostras foi preparado associando-se bombardeamento iônico de mais alta energia, pela aplicação de pulsos de 1200 V (20% ciclo de trabalho) ao porta-amostras e também reduzindo a pressão da atmosfera de deposição para 4,0 Pa. Nesta condição, o tempo de deposição foi de 60 minutos. Comparou-se os resultados obtidos nestes experimentos aos equivalentes obtidos anteriormente. A espessura da camada foi obtida por meio de um perfilômetro e a taxa de deposição pela razão entre espessura e tempo de deposição. A composição elementar e a estrutura molecular dos filmes foram investigadas através das técnicas de espectroscopia de retroespalhamento Rutherford e de absorção no infravermelho, respectivamente. Difração de raios X foi utilizada para investigar a microestrutura dos filmes. Inspeções na morfologia e composição química das superfícies foram conduzidas associando microscopia eletrônica de varredura e espectroscopia de energia dispersiva. A rugosidade foi derivada de perfis topográficos adquiridos por perfilometria e microscopia de força atômica, enquanto a molhabilidade da superfície foi determinada através da técnica de gota séssil. De forma geral, os filmes depositados apresentaram contribuições de grupos orgânicos e de inorgânicos relacionados à alumina amorfa. O aumento da O2% afetou a cinética do plasma, proporcionando alterações na taxa de deposição (1 a 25 nm/min), rugosidade (1 a 13 nm) e redução na concentração de carbono proveniente do precursor, de 43% (O2% = 0%) para 6% (O2% = 100%). Com o aumento na O2% também foram encontradas variações na densidade dos filmes, dentro da faixa de 0,7 a 1,9 g/cm³, e tendência de queda no ângulo de contato de 53 para 17°. Por sua vez, quando o bombardeamento iônico é associado ao processo de deposição, altera-se a taxa de crescimento dos filmes (3 a 29 nm/min) e a morfologia da superfície, por meio do alívio de tensões internas e aumento da estabilidade física da estrutura resultante. A composição química não sofreu alterações, devido as condições do sputtering reativo permanecerem inalteradas nas deposições. Já a rugosidade e a molhabilidade da superfície apresentaram comportamentos condizentes com os resultados da morfologia e topografia. Quando condições mais energéticas de deposição foram empregadas, filmes óxidos com contaminações orgânicas foram obtidos para a deposição que empregou aquecimento resistivo do porta-amostras. Nesta condição, devido a redução na pressão total, mesmo com o aquecimento resistivo a taxa de deposição foi maior (~ 6 vezes) que aquela obtida sem aquecimento. Para a situação em que bombardeamento iônico de alta energia foi utilizado, estrutura e composição química similares ao do composto precursor foram obtidas. Os resultados são interpretados em termos dos processos predominantes em cada uma das metodologias empregadas. / Alumina films were deposited by a new plasma deposition method using aluminum acetylacetonate (AAA) powder as precursor. In a previous study by our group, the feasibility of AAA sputtering in argon plasmas for thin films deposition was demonstrated. The good results obtained stimulated the development of this work, aiming at the improvement of the deposition methodology. For this, the effects of modifications in the chemical composition of the plasma atmosphere were first investigated, through the use of different oxygen to argon proportions (O2%), making the process a reactive sputtering. The depositions were performed by spreading the AAA powder on the lower electrode of a capacitively coupled plasma system. Argon, oxygen or a mixture of both were admitted up to a pressure of 11.0 Pa. Application of radiofrequency power (13.56 MHz, 150 W) to the powder covered electrode generated the plasma, keeping the upper electrode, also used as a sample holder, grounded. Deposition times of 90 minutes were used. The effects of varying the oxygen proportion from 0 to 100%, on the film properties were studied. In the subsequent stage, films were deposited by reactive sputtering using the condition considered best in the last stage of the work (O2% = 25%) and keeping the pressures, power and treatment time constant. Instead of grounding the sample holder, however, negative rectangular pulses (600 V, 2 kHz, 1-100% duty cycle) were applied, promoting ion bombardment during the deposition by reactive sputtering. The influence of the pulse duty cycle on the properties of the films was evaluated. In the last study stage, films were deposited by reactive sputtering from atmospheres containing 25% O2 and 75% Ar and with more energetic conditions than those used in previous cycles. For such, a first samples set was prepared by resistive heating of the sample holder (410ºC) in a lower plasma pressure (4.0 Pa) than that previously used. The deposition time was 28 minutes. A second samples set was carried out associating ion bombardment of the highest energy, by the application of the 1200 V pulses (20% duty cycle) to the sample holder and also reducing the pressure of the deposition atmosphere to 4.0 Pa. In this condition, the deposition time was 60 minutes. The results obtained in these experiments were compared to the equivalent obtained previously. Film thickness was obtained by profilometry and the deposition rate calculated as the ratio between the thickness and deposition time. Elemental composition and molecular structure of the films were investigated using Rutherford backscattering and infrared absorption spectroscopy, respectively. X-ray diffraction was used to investigate the microstructure of the films. Surface morphology and chemical composition were studied using scanning electron microscopy and energy dispersive spectroscopy. Roughness was derived from topographic profiles acquired by profilometry and atomic force microscopy, whereas the surface wettability was determined using the sessile drop technique. In general, the deposited films showed contributions from organic and inorganic groups related to amorphous alumina. Increasing in O2% affected the plasma kinetics, providing changes in deposition rate (1 to 25 nm/min), roughness (1 to 13 nm) and reduction in the carbon concentration coming from the precursor, 43% (O2% = 0%) to 6% (O2% = 100%). With the increase in O2% it was also found variations in the films density, within the range from 0.7 to 1.9 g/cm³, and a downward trend in the contact angle of 53 to 17°. In turn, when the ion bombardment is associated with the deposition process, changes are found in the deposition rate (3 to 29 nm/min) and the surface morphology, through the internal strains relief and increased in the physical stability of resulting structure. The chemical composition did not suffer changes because the conditions of the reactive sputtering remain unchanged in the depositions. The roughness and surface wettability showed behavior consistent with the morphology and topography results. When more energetic conditions were employed, oxide films with organic contaminations were obtained for the deposition which applied the sample holder resistive heating. In this condition, due to the reduction in the total pressure, even with the resistive heating the deposition rate was higher (~ 6 times) than that obtained without heating. For the situation which high-energy ion bombardment was used, the structure and chemical composition similar to the precursor were obtained. The results are interpreted in terms of the prevailing processes in each of the methodologies applied.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of this study was to compare the efficacy of genetic transformation of potato, cultivar Bintje, using two methods: Agrobacterium tumefaciens mediated transformation and microprojectile bombardment. The same plasmid p35SGUSint, which cosists of 35S CaMV promoter, gus and nptII genes, was used for both transformations of internodal potato explants. Kamamycin selection, transient and stable expressions of {$\beta$}-glucuronidase and PCR amplification of gus and nptII transgenes were used for transgenic plant selection, identification and analysis.

No description available.

This thesis describes and evaluates the successes and failures of the use of strategic bombers through the abilities of one heavy bombardment group, the 389th. It examines the different missions that determined the effectiveness of the Group. When employed in a strategic bombing role, the 389th contributed significantly to the destruction of the German war industries and transportation system. When used as a tactical bomber, a mission for which it had neither proper training nor equipment, the 389th was generally a failure.

Schistosomen verursachen die Tropenparasitose Schistosomiasis mit 250-300 Millionen infizierten Menschen weltweit. Zur Analyse von Genfunktionen, durch die wirksame Medikamente entwickelt werden könnten, wird ein System zur Herstellung transgener Schistosomen benötigt. In dieser Arbeit wurde daher versucht, ein System zur Transfektion von Schistosoma mansoni von der transienten Expression von Reportergenen ausgehend zu entwickeln. Die Funktionalität der hergestellten Plasmide zur transienten Transfektion wurde durch die Expression der Reporter Enhanced Green Fluorescent Protein (EGFP) und beta-Galatosidase unter der Kontrolle der schistosomalen Promotoren für das Calreticulin, die 28 kDa-Glutathion-S-Transferase und das 70 kDa-Hitzeschockprotein in cos7-Zellen nachgewiesen. Für den Gentransfer in S. mansoni wurden Mikroinjektion, Elektroporation und Mikroprojektilbeschuss eingesetzt. Der Mikroprojektilbeschuss erwies sich als geeignete Methode, Plasmid-DNA zur transienten Expression von EGFP und beta-Galaktosidase in adulte und larvale Stadien zu transferieren. Da die beobachtete Reportergenexpression schwach war, wurde durch den Einsatz von Discosoma Red Fluorescent Protein (DsRed) als Reportergen versucht, das Transfektionsssytem zu optimieren. Außerdem wurden die potentiellen Promotoren für das Cathepsin D und für ein Aktin durch inverse PCR aus genomischer DNA isoliert. Die Funktionalität dieser Promotoren wurde in cos7- bzw. HeLa-Zellen nachgewiesen, eine verbesserte Expression in S. mansoni dagegen wurde mit diesen zwei Promotoren und unter Einsatz von DsRed nicht erreicht. Da S. mansoni sich in vitro nicht kultivieren lässt, muss für die Etablierung eines Transfektionssystems ein in seinen Keimzellen transgenes Stadium in den Lebenszyklus eingeschleust werden. Durch Mikroprojektilbeschuss transfizierte Mirazidien (freilebende Stadien des Parasiten) infizierten Zwischenwirtsschnecken und entwickelten sich zu transgenen Sporozysten, was durch die Transkription von EGFP nachgewiesen wurde. Für eine stabile Integration in das Genom werden unter anderem mobile genetische Elemente eingesetzt. Boudicca ist ein endogenes long terminal repeat (LTR) Retroelement. In dieser Arbeit wurde gezeigt, dass es in adulten Würmern, Sporozysten und Zerkarien transkribiert wird. Das LTR kann in vitro als Promotor zur Expression von EGFP eingesetzt werden. Außerdem wurde eine transkribierte Kopie kloniert und analysiert. Die in dieser Arbeit gewonnen Ergebnisse weisen darauf hin, dass Boudicca als Vektor zur stabilen Transfektion von S. mansoni eingesetzt werden kann. / Schistosomiasis caused by schistosomes affects about 250-300 million people world wide. The establishment of a transgenesis system for schistosomes is a pre-requisite to the identification of new drug targets and to the analysis of gene regulation and will add to our knowledge of parasite physiology and development. In this study, plasmids expressing Enhanced Green Fluorescent Protein (EGFP) and beta-galactosidase driven by the schistosomal promoters of the calreticulin, the 28kDa-glutathione-S-transferase and the 70kDa-heatshock protein were cloned and successfully tested in cos7-cells. Microinjection, electroporation and particle bombardment were used to introduce plasmid DNA into Schistosoma mansoni. Adult and larval stages of S. mansoni subjected to particle bombardment transiently expressed the reporter genes, although the observed transfection rates were very low. To optimize the transfection system Discosoma Red Fluorescent Protein (DsRed) was used as a reporter gene. Furthermore, the schistosomal promoters of the aspartic protease cathepsin D and a cytoplasmatic actin gene were isolated from genomic DNA using inverse PCR. The isolated promoters were able to drive EGFP and DsRed expression in cos7- or HeLa-cells, but improved expression could not be observed in S. mansoni. Due to the complexity of the life cycle S. mansoni cannot be cultured in vitro. In order to develop a transgenesis system for schistosomes, the life cycle must consequently be completed by genetically modified parasites. In this study it was shown that the free-living and mobile miracidium that infects the intermediate host snail could be transformed by particle bombardment and reintroduced in the life cycle using the natural path of infection. Development into transgenic sporocysts was shown through the isolation of EGFP transcripts from intermediate host snails. Mobile genetic elements are used as molecular tools to achieve stable integration of a foreign gene into a genome. Boudicca is an endogeneous long-terminal repeat (LTR) transposon in the schistosomal genome. In this study it was shown that it is transcribed in adult worms, sporocysts and cercariae. The LTR drives EGFP expression in cell cultures. Furthermore, a Boudicca transcript was cloned and analyzed by sequencing. The results suggest that Boudicca can be used as a molecular tool for stable integration of transgenes into the schistosomal genome.

Les travaux menés durant cette thèse s’inscrivent dans le cadre du développement de la mission spatiale Solar Probe Plus (NASA). Cette sonde d’exploration, dont la vocation est l’étude du Soleil, pénétrera la couronne solaire pour y faire des mesures in-situ. Ce travail de thèse a consisté à mener l’étude expérimentale des matériaux constitutifs de la sonde: métaux réfractaires (W, Re, Ta, Mo, Nb, Ti), composite C/C, un revêtement de nitrure de bore pyrolytique (pBN) ainsi que des dépôts métalliques texturés à forte émissivité. L’environnement à l’approche du soleil fut reproduit expérimentalement au sol au laboratoire PROMES-CNRS en associant le four solaire d’1 MW à Odeillo au moyen d’essai MEDIASE (Moyen d’Essai et de Diagnostic en Ambiance Spatiale Extrême). Grâce à ces moyens expérimentaux, ces matériaux candidats ont pu être testés sous formes d’échantillons, à très haute température (1100-2500 K), sous vide (10-4 Pa), ainsi que sous bombardement de protons (1-4 keV, jusqu'à 1018 ions m-2 s-1, pour la simulation du vent solaire). La propriété matériau sur laquelle cette étude expérimentale s’est concentrée est l’émissivité, dans la mesure où celle-ci va conditionner la température des surfaces de la sonde faisant face au Soleil. Celle-ci a été mesurée in-situ dans MEDIASE pendant les différents traitements. Dans le cadre de l’étude des métaux réfractaires, il a s’agit de comprendre la relation entre état de surface et émissivité, ainsi que d’étudier les modifications induites par les traitements. En ce qui concerne les dépôts métalliques texturés ainsi que le dépôt de pBN, leur étude a consisté à évaluer leurs performances radiatives et leurs comportements à haute température. / Solar Probe Plus (NASA) will be a historic mission of space exploration as it will consist in the first spacecraft to enter the solar corona. The spacecraft will face harsh environmental conditions that no other spacecraft has ever encountered in the past. One of the most critical technology developments for this mission is thus material science related: the materials constituting all the surfaces directly facing the Sun must be studied and tested in a relevant environment. The study of the candidate materials has been carried out at PROMES-CNRS: refractory metals for the instruments (W, Re, Ta, Mo, Nb, Ti, and their alloys), and refractory ceramics for the thermal protection shield (C/C composite and pyrolytic boron nitride - pBN). Samples of these materials were tested experimentally in a simulated near-Sun environment. This environment was simulated on ground thanks to the association of the 1 MW solar furnace in Odeillo, to the MEDIASE facility. This way materials were tested at very high temperature (1100-2500 K) in high vacuum (10-4 Pa), with the solar wind being reproduced via a proton bombardment (1-4 keV, up to 1018 ions m-2 s-1). The material properties that we have studied the most are the thermal radiative properties as they fully determine the temperature of a free-standing surface exposed to an intense radiative flux in vacuum. For refractory metals, it mainly consisted in studying the relationship between surface state and radiative properties, as well as the effect of the treatments. As for the textured metallic coatings and pBN, it consisted mainly in determining their efficiencies and understanding their behaviors.

Depuis la première synthèse du graphène, la spectroscopie Raman s'est imposée comme un standard pour la caractérisation de celui-ci. Le dopage et les défauts du graphène ont tous deux été étudiés abondamment à l'aide de spectromètres Raman en configuration confocal. Cependant, l'origine de toutes les bandes dans le spectre Raman du graphène n'est pas encore totalement comprise. Les bandes liées à la présence de défauts impliquent des processus de deuxièmes ordres qui se complexifient davantage avec le dopage du graphène. Aussi, la configuration confocal n'offre aucune information spatiale sur l'échantillon. Il est possible de générer une carte Raman par la prise de mesure point par point, mais ceci est un processus long et laborieux. Le RIMA, un appareil conçu dans le laboratoire du professeur Martel en collaboration avec Photon Etc, permet de pallier à ce problème. Avec un peu plus d'un million de spectres Raman mesurés en quelques heures sur une grande région, le RIMA permet de générer des cartes Raman d'une qualité exceptionnelle. Le RIMA est alors l'outil de choix pour l'étude présentée dans ce mémoire. L'objectif de ce mémoire est de mettre au point une méthode pour étudier l'effet du dopage et des défauts sur le spectre Raman du graphène. Pour obtenir des échantillons propices à l'étude, certains paramètres de la croissance du graphène par dépôt chimique en phase vapeur ont été étudiés. Le graphène a été dopé avec le couple oxydoréducteur Pt/PtO et des solutions tampons. Des cartes Raman ont été produites avec le RIMA. Ces cartes ont été prises à trois niveaux de dopage avant et après bombardement par ions d'argon. Les expériences sur la croissance ont permis d'obtenir des échantillons qui contiennent peu de bicouches, ce qui a permis de prendre des mesures sans tenir compte de leurs effets. Le dépôt de platine a été optimisé pour obtenir des particules dispersées de façon uniforme sur l'échantillon. Le dopage par le couple oxydoréducteur n'a pas été aussi efficace qu'attendu. L'analyse des cartes Raman a permis de révéler que le bombardement ne donne pas un résultat uniforme sur l'échantillon. L'hypothèse proposée pour expliquer l'écart du dopage et la non-uniformité des défauts est la présence de résidus de polymères à la surface du graphène avant le dépôt de platine. Ces résidus affecteraient le transfert de charge lors du dopage du graphène et protégeraient l'échantillon des ions d'argon lors du bombardement. / Since the first synthesis of graphene, Raman spectroscopy has become a standard for its characterization. Both doping and defects in graphene have been studied extensively using Raman spectrometers in confocal configuration. However, the origin of all bands in the Raman spectrum of graphene is not yet fully understood. The bands related to the presence of defects involve second order processes that become more complex with the doping of graphene. Also, the confocal configuration does not offer any spatial information on the sample. It is possible to generate a Raman map by taking point by point measurements, but this is a long and laborious process. The RIMA, a device designed in Professor Martel's laboratory in collaboration with Photon Etc, overcomes this problem. With a little more than a million Raman spectra measured in a few hours over a large area, the RIMA allows to generate Raman maps of exceptional quality. The RIMA is then the tool of choice for the study presented in this master's thesis. The objective of this master's thesis is to develop a method to study the effect of doping and defects on the Raman spectrum of graphene. To obtain suitable samples for the study, some parameters of graphene growth by chemical vapor deposition were studied. Graphene was doped with the redox couple Pt/PtO and buffer solutions. Raman maps were produced with the RIMA. These maps were taken at three doping levels before and after argon ion bombardment. Growth experiments yielded samples with few bilayers, allowing measurements to be taken without regard to their effects. Platinum deposition was optimized to obtain uniformly dispersed particles on the sample. Doping with the redox couple was not as effective as expected. The analysis of the Raman maps revealed that the bombardment does not give a uniform result on the sample. The hypothesis proposed to explain the doping discrepancy and the non-uniformity of the defects is the presence of polymer residues on the graphene surface before the platinum deposition. These residues would affect the charge transfer during the doping of the graphene and would protect the sample from argon ions during the bombardment.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

No description available.