Characterization of the Sterile20 kinase Slik : A regulator of growth in Drosophila

La prolifération cellulaire et la croissance tissulaire sont étroitement contrôlées au cours du développement. Chez la Drosophila melanogaster, ces processus sont régulés en partie par la kinase stérile-20 Slik (SLK et LOK chez les mammifères) et le suppresseur de tumeur Hippo (Hpo, MST1/2 chez les mammifères) dans les cellules épithéliales. La surexpression de la kinase Slik augmente la taille des tissus chez les mouches adultes. Cependant, les mutants slik-/- meurent avant d'avoir terminé leur développement. Lorsqu’elle est surexprimée dans les cellules épithéliales des ailes en voie de développement, cette protéine favorise la prolifération cellulaire. En outre, l'expression de Slik dans une population de cellules conduit à une surprolifération des cellules voisines, même quand elles sont physiquement séparées. Ceci est probablement dû à la sécrétion de facteurs de croissance qui stimulent la prolifération de manière paracrine. En utilisant des méthodes génétiques et transcriptomiques, nous essayons de déterminer les molécules et les mécanismes impliqués. Contrairement à ce qui a été publié, nous avons constaté que Slik ne transmet pas de signal prolifératif en inhibant le suppresseur de tumeur Merlin (Mer, NF2 chez les mammifères), un composant en amont de la voie Hippo. Plutôt, elle favorise la prolifération non-autonome et la croissance des tissus en signalisation par la kinase dRaf (la seule kinase de la famille Raf chez la drosophile). Nous prouvons que dRaf est nécessaire chez les cellules voisines pour conduire la prolifération chez ces cellules. De plus, nous avons utilisé le séquençage du transcriptome pour identifier de nouveaux effecteurs en aval de Slik. Ce qui permettra de mieux comprendre les effets de SLK et LOK chez les humains.

Régulation de la kinase Ste20 Slik de Drosophila par phosphorylation de la boucle d'activation

Les kinases constituent une famille majeure de protéines qui régulent divers processus par la phosphorylation de leurs substrats, mais aussi par leur activité non- catalytique. Ce rôle indépendant de l’activité kinase a été observé chez quelques protéines dont des membres de la famille Sterile-20. La kinase Ste20 Slik de Drosophila aide au maintien de l’intégrité des tissus épithéliaux en phosphorylant l’ERM Moesin et peut aussi induire une prolifération cellulaire non-autonome indépendamment de son activité catalytique. La méthode de régulation de ces deux rôles était jusqu’ici inconnue. Nous avons identifié 19 sites de phosphorylation chez Slik par spectrométrie de masse. À l’aide de mutants, nous démontrons que les deux fonctions de Slik sont régulées par la phosphorylation d’au moins 2 résidus conservés de son segment d’activation par un mécanisme d’auto- et/ou trans-phosphorylation. Cette étude amène une meilleure compréhension de la régulation de l’intégrité épithéliale et de la croissance, deux processus clés qui sont souvent déréglés dans le cancer et certaines maladies génétiques.

Spectacle and the one-man band : technology, performing bodies, and imaginary spaces

L’étiquette « homme-orchestre » est apposée à une grande variété de musiciens qui se distinguent en jouant seuls une performance qui est normalement interprétée par plusieurs personnes. La diversité qu’a pu prendre au cours du temps cette forme n’est pas prise en compte par la culture populaire qui propose une image relativement constante de cette figure tel que vue dans les films Mary Poppins (1964) de Walt Disney et One-man Band (2005) de Pixar. Il s’agit d’un seul performeur vêtu d’un costume coloré avec une grosse caisse sur le dos, des cymbales entre les jambes, une guitare ou un autre instrument à cordes dans les mains et un petit instrument à vent fixé assez près de sa bouche pour lui permettre d’alterner le chant et le jeu instrumental. Cette thèse propose une analyse de l’homme-orchestre qui va au-delà de sa simple production musicale en situant le phénomène comme un genre spectaculaire qui transmet un contenu symbolique à travers une relation tripartite entre performance divertissante, spectateur et image. Le contenu symbolique est lié aux idées caractéristiques du Siècle des lumières tels que la liberté, l’individu et une relation avec la technologie. Il est aussi incarné simultanément par les performeurs et par la représentation de l’homme-orchestre dans l’imaginaire collectif. En même temps, chaque performance sert à réaffirmer l’image de l’homme-orchestre, une image qui par répétitions est devenue un lieu commun de la culture, existant au-delà d’un seul performeur ou d’une seule performance. L’aspect visuel de l’homme-orchestre joue un rôle important dans ce processus par une utilisation inattendue du corps, une relation causale entre corps, technologie et production musicale ainsi que par l’utilisation de vêtements colorés et d’accessoires non musicaux tels des marionnettes, des feux d’artifice ou des animaux vivants. Ces éléments spectaculaires divertissent les spectateurs, ce qui se traduit, entre autres, par un gain financier pour le performeur. Le divertissement a une fonction phatique qui facilite la communication du contenu symbolique.

Bodies, stories, cities : learning to read and write (in) Montréal with Gail Scott

Cette lecture, tant critique, comparative, et théorique que pédagogique, s’ancre dans le constat, premièrement, qu’il advient aux étudiantEs en littérature de se (re)poser la question des coûts et complicités qu’apprendre à lire et à écrire présuppose aujourd’hui; deuxièmement, que nos pratiques littéraires se trament au sein de lieux empreints de différences, que l’on peut nommer, selon le contexte, métaphore, récit, ville; et, troisièmement, que les efforts et investissements requis sont tout autant couteux et interminable qu’un plaisir et une nécessité politique. Ces conclusions tendent vers l’abstrait et le théorique, mais le langage en lequel elles sont articulées, langage corporel et urbain, de la dépendance et de la violence, cherche d’autant plus une qualité matérielle et concrète. Or, l’introduction propose un survol des lectures et comparaisons de Heroine de Gail Scott qui centre ce projet; identifie les contextes institutionnels, historiques, et personnels qui risquent, ensuite, de décentrer celui-ci. Le premier chapitre permet de cerner le matérialisme littéraire qui me sert de méthode par laquelle la littérature, à la fois, sollicite et offre une réponse à ces interrogations théoriques. Inspirée de l’œuvre de Gail Scott et Réjean Ducharme, premièrement, et de Walter Benjamin, Elisabeth Grosz, et Pierre Macherey ensuite, ‘matérialisme’ fait référence à cette collection de figures de pratiques littéraires et urbaines qui proviennent, par exemple, de Georges Perec, Michel DeCerteau, Barbara Johnson, et Patricia Smart, et qui invitent ensuite une réflexions sur les relations entre corporalité et narrativité, entre la nécessité et la contingence du littéraire. De plus, une collection de figures d’un Montréal littéraire et d’une cité pédagogique, acquis des œuvres de Zygmunt Bauman, Partricia Godbout, et Lewis Mumford, constitue en effet un vocabulaire nous permettant de mieux découvrir (et donc enseigner) ce que lire et apprendre requiert. Le deuxième chapitre propose une lecture comparée de Heroine et des romans des auteures québécoises Anne Dandurand, Marie Gagnon, et Tess Fragoulis, dans le contexte, premièrement, les débats entourant l’institutionnalisation de la littérature (anglo)Québécoise et, deuxièmement, des questions pédagogiques et politiques plus larges et plus urgentes que nous pose, encore aujourd’hui, cette violence récurrente qui s’acharna, par exemple, sur la Polytechnique en 1989. Or, cette intersection de la violence meurtrière, la pratique littéraire, et la pédagogie qui en résulte se pose et s’articule, encore, par le biais d’une collection de figures de styles. En fait, à travers le roman de Scott et de l’œuvre critique qui en fait la lecture, une série de craques invite à reconnaître Heroine comme étant, ce que j’appelle, un récit de dépendance, au sein duquel se concrétise une temporalité récursive et une logique d’introjection nous permettant de mieux comprendre la violence et, par conséquent, le pouvoir d’une pratique littéraire sur laquelle, ensuite, j’appuie ma pédagogie en devenir. Jetant, finalement, un regard rétrospectif sur l’oeuvre dans son entier, la conclusion de ce projet se tourne aussi vers l’avant, c’est-à-dire, vers ce que mes lectures dites matérialistes de la littérature canadienne et québécoise contribuent à mon enseignement de la langue anglaise en Corée du Sud. C’est dans ce contexte que les propos de Jacques Rancière occasionnent un dernier questionnement quant à l’historique des débats et des structures pédagogiques en Corée, d’une part, et, de l’autre, les conclusions que cette lecture de la fiction théorique de Gail Scott nous livre.

Acoustic radiation and scattering from cylindrical bodies and analysis of transducer arrays

New mathematical methods to analytically investigate linear acoustic radiation and scattering from cylindrical bodies and transducer arrays are presented. Three problems of interest involving cylinders in an infinite fluid are studied. In all the three problems, the Helmholtz equation is used to model propagation through the fluid and the beam patterns of arrays of transducers are studied. In the first problem, a method is presented to determine the omni-directional and directional far-field pressures radiated by a cylindrical transducer array in an infinite rigid cylindrical baffle. The solution to the Helmholtz equation and the displacement continuity condition at the interface between the array and the surrounding water are used to determine the pressure. The displacement of the surface of each transducer is in the direction of the normal to the array and is assumed to be uniform. Expressions are derived for the pressure radiated by a sector of the array vibrating in-phase, the entire array vibrating in-phase, and a sector of the array phase-shaded to simulate radiation from a rectangular piston. It is shown that the uniform displacement required for generating a source level of 220 dB ref. μPa @ 1m that is omni directional in the azimuthal plane is in the order of 1 micron for typical arrays. Numerical results are presented to show that there is only a small difference between the on-axis pressures radiated by phased cylindrical arrays and planar arrays. The problem is of interest because cylindrical arrays of projectors are often used to search for underwater objects. In the second problem, the errors, when using data-independent, classical, energy and split beam correlation methods, in finding the direction of arrival (DOA) of a plane acoustic wave, caused by the presence of a solid circular elastic cylindrical stiffener near a linear array of hydrophones, are investigated. Scattering from the effectively infinite cylinder is modeled using the exact axisymmetric equations of motion and the total pressures at the hydrophone locations are computed. The effect of the radius of the cylinder, a, the distance between the cylinder and the array, b, the number of hydrophones in the array, 2H, and the angle of incidence of the wave, α, on the error in finding the DOA are illustrated using numerical results. For an array that is about 30 times the wavelength and for small angles of incidence (α<10), the error in finding the DOA using the energy method is less than that using the split beam correlation method with beam steered to α; and in some cases, the error increases when b increases; and the errors in finding the DOA using the energy method and the split beam correlation method with beam steered to α vary approximately as a7 / 4 . The problem is of interest because elastic stiffeners – in nearly acoustically transparent sonar domes that are used to protect arrays of transducers – scatter waves that are incident on it and cause an error in the estimated direction of arrival of the wave. In the third problem, a high-frequency ray-acoustics method is presented and used to determine the interior pressure field when a plane wave is normally incident on a fluid cylinder embedded in another infinite fluid. The pressure field is determined by using geometrical and physical acoustics. The interior pressure is expressed as the sum of the pressures due to all rays that pass through a point. Numerical results are presented for ka = 20 to 100 where k is the acoustic wavenumber of the exterior fluid and a is the radius of the cylinder. The results are in good agreement with those obtained using field theory. The directional responses, to the plane wave, of sectors of a circular array of uniformly distributed hydrophones in the embedded cylinder are then computed. The sectors are used to simulate linear arrays with uniformly distributed normals by using delays. The directional responses are compared with the output from an array in an infinite homogenous fluid. These outputs are of interest as they are used to determine the direction of arrival of the plane wave. Numerical results are presented for a circular array with 32 hydrophones and 12 hydrophones in each sector. The problem is of interest because arrays of hydrophones are housed inside sonar domes and acoustic plane waves from distant sources are scattered by the dome filled with fresh water and cause deterioration in the performance of the array.

Role of academic bodies with special references to the academic programmes in the universities in Kerala

This makes a thorough study of Role of Academic Bodies with Special Reference to the Academic Programmes in the Universities in Kerala. The present study has dealt with the constitution, powers and functions of the authorities of the universities in Kerala, especially the academic bodies which have well-defined powers and function. This thesis gives in detail the role and functions of the academic bodies in the four universities in Kerala, under study: and examines the role-effectiveness of these bodies. To sum up, it can be concluded that the academic bodies in the universities in Kerala do not function as effectively as envisaged in the laws of the universities. They have a maintenance-oriented approach rather than a dynamic one, without thinking in terms of introducing innovations. They need improvement in the various aspects of their constitution, selection of members and their mode of functioning

Studies on physicochemical and biological properties of protease inhibitor from edible mushroom Pleurotus floridanus

Protease inhibitors are found abundantly in numerous plants, animals and microorganisms, owing their significance to their application in the study of enzyme structures, reaction mechanisms and also their utilization in pharmacology and agriculture. They are (synthetic/natural) substances that act directly on proteases to lower the catalytic rate. Although most of these inhibitory proteins are directed against serine proteases, some target cysteine, aspartyl or metalloproteases (Bode and Huber, 1992). Protease inhibitors are essential for regulating the activity of their corresponding proteases and play key regulatory roles in many biological processes. Applications of protease inhibitors are intimately connected to the proteases they inhibit; an overview of proteases with the modes of regulation of their proteolytic activity is discussed

“Studies on extracellular enzyme production during growth of Pleurotus sp. on lignocellulosic agriwaste and the utilization of spent mushroom substrate”

Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043×10−10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases

A crack on the interface of piezo-electric bodies

Singularities of elastic and electric fields are investigated at the tip of a crack on the interface of two anisotropic piezo-electric media under various boundary conditions on the crack surfaces. The Griffith formulae are obtained for increments of energy functionals due to growth of the crack and the notion of the energy release matrix is introduced. Normalization conditions for bases of singular solution are proposed to adapt them to the energy, stress, and deformation fracture criteria. Connections between these bases are determined and additional properties of the deformation basis related to the notion of electric surface enthalpy are established.

Not the outside but the inside matters : functional analysis of Capricious and Tartan during Drosophila tracheal morphogenesis

The tubular structures, which transport essential gases, liquids, or cells from one site to another, are shared among various divergent organisms. These highly organized tubular networks include lung, kidney, vasculature and mammary gland in mammals as well as trachea and salivary gland in Drosophila melanogaster. Many questions regarding the tubular morphogenesis cannot be addressed sufficiently by investigating the mammalian organs because their structures are extremely complex and therefore, systematic analyses of genetic and cellular programs guiding the development is not possible. In contrast, the Drosophila tracheal development provides an excellent model system since many molecular markers and powerful tools for genetic manipulations are available. Two mechanisms were shown to be important for the outgrowth of tracheal cells: the FGF signaling pathway and the interaction between the tracheal cells and the surrounding mesodermal cells. The Drosophila FGF ligand encoded by branchless (bnl) is localized in groups of cells near tracheal metameres. The tracheal cells expressing the FGF receptor breathless (btl) respond to these sources of FGF ligand and extend towards them. However, this FGF signaling pathway is not sufficient for the formation of continuous dorsal trunk, the only muticellular tube in tracheal system. Recently, it was found out that single mesodermal cells called bridge-cells are essential for the formation of continuous dorsal trunk as they direct the outgrowth of dorsal trunk cells towards the correct targets. The results in this PhD thesis demonstrate that a cell adhesion molecule Capricious (Caps), which is specifically localized on the surface of bridge-cells, plays an essential role in guiding the outgrowing dorsal trunk cells towards their correct targets. When caps is lacking, some bridge-cells cannot stretch properly towards the adjacent posterior tracheal metameres and thus fail to interconnect the juxtaposing dorsal trunk cells. Consequently, discontinuous dorsal trunks containing interruptions at several positions are formed. On the other hand, when caps is ectopically expressed in the mesodermal cells through a twi-GAL4 driver, these mesodermal cells acquire a guidance function through ectopic caps and misguide the outgrowing dorsal trunk cells in abnormal directions. As a result, disconnected dorsal trunks are formed. These loss- and gain-of-function studies suggest that Caps presumably establishes the cell-to-cell contact between the bridge-cells and the tracheal cells and thereby mediates directly the guidance function of bridge-cells. The most similar protein known to Caps is another cell adhesion molecule called Tartan (Trn). Interestingly, trn is expressed in the mesodermal cells but not in the bridge-cells. When trn is lacking, the outgrowth of not only the dorsal trunks but also the lateral trunks are disrupted. However, in contrast to the ectopic expression of caps, the misexpression of trn does not affect tracheal development. Whereas Trn requires only its extracellular domain to mediate the matrix function, Caps requires both its extracellular and intracellular domains to function as a guidance molecule in the bridge-cells. These observations suggest that Trn functions differently from Caps during tracheal morphogenesis. Presumably, Trn mediates a matrix function of mesodermal cells, which support the tracheal cells to extend efficiently through the surrounding mesodermal tissue. In order to determine which domains dictate the functional specificity of Caps, two hybrid proteins CapsEdTrnId, which contains the Caps extracellular domain and the Trn intracellular domain, and TrnEdCapsId, which consists of the Trn extracellular domain and the Caps intracellular domain, were constructed. Gain of function and rescue experiments with these hybrid proteins suggest on one hand that the extracellular domains of Caps and Trn are functionally redundant and on the other hand that the intracellular domain dictates the functional specificity of Caps. In order to identify putative interactors of Caps, yeast two-hybrid screening was performed. An in vivo interaction assay in yeast suggests that Ras64B interacts specifically with the Caps intracellular domain. In addition, an in vitro binding assay reveals a direct interaction between an inactive form of Ras64B and the Caps intracellular domain. ras64B, which encodes a small GTPase, is expressed in the mesodermal cells concurrently as caps. Finally, a gain-of-function study with the constitutively active Ras64B suggests that Ras64B presumably functions downstream of Caps. All these results suggest consistently that the small GTPase Ras64B binds specifically to the Caps intracellular domain and may thereby mediate the guidance function of Caps.

Functional genomics of the Drosophila melanogaster X chromosome and the role of DWnt5 during development

The collection of X chromosome insertions (PX) lethal lines, which was isolated from a screen for essential genes on the X chromosome, was characterized by means of cloning the insertion sites, mapping the sites within genomic DNA and determination of the associated reporter gene expresssion patterns. The established STS flanking the P element insertion sites were submitted to EMBL nucleotide databases and their in situ data together with the enhancer trap expression patterns have been deposited in the FlyView database. The characterized lines are now available to be used by the scientific community for a detailed analysis of the newly established lethal gene functions. One of the isolated genes on the X chromosome was the Drosophila gene Wnt5 (DWnt5). From two independent screens, one lethal and three homozygous viable alleles were recovered, allowing the identification of two distinct functions for DWnt5 in the fly. Observations on the developing nervous system of mutant embryos suggest that DWnt5 activity affects axon projection pattern. Elevated levels of DWNT5 activity in the midline cells of the central nervous system causes improper establishment and maintenance of the axonal pathways. Our analysis of the expression and mutant phenotype indicates that DWnt5 function in a process needed for proper organization of the nervous system. A second and novel function of DWnt5 is the control of the body size by regulation of the cell number rather than affecting the size of cells. Moreover, experimentally increased DWnt5 levels in a post-mitotic region of the eye imaginal disc causes abnormal cell cycle progression, resulting in additional ommatidia in the adult eye when compared to wild type. The increased cell number and the effects on the cell cycle after exposure to high DWNT5 levels is the result of a failure to downregulate cyclin B and therefore the unsuccessful establishment of a G1 arrest.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

Studies of cap-independent mRNA translation in Drosophila melanogaster

Control of protein synthesis is a key step in the regulation of gene expression during apoptosis and the heat shock response. Under such conditions, cap-dependent translation is impaired and Internal Ribosome Entry Site (IRES)-dependent translation plays a major role in mammalian cells. Although the role of IRES-dependent translation during apoptosis has been mainly studied in mammals, its role in the translation of Drosophila apoptotic genes has not been yet studied. The observation that the Drosophila mutant embryos for the cap-binding protein, the eukaryotic initiation factor eIF4E, exhibits increased apoptosis in correlation with up-regulated proapoptotic gene reaper (rpr) transcription constitutes the first evidence for the existence of a cap-independent mechanism for the translation of Drosophila proapoptotic genes. The mechanism of translation of rpr and other proapoptotic genes was investigated in this work. We found that the 5 UTR of rpr mRNA drives translation in an IRES-dependent manner. It promotes the translation of reporter RNAs in vitro either in the absence of cap, in the presence of cap competitors, or in extracts derived from heat shocked and eIF4E mutant embryos and in vivo in cells transfected with reporters bearing a non functional cap structure, indicating that cap recognition is not required in rpr mRNA for translation. We also show that rpr mRNA 5 UTR exhibits a high degree of similarity with that of Drosophila heat shock protein 70 mRNA (hsp70), an antagonist of apoptosis, and that both are able to conduct IRES-mediated translation. The proapoptotic genes head involution defective (hid) and grim, but not sickle, also display IRES activity. Studies of mRNA association to polysomes in embryos indicate that both rpr, hsp70, hid and grim endogenous mRNAs are recruited to polysomes in embryos in which apoptosis or thermal stress was induced. We conclude that hsp70 and, on the other hand, rpr, hid and grim which are antagonizing factors during apoptosis, use a similar mechanism for protein synthesis. The outcome for the cell would thus depend on which protein is translated under a given stress condition. Factors involved in the differential translation driven by these IRES could play an important role. For this purpose, we undertook the identification of the ribonucleoprotein (RNP) complexes assembled onto the 5 UTR of rpr mRNA. We established a tobramycin-affinity-selection protocol that allows the purification of specific RNP that can be further analyzed by mass spectrometry. Several RNA binding proteins were identified as part of the rpr 5 UTR RNP complex, some of which have been related to IRES activity. The involvement of one of them, the La antigen, in the translation of rpr mRNA, was established by RNA-crosslinking experiments using recombinant protein and rpr 5 UTR and by the analysis of the translation efficiency of reporter mRNAs in Drosophila cells after knock down of the endogenous La by RNAi experiments. Several uncharacterized proteins were also identified, suggesting that they might play a role during translation, during the assembly of the translational machinery or in the priming of the mRNA before ribosome recognition. Our data provide evidence for the involvement of La antigen in the translation of rpr mRNA and set a protocol for purification of tagged-RNA-protein complexes from cytoplasmic extracts. To further understand the mechanisms of translation initiation in Drosophila, we analyzed the role of eIF4B on cap-dependent and cap-independent translation. We showed that eIF4B is mostly involved in cap-, but not IRES-dependent translation as it happens in mammals.

Characterization of the role of Drosophila JAK/STAT signalling in cellular proliferation

Der Janus Kinase / signal transducer and activator of transcription (JAK/STAT) Signal- transduktionsweg wird für viele Entwicklungsvorgänge benötigt und spielt eine zentrale Rolle bei der Hämatopoese und bei der Immunantwort. Obwohl der JAK/STAT-Signalweg in den vergangenen Jahren Gegenstand intensiver Forschung war, erschwert die Redundanz des Signalwegs bei Wirbeltieren genetische Untersuchungen zur Identifizierung derjenigen Mechanismen, die den JAK/STAT-Signalweg regulieren. Der JAK/STAT-Signaltransduktionsweg ist evolutionär konserviert und ebenfalls bei der Taufliege Drosophila melanogaster vorhanden. Im Gegensatz zu Wirbeltieren ist der Signaltransduktionsweg von Drosophila weniger redundant und beinhaltet folgende Hauptkomponenten: den Liganden Unpaired (Upd), den Transmembranrezeptor Domeless (Dome), die einzige JAK-Tyrosinkinase Hopscotch (hop), sowie den Transkriptionsfaktor STAT92E. In der vorliegenden Arbeit wird die Rolle des JAK/STAT-Signalwegs bei der zellulären Proliferation mithilfe der Modellsysteme der Flügel- und der Augen-Imaginalscheiben von Drosophila charakterisiert. "Loss-of-function"- und "Gain-of-function"-Experimente zur Verminderung beziehungs-weise Erhöhung der Signalaktivität zeigten, dass der JAK/STAT-Signalweg eine Rolle bei der zellulären Proliferation der Flügel-Imaginalscheiben spielte, ohne die Zellgröße oder Apoptose zu verändern. Bei der Flügelentwicklung während des zweiten und des frühen dritten Larvalstadiums war die Aktivität des JAK/STAT-Signalwegs sowohl notwendig für die zelluläre Proliferation als auch hinreichend, um Überproliferation anzutreiben. Allerdings änderte sich während der späten dritten Larvalstadien die JAK/STAT-Signalaktivität, sodass endogene STAT92E-Mengen einen anti-proliferativen Effekt im gleichen Gewebe aufwiesen. Weiterhin reichte die ektopische Aktivierung des JAK/STAT-Signalwegs zu diesem späten Entwicklungszeitpunkt aus, um die Mitose zu inhibieren und die Zellen in der Phase G2 des Zellzyklus zu arretieren. Diese Ergebnisse legen den Schluss nahe, dass der JAK/STAT-Signalweg sowohl pro-proliferativ in frühen Flügelscheiben als auch anti-proliferativ zu späten Stadien der Flügelscheiben-Entwicklung wirken kann. Dieser späte anti-proliferative Effekt wurde durch einen nicht-kanonischen Mechanismus der STAT92E-Aktivierung vermittelt, da späte hop defiziente Zellverbände im Vergleich zu Wildtyp-Zellen keine Veränderungen im Ausmaß der zellulären Proliferation aufwiesen. Ferner konnte gezeigt werden, dass eine während der Larvalstadien exprimierte dominant-negative und im N-Terminus deletierte Form von STAT92E (?NSTAT92E) nicht für den anti-proliferativen Effekt verantwortlich ist. Diese Tatsache ist ein weiteres Indiz dafür, dass das vollständige STAT92E den späten anti-proliferativen Effekt verursacht. Um Modulatoren für die von JAK/STAT vermittelte zelluläre Proliferation zu identifieren, wurde ein P-Element-basierter genetischer Interaktions-Screen in einem sensibilisierten genetischen Hintergrund durchgeführt. Insgesamt wurden dazu 2267 unabhängige P-Element-Insertionen auf ihre Wechselwirkung mit der JAK/STAT-Signalaktivität untersucht und 24 interagierende Loci identifiziert. Diese Kandidaten können in folgende Gruppen eingeordnet werden: Zellzyklusproteine, Transkriptionsfaktoren, DNA und RNA bindende Proteine, ein Mikro-RNA-Gen, Komponenten anderer Signaltransduktionswege und Zelladhäsionsproteine. In den meisten Fällen wurden mehrere Allele der interagierenden Kandidatengene getestet. 18 Kandidatengene mit übereinstimmend interagierenden Allelen wurden dann zur weiteren Analyse ausgewählt. Von diesen 18 Kandidaten-Loci wurden 7 mögliche JAK/STAT-Signalwegskomponenten und 6 neue Zielgene des Signalwegs gefunden. Zusammenfassend wurde das Verständnis um STAT92E verbessert. Dieses Protein hat die gleiche Funktion wie das STAT3-Protein der Wirbeltiere und treibt die zelluläre Proliferation voran. Analog zu STAT1 hat STAT92E aber auch einen anti-proliferativen Effekt. Ferner wurden 24 mögliche Modulatoren der JAK/STAT-Signalaktivität identifiziert. Die Charakterisierung dieser Wechselwirkungen eröffnet vielversprechende Wege zu dem Verständnis, wie JAK/STAT die zelluläre Proliferation reguliert und könnte bei der Entwicklung von neuartigen therapeutischen Targets zur Behandlung von Krebskrankheiten und Entwicklungsstörungen beitragen.