Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.

No description available.

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana. / Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime. / Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

No description available.

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. / Dissertation

Les virus géants sont un groupe de virus ADN double brin caractérisés par une taille géante du virion et du génome, et un répertoire de gènes qui comprend environ 450 à 2500 gènes prédits. Une proportion importante de ces gènes (jusqu'à 93%) sont des 'ORFans', ou codent pour des protéines de fonction inconnue. Acanthamoeba polyphaga mimivirus est le premier virus géant découvert, il y a une décennie, par co-culture sur Acanthamoeba spp. Il est le membre prototype de la famille Mimiviridae. Le génome de Mimivirus code pour environ 1000 protéines, parmi lesquelles ~50% n'ont pas d'homologue connu dans les banques de séquences publiques. La capside de Mimivirus a un diamètre d'environ 500 nm et est couverte par une couche dense de fibres, à l'exception de l'un de ses sommets. Ces fibres sont d'environ 130 nm de longueur et se composent d'une tige souple et d'une tête de forme globulaire.Dans ce travail de thèse, nous avons cherché à étudier les gènes impliqués dans la formation des fibres de Mimivirus. Dans ce but, nous avons notamment exprimé des gènes candidats dans E. coli, et nous avons mis au point une stratégie qui a utilisé l'interférence ARN afin d'étudier la fonction et la structure des protéines de Mimivirus. Nous avons annoté quatre protéines associées aux fibres. La stratégie utilisant les petits ARN interférant appliquée ici est originale et a été utilisée pour la première fois pour les virus géants qui infectent les amibes. Elle pourrait permettre de décrypter la fonction des gènes des mimivirus et d'annoter potentiellement des centaines de protéines présentes dans les bases de données publiques, et de différencier l'ADN poubelle des gènes réellement utilisés. / Giant viruses are a group of double stranded DNA viruses that are characterized by a giant virion and genome size, and gene repertoires encompassing approximately 450 to 2500 predicted genes. A substantial proportion of these genes (up to 93%) consists in ORFans, or encodes proteins with unknown functions. Acanthamoeba polyphaga mimivirus is the first giant virus that was discovered, a decade ago, after co-culturing on Acanthamoeba spp. It is the prototype member of the family Mimiviridae. Mimivirus encodes about 1000 proteins, among which ~50% have no known homolog in public sequence databases. The Mimivirus capsid is about 500 nm in diameter and is covered by a dense layer of fibers, except at one of its vertices. These fibers are about 130 nm in length and consist of a soft shaft and a globular shaped head.In this thesis work, we aimed to study the genes involved in the formation of the Mimivirus fibers. For this purpose, we have expressed candidate genes in E. coli, and implemented a strategy that used RNA interference to study the function and structure of Mimivirus proteins. We then succeeded in annotating four proteins as fiber associated proteins. The short interfering RNA strategy that we applied here is original and has been used for the first time in giant viruses that infect amoeba. It could allow deciphering the function of the mimivirus gene repertoires and help annotating hundreds of proteins without known function found in public databases and differentiate between junk DNA and truly used genes.

La différenciation sexuelle des Isopodes dépend d'une hormone sexuelle protéique, l'hormone androgène (HA), caractéristique des Malacostracés. Cet Insulin-Like Peptide suffit à induire par sa présence la différenciation mâle de ces Crustacés. Nous avons identifié in silico le transporteur circulant de l'HA, l'IGFBP-rP1, chez de nombreuses espèces d'Isopodes ainsi qu'à l'échelle des Crustacés. De la même façon, nous avons identifié deux récepteurs transmembranaires, l'IR1 et l'IR2, issus d'une duplication de gène spécifique des Malacostracés. Les patrons d'expression de ces gènes ont été étudiés sur notre espèce modèle, Armadillidium vulgare. Av-IGFBP-rP1 et Av-IR1 sont exprimés de manière ubiquiste et tout au long du développement. Av-IR2 est aussi exprimé à chaque stade de la différenciation mais ce transcrit est quasi-spécifique des glandes androgènes et ovaires. Une approche par ARNi a confirmé l'implication de ces trois protéines dans la voie de signalisation de l'HA. En effet, l'inhibition de l'HA, Av-IGFBP-rP1 et Av-IR1 provoquent l'hypertrophie des glandes androgènes, suggérant leur implication dans une boucle de rétro-contrôle de l'HA. L'inhibition de Av-IR2 semble seulement provoquer la différenciation d'ouvertures génitales femelles. Ces phénotypes sont comparables à ceux des intersexués mâles induits par la bactérie féminisante endogène Wolbachia. Nous montrons cependant que la bactérie altère seulement l'expression de l'HA et pas celle des récepteurs. Enfin, nous avons testé l'effet du bisphénol A mais nous n'observons pas d'altération de la différenciation sexuelle des larves lors d'expositions à ce perturbateur endocrinien exogène. / Sexual differentiation in Isopods relies on a proteinaceous sex hormone called androgenic hormone (AH), specific to Malacostracans. This Insulin-Like Peptide induces male differentiation by its mere presence in these Crustaceans. We identified in silico the circulating carrier of the AH, called IGFBP-rP1, in many Isopod species, but also on the crustacean scale. Similarly, we identified two transmembrane receptors, IR1 and IR2, coming from a gene duplication specific to Malacostracans. The expression patterns of these genes were investigated in our model species, Armadillidium vulgare. Av-IGFBP-rP1 and Av-IR1 are broadly expressed in the animal and throughout development. Av-IR2 is also expressed at each developmental stage but this transcript is almost specific to androgenic glands and ovaries. An RNAi approach has confirmed the implication of these three proteins in the AH signalling pathway. Indeed, the inhibition of AH, Av-IGFBP-rP1 and Av-IR1 induces androgenic gland hypertrophy, suggesting their implication in an AH feedback loop. Av-IR2 inhibition seems to provoke the differentiation of female genital apertures only. These phenotypes are similar to those of male intersexes induced by the endogenous feminizing bacterium Wolbachia. Yet, we show that the bacterium alters the expression of the AH only and not the one of its receptors. Finally, we have tested the effect of bisphenol A but we observe no alteration of the sexual differentiation in larvae upon exposition to this exogenous endocrine disruptor.

O carcinoma de células escamosas de vulva (CECV) corresponde a cerca de 95% dos tumores vulvares. Apresenta bom prognóstico quando diagnosticado precocemente. O tratamento cirúrgico, embora eficaz, pode ser mutilante e acarreta em sérios danos psicossociais para as pacientes. Embora algumas pesquisas sobre os mecanismos que determinam os comportamentos clínico e biológico dos CECV tenham sido realizadas, ainda há muito a ser investigado. As tetraspaninas (TSPANs) são proteínas de membrana que interagem com diversas moléculas e estão envolvidas em diferentes processos fisiológicos como proliferação e migração celular. Vários estudos associam sua expressão desregulada ao desenvolvimento de cânceres. Resultados anteriores de nosso grupo mostraram maior expressão de CD63 em amostras de pacientes com CECV, e menor expressão de KAI1/CD82, em relação ao tecido normal adjacente. Porém, seu papel nesses tumores permanece incerto. Assim, o objetivo deste trabalho foi caracterizar os perfis de expressão gênica e proteica de CD63 e KAI1/CD82, bem como avaliar os efeitos de sua manipulação genética no comportamento de células de CECV metastático e não metastático. As linhagens utilizadas neste trabalho foram SW954 (ATCC® HTB-117(TM), não metastática) e SW962 (ATCC® HTB-118(TM), metastática). Os ensaios de qRT PCR mostraram maior expressão de CD63, e menor de KAI1/CD82, nas células metastáticas, em relação as não metastáticas. Assim, optou-se pela manipulação transiente somente de CD63 por RNA interferente (RNAi). Os resultados mostraram efeitos significativos de inibição de expressão gênica e proteica de CD63 em ambas linhagens, por qRT PCR e imunocitoquímica (ICQ), respectivamente, porém, a inibição da TSPAN foi mais proeminente nas células metastáticas. A deleção da tetraspanina acarretou em redução significativa da proliferação das células não metastáticas (*p < 0.05) e metastáticas (**p=0.0022), bem como na migração das células metastáticas (*p < 0.05). Assim sendo, os resultados apontam CD63 com relevante papel nos CECV, uma vez que sua inibição compromete a proliferação e capacidade de migração das células tumorais. Além disso, a perda de expressão do supressor de metástases KAI1/CD82 corrobora os dados para outros tipos de canceres. Em conjunto, essas TSPAN, podem ser considerados não só importantes fatores prognósticos no CECV, mas potenciais alvos terapêuticos / Vulvar squamous cell carcinoma (VSCC) accounts about 95% of vulvar tumors. When diagnosed at an early stage, prognosis is usually good. Although effective, surgical treatment can be mutilating and entails serious psychosocial damage to patients. Whilst molecular aspects in VSCCs have been investigated, mechanisms underlying the VSCC clinical and biological behavior are poorly understood. Tetraspanins (TSPANs) are membrane proteins, which can interact with several molecules. In addition, they are involved in physiological processes such as proliferation and migration. Besides that, several studies show the deregulated expression of TSPANs associated with cancer development. Previous results of our group showed higher expression of CD63, and lower expression of KAI1/CD82, in VSCC patient samples, compared to adjacent normal tissue. However, the role of these proteins in vulvar tumors remains uncertain. Thus, the aim of this work was to characterize CD63 and KAI1/CD82 gene and protein expression profile in VSCC metastatic and non-metastatic cells lines, and to investigate the effects of genetic manipulation on these cells behavior. The cell lines used in this work were SW954 (Non-metastatic) and SW962 (metastatic). The qRT PCR assays showed CD63 overexpression and KAI1/CD82 downexpression in the metastatic cells. Thus, we chose to perform transient manipulation of CD63 by interfering RNA (RNAi). The RNAi assays showed significant inhibitory effects of gene and protein expression of CD63 in both cell lines, by qRT PCR and immunocytochemistry (ICC), respectively, however, inhibition of TSPAN was more prominent in metastatic cells. When inhibited, CD63 showed a significant decrease in proliferation of non-metastatic (*p < 0.05) and metastatic cells (**p=0.0022), as well as migration of metastatic cells (*p < 0.05). Therefore, the results point to CD63 playing a relevant role in VSCC, since its inhibition compromises the proliferation and migration capacity of tumor cells. In addition, decrease in the expression of KAI1/CD82 metastasis suppressor corroborates with other data in different types of cancers. Taken together, these TSPANs may be considered not only important prognostic factors in VSCC, but potential therapeutic targets

Tumormodelle, in denen die maligne Transformation durch definierte Onkogene experimentell ausgelöst und unterhalten wird, bieten vielfältige Möglichkeiten, die komplexen Mechanismen der Tumorentstehung und Therapieresistenz zu untersuchen und neue Ansätze für Diagnostik und Therapie auszuarbeiten. KRAS-Onkogen-„getriebene“ Transformationsmodelle spiegeln neben anderen tumorspezifischen Veränderungen insbesondere die charakteristischen Änderungen des Transkriptoms wider. In der vorliegenden Arbeit wird ein Modell für Ovarialtumore auf Grundlage von Rose Zellen („Rat ovarian surface epithelium“) verwendet, um die Rolle von Transkriptionsfaktoren, welche durch die KRAS-vermittelte Signaltransduktion hoch reguliert werden, zu untersuchen. Die KRAS-transfomierten Derivate der normalen Rose Zellen zeigen die typischen Merkmale von ankerunabhängigen und invasiven Tumorzellen. Aufgrund der hohen Komplexität sind die Interaktionen zwischen der zytoplasmatischen Signaltransduktion und dem durch sie regulierten Transkriptionsfaktornetzwerk noch weitgehend unverstanden. Die Transkriptionsfaktoren Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 und RelA wurden systematisch mittels RNA-Interferenz in KRAS-transformierten Rose Zellen transient ausgeschaltet. Danach wurden Proliferation, Morphologie (epithelial-mesenchymale Transition, EMT) und Ankerunabhängigkeit der Zellen bestimmt. Alle untersuchten Transkriptionsfaktoren beeinflussten die KRAS-induzierten morphologischen Veränderungen teilweise, belegt durch die Abnahme der EMT-Merkmale nach siRNA-vermittelter Ausschaltung. Der Knock-down der Transkriptionsfaktoren Otx1, Gfi1 und RelA hemmte die Proliferation, während Fosl1, Hmga2, Klf6 und JunB die generelle Proliferationsfähigkeit nicht beeinflussten, jedoch spezifisch die ankerunabhängige Proliferation blockierten. Diesen Faktoren kommt daher eine spezifische Funktion in der neoplastischen Transformation zu, da die Ankerunabhängigkeit sehr gut mit der Tumorigenität korreliert ist. Um die Beteiligung der Transkriptionsfaktoren an der Deregulation von Zielgenen zu erfassen, wurden Genexpressionsmuster aller Zellen, in denen jeweils ein Faktor durch siRNA ausgeschaltet war, mittels Microarray-Analyse identifiziert. Auf dieser Grundlage wurde ein Netzwerk-Modell der regulatorischen Interaktionen zwischen den Transkriptionsfaktoren berechnet. Die Existenz der beiden funktionellen Gruppen wurde im Modell bestätigt. Darüber hinaus zeigte sich eine gegenseitige Abhängigkeit des transkriptionellen Netzwerks und der zytoplasmatischen Signaltransduktion, gemessen mittels Proteinanalyse der mitogenabhängigen Signalkinasen (MAPK). Diese wird als kompensatorische Regulation interpretiert, welche trotz Pertubation, experimentell durch siRNA, das effiziente Überleben der transformierten Zellen sicherstellt. Die vorliegende Studie schafft somit die Voraussetzung und Motivation, das reduzierte Netzwerk aus sieben Komponenten auf alle differentiell exprimierten Transkriptionsfaktoren zu erweitern. Möglicherweise behindern solche Regulationskreise in der klinischen Situation die effektive Wirkung zielgerichteter Therapien. / Tumor models, in which malignant transformation was experimentally triggered and maintained through defined oncogenes, offer manifold opportunities to determine the complex mechanisms of tumor progression and resistance to therapies, and to develop new strategies for diagnosis and therapy. Particularly, KRAS oncogene driven models of transformation reflect the characteristic alterations of the transcriptome, among other tumor specific changes. In the present work a model for ovarian cancer based on Rose („Rat ovarian surface epithelium“) cells has been used to evaluate the role of transcription factors, which are up-regulated through KRAS dependent signaling. The KRAS transformed derivates of normal ROSE cells exhibit typical characteristics of anchorage-independent and invasive tumor cells. Due to the high complexity of cellular networks, the interactions between cytoplasmic signalling and their regulated transcription factors are not well understood. The transcription factors Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA were systematically eliminated by transient RNA interference in KRAS transformed ROSE cells. The proliferation, morphology (epithelial-mesenchymal transition, EMT) and anchorage-independence of the cells were determined. All of the selected transcription factors had partial effect on the KRAS induced morphologic changes, documented by reduction of EMT-properties after siRNA treatment. The knock-down of the transcription factors Otx1, Gfi1 and RelA blocked proliferation in general, whereas Fosl1, Hmga2, Klf6 and JunB had no influence on proliferation but specifically blocked the anchorage-independence. Thus, these factors exhibited essential functions in the process of neoplastic transformation, because the anchorage-independence correlates very well with tumorigenicity. In order to elucidate the involvement of the transcription factors in the genetic deregulation of their target genes, microarray based gene expression profiles were determined from all cells in which one factor was eliminated by siRNA. Based on these data, a network model of regulatory interactions among these transcription factors was calculated. The existence of both functional groups was confirmed by the model. Furthermore, an interdependence of the transcriptional networks and cytoplasmatic signaling was observed by protein analysis of the mitogen dependent signal kinases (MAPK). This was interpreted as compensatory regulation, which in spite of experimental perturbation by siRNA, permitted efficient survival of the transformed cells. Thus, the present work provides the basis and motivation to extend the reduced network composed of seven components to all regulated transcription factors. Potentially, such regulatory networks diminish the efficacy of targeted therapies in clinical situations.