Der Gen-Knockdown mittels RNAi hat sich als essentiell erwiesen, um Inhibitoren der induzierten Transdifferenzierung in C. elegans zu identifizieren (Tursun et al., 2011). Bakterienstämme, die dsRNA exprimieren, das die Expression spezifischer Gene mindert, können dem Wurm direkt zugefüttert werden, um einen genomweiten RNAi-screen der insgesamt 20.000 Gene in C. elegans durchzuführen. Allerdings werden die meisten biologischen Prozese durch mehr als ein Gen reguliert, was den Bedarf nach einer Methode generiert, die es erlaubt, zwei oder mehr Gene gleichzeitig herunter zu regulieren, um die Steuerung biologischer Prozesse studieren zu können. Die derzeitig vorhandenen Methoden liefern entweder nicht reproduzierbare Ergebnisse oder sind nicht skalierbar. Wir nutzen baktierelle Konjugation, die es durch ein konjugatives Plasmid ermöglicht Bakterienzellen zu generieren, die zwei verschiedene RNAi-Plasmide enthalten. Das Ziel war es, modifizierte RNAi-Donor-Plasmide mittels bakterieller Konjugation an eine Vielzahl anderer Bakterienzellen zu übertragen, die bereits ein anderes RNAi-Plasmid enthalten und dies dann im Hochdurchsatzverfahren durchführen zu können. Um Enhancer induzierter Expression von unc-25::gfp in der Keimbahn, ermöglicht durch den Knockdown des Histonchaperons LIN-53 (RbAp46/48 in Menschen), zu finden, wurden RNAi-Klone generiert, die gleichzeitig lin-53 als auch eines von insgesamt 800 verschiedenen Chromatin-bezogenen Gene herunter regulieren. Dabei identifizierten wir RBBP-5, Mitglied des Set1/ MLL-Methyltransferase-Komplexes, als neuen Barrierefaktor der induzierten Transdifferenzierung. RBBP-5 agiert dabei mutmaßlich parallel zu LIN-53. Doppelte RNAi, ermöglicht durch bakterielle Konjugation, erlaubt den simultanen Knockdown zweier oder mehr Gene, um genetische Interaktionen studieren zu können und erweitert damit die Einsatzmöglichkeiten von RNAi-Screens, um untereinander verbundene biologische Prozesse zu studieren. / The knock down of genes by RNAi has been fundamental to identify inhibitors of induced cell transdifferentiation in C. elegans (Tursun et al., 2011). Bacteria strains expressing dsRNA that target specific genes can be fed to the worm allowing straightforward whole-genome RNAi screens of the 20,000 genes in theC. elegans genome. However, many biological processes are regulated by more than one gene raising the need for simultaneous knock down of two or more genes to more fully interrogate the regulation of complex biological processes. Two approaches are currently available for double RNAi knockdown, − two bacteria strains expressing specific dsRNA can be mixed and grown together and fed simultaneously, which gives highly variable results. Alternatively, a new bacterial clone can be generated carrying a plasmid on which two RNAi targets of interest are 'stitched' together, which is not scalable. To address this challenge, we have developed a protocol using bacterial conjugation mediated by the 'Fertility Factor' (F) Episome in order to combine two different RNAi plasmids in a single bacterium. The objective was to be able to transfer a single RNAi plasmid to a large number of bacterial cells carrying different RNAi clones in one step in a high-throughput manner for large scale 'double' or even 'triple' RNAi screens. To find enhancers of induced unc-25::gfp expression in the germ line enabled by the depletion of histone chaperone LIN-53 (RbAp46/48 in humans), double RNAi clones targeting lin-53 and a total of 800 chromatin-related genes were generated and screened. We identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel reprogramming barrier that putatively acts in a parallel pathway to LIN-53. Double RNAi by conjugation permits to reliably knock down two genes simultaneously in order to study genetic interactions at a genome-wide level, thus further increasing the versatility of RNAi screens to investigate interconnected biological processes.

O agente etiológico da Febre Maculosa das Montanhas Rochosas (RMSF), conhecida no Brasil como Febre Maculosa Brasileira, é a bactéria Rickettsia rickettsii. Essa bactéria é transmitida ao homem pela picada de diferentes espécies de carrapatos ixodídeos. No Brasil, os vetores são Amblyomma cajennense e A. aureolatum. As taxas de prevalência de R. rickettsii nas populações de carrapatos de áreas endêmicas para RMSF são baixas, em geral abaixo de 1%. Essa baixa prevalência parece estar associada a menores taxas reprodutivas e de sobrevivência de linhagens infectadas, sugerindo que R. rickettsii seja patogênica também para os seus vetores. Infecções experimentais demonstraram que 80-100% dos indivíduos de uma colônia de A. aureolatum mantida em laboratório são infectados por R. rickettsii, enquanto apenas 10-60% de A. cajennense adquirem a bactéria. Esses dados indicam que as respostas dessas duas espécies de carrapatos à infecção sejam diferentes, resultando em diferentes taxas de prevalência da bactéria. Dessa maneira, a caracterização molecular das interações entre carrapatos do gênero Amblyomma e a bactéria R. rickettsii é importante, podendo gerar informações não somente para o esclarecimento acerca dos mecanismos de patogenicidade de R. rickettsii para os carrapatos, mas também para um melhor entendimento dos mecanismos responsáveis pela aparente restringência de A. cajennense à infecção. Assim, os objetivos do presente estudo foram: (i) analisar os efeitos da infecção por R. rickettsii sobre o perfil de expressão gênica de carrapatos A. cajennense por hibridação subtrativa por supressão (SSH), (ii) validar os dados de SSH por reação em cadeia de polimerase quantitativa precedida por transcrição reversa (RT-qPCR) e (iii) caracterizar funcionalmente dois genes com expressão induzida pela infecção por RNA de interferência (RNAi). Após a análise bioinformática dos dados de SSH, 44 sequências únicas foram obtidas, das quais 36 representam genes com expressão induzida e 8 genes com expressão reprimida pela infecção. A indução dos genes codificadores da subunidade I da citocromo c oxidase (COX1), da subunidade IV da NADH desidrogenase, de uma proteína com domínio de inibidor de serina-proteases Kunitz-type (papilina-like), identificados por SSH, e de um peptídeo antimicrobiano (hebraeína), foi confirmada por RT-qPCR. O silenciamento gênico da hebraeína e da papilina-like não teve nenhum efeito na aquisição de R. rickettsii pelo vetor, indicando que, isoladamente, não são responsáveis pela proteção de A. cajennense contra a infecção. Os dados gerados pelo presente estudo abrem perspectivas para que outros genes sejam avaliados quanto ao seu papel na aquisição de R. rickettsii, os quais, no futuro, podem ser considerados como alvos para o desenvolvimento de vacinas. / The etiologic agent of the Rocky Mountain Spotted Fever (RMSF), also known as Brazilian Spotted Fever in Brazil, is the bacterium Rickettsia rickettsii. This rickettsia is transmitted to humans by the bite of various tick species. In Brazil, Amblyomma cajennense and A. aureolatum are known as vectors. The prevalence rates of R. rickettsii infected ticks in RMSF endemic areas are low, oscillating around 1%. These low prevalence rates seems to be associated with lower reproductive and survival rates of infected ticks, suggesting that R. rickettsii is also pathogenic to its vectors. Experimental infections with R. rickettsii have demonstrated that 80 to 100% of A. aureolatum ticks from a laboratory colony acquire this bacterium, whereas only 10 to 60% of A. cajennense ticks become infected. These results indicate that the responses of these two tick species against infection are different, resulting in different prevalence rates of the bacterium. Therefore, the elucidation of the interactions between ticks of the genera Amblyomma and the bacterium R. rickettsii at a molecular level is important to provide information to better understand the mechanisms of pathogenicity of R. rickettsii against ticks as well as for the elucidation of the mechanisms responsible for the apparent refractoriness of A. cajennense against infection. Therefore, the objectives of the current study were: (i) analyze the effets of the infection with R. rickettsii on the gene expression of ticks A. cajennense by suppression subtractive hybridization (SSH), (ii) validate SSH data by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and (iii) functionally characterize two genes induced by infection using RNA interference (RNAi). After bioinformatics analysis of SSH data, 44 unique sequences were obtained, among which 36 represent genes with expression induced and 8 repressed genes by infection. The induction of genes encoding subunit I of cytochrome c oxidase (COX1), the NADH dehydrogenase subunit IV, a protein containing Kunitz-type inhibitor domain (papilin-like), identified by SSH, and an antimicrobial peptide (hebraein), was confirmed by RT-qPCR. The effects of knockdown of hebraein and papilin-like encoding genes had no effect on the acquisition of R. rickettsii by the vector. Data of the current study may be used to evaluate the role of other genes in acquisition of R. rickettsii, which, in the future, may be considered as target for vaccine development.

Caenorhabditis elegans possui quatro genes de kpcs (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Em C. elegans, dois dos quatro polipeptídeos de vitelogenina encontrados dentro dos ovócitos, YP115 e YP88, se originam a partir de um precursor polipeptídico (VIT-6) clivado pós-traducionalmente após o motivo RGKR. Nematóides transgênicos foram produzidos com construções repórteres transcricionais de GFP. Foi verificada expressão de kpc-1 tanto em neurônios quanto em células musculares e intestinais. Esses dados, aliados aos dados da literatura para os outros genes kpc de C. elegans, sugerem o envolvimento de KPC-1 no processamento de VIT-6, que é secretada por células intestinais. Ensaios de Western-blot compararam o processamento de VIT-6 em nematóides selvagens, mutantes e knock-down por RNAi para os diferentes genes kpc. A análise de nematóides mutantes e knock-down por RNAi combinado para os outros três genes de convertase de C. elegans confirmou a redundância da atividade dessas enzimas no processamento de VIT-6. / Four kpc genes are found in the Caenorhabditis elegans genome: (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Two of the four vitellogenin polypeptides, YP115 and YP88, originate from a precursor, VIT-6. VIT-6 is cleaved post-translationally after the RGKR motif. Transgenic worms carrying GFP transcription reporter constructs were produced. Expression of kpc-1 has been localized to neurons as well as muscular and intestinal cells. These data, together with the ones available from the literature for the other kpc genes, suggest the involvement of KPC-1 in the processing of VIT-6, which is secreted from intestinal cells. Western-blot analysis compared the pattern of VIT-6 processing in wild-type, mutants and RNAi-treated worms for the other kpcs. Analysis of worms treated by combined RNAi confirmed the redundancy of KPCs in VIT-6 processing.

A região N-terminal de OTI-VIT-1 foi expressa e os polipeptídeos recombinantes foram purificados. OTI-VIT-1 pode ser homólogo da vitelina YP170B de C. elegans. Foram identificados um intron na região 5´ e dois na região 3´ do gene Oti-vit-1. Antissoro monoespecífico para PVIT1HisC confirmou que o gene Oti-vit-1 codifica VT1. O polipeptídeo recombinante P40-H, correspondente à região N-terminal da proteína OTI-VIT-6 interage com um polipeptídeo de aproximadamente 100 kDa (P100) presente em extratos proteicos totais de O. tipulae. Estudamos também o papel da Proteína Microssômica Transportadora de Triglicerídeos (MTP) na biossíntese de Vitelogenina do nematoide C. elegans. Ensaios de RNAi em C. elegans, utilizando parte da sequência do gene da MTP (Cel-dsc-4) foram realizados nas linhagens N2 e DH1033. Microscopia de fluorescência de vermes adultos da linhagem DH1033, submetidos a RNAi, mostrou acúmulo de YP170B::GFP no interior dos enterócitos. Este acúmulo sugere a participação da MTP na secreção de VTG. Análise imunológica da vitelogenina nestes mesmos vermes não detectaram alterações no processamento de CEL-VIT-6, sugerindo que o mesmo ocorra não só no pseudoceloma, mas também no interior dos enterócitos. / The N-terminal region of OTI-VIT-1 was expressed and the recombinant polypeptides were purified. OTI-VIT-1 may be homologous to the vitellin YP170B from C. elegans. We identified an intron in the 5 \'region and two in 3\' region from Oti-vit-1. Monospecific antisera to PVIT1HisC confirmed that the gene Oti-vit-1 encodes VT1. The recombinant polypeptide P40-H, corresponding to the N-terminal region of the protein OTI-VIT-6, interacts with a polypeptide of approximately 100 kDa (P100) present in total protein extracts of O. tipulae. The role of microsomal triglyceride transfer protein (MTP) in the biosynthesis of vitellogenin was studied in the nematode C. elegans. Trials of RNAi in C. elegans, using the sequence of the MTP gene (Cel-dsc-4) were performed in the strains N2 and DH1033. Fluorescence microscopy of adult worms of strain DH1033, subjected to RNAi, showed accumulation of YP170B:: GFP within the enterocytes. This accumulation suggests the involvement of MTP in the secretion of VTG. Analysis using anti-vitellogenin immune serum did not detect changes in the processing of CEL-VIT-6, suggesting that it occurs not only in pseudocoelom but also within the enterocytes.

Le réseau de signalisation qui répond à l’insuline et aux nutriments est conservé chez les métazoaires, où il joue un rôle central dans le contrôle du métabolisme et de la croissance. Les nutriments assimilés sont soit directement utilisés pour la croissance tissulaire, soit stockés principalement sous forme de triglycérides. Chez la drosophile, l’activation de ce réseau de signalisation dans le corps gras, un organe qui remplit à la fois les fonctions hépatiques et destockage, induit une augmentation du stockage de lipides sous forme de nombreuses gouttelettes lipidiques (LDs). A l’inverse, la carence alimentaire se traduit par une augmentation de la taille des LDs et une diminution de lipides stockés. La kinase TOR (TargetOf Rapamycine) et son substrat S6 Kinase (S6K) jouent un rôle central dans cette régulation.Chez la drosophile, ces 2 kinases (dTOR et dS6K) contrôlent les aspects autonome-cellulaireset hormonaux de la croissance. En dépit de nombreuses études sur divers organismes modèles,destinées à comprendre les mécanismes régulateurs de S6K, rien n’est connu à ce jour sur lecontrôle de sa dégradation.Nous avons utilisé une banque de lignées exprimant des ARN interférant (RNAi) contre unegrande quantité de gènes de la drosophile, pour réaliser 3 des cribles génétiques destinés à identifier de nouveaux régulateurs du métabolisme et de la croissance. Dans le premier crible,les RNAi ont été induits dans la glande prothoracique, siège de la production de l’hormonestéroïde ecdysone connue pour réguler la croissance et les étapes du développement, souscontrôle de la nutrition et de la signalisation dTOR. Sur 7000 gènes criblés, 620 ont étéidentifiés comme nécessaire à la production d’ecdysone. Dans le second crible, nous avonsexprimé les RNAi de 4000 gènes dans le corps gras pour rechercher ceux qui induisaient uneaugmentation de la taille des LDs. L’objectif était d’identifier des gènes impliqués dans la réponse à la carence alimentaire, et nous avons ainsi retenu 24 candidats intéressants. Le troisième crible représente la majeure partie du travail de thèse, où nous avons criblé les RNAi susceptibles de modifier un phénotype de croissance induit par dS6K. Sur 7000 gènes testés,nous en avons retenu 45 qui ont ensuite été utilisés pour générer un diagramme d’interaction en utilisant les informations disponibles dans les banques de données. Les candidats les plus intéressants ont ensuite été analysés en culture de cellules pour identifier ceux qui régulent l’activité de dS6K et ceux qui régulent sont niveau d’expression. Parmi ces derniers, nousavons identifié le gène codant pour Archipelago (Ago), connue pour contrôler la dégradationrégulée des protéines-cibles au niveau du protéasome. Nous avons réalisé de nombreusesexpériences qui montrent que ago et dS6K interagissent génétiquement. En outre, il est indiquédans les banques de données que ces protéines interagissent entre elles par la technique des 2-hybrides en levure. Tous ces résultats révèlent que Ago régule la dégradation de dS6K, etposent les premières pierres de ce niveau de régulation. / The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.

No description available.

No description available.

No description available.

KRAS is a gene that plays a very important role in the initiation and development of several types of cancer. In particular, 90% of human pancreatic cancers are due to KRAS mutations. KRAS is difficult to target directly and a promising therapeutic path is its indirect inactivation by targeting one of its Synthetic Lethal Partners (SLPs). A gene G is a Synthetic Lethal Partner of KRAS if the simultaneous perturbation of KRAS and G leads to cell death. In the past, efforts to identify KRAS SLPs with high-throughput RNAi screens have been performed. These studies have reported only few top-ranked SLPs. To our knowledge, these screens have never been considered in combination for further examination. This thesis employs integrative analysis of the published screens, utilizing additional, independent data aiming at the detection of more robust therapeutic targets. To this aim, RankSLP, a novel statistical analysis approach was implemented, which for the first time i) consistently integrates existing KRAS-specific RNAi screens, ii) consistently integrates and normalizes the results of various ranking methods, iii) evaluates its findings with the use of external data and iv) explores the effects of random data inclusion. This analysis was able to predict novel SLPs of KRAS and confirm some of the existing ones.

Caenorhabditis elegans possui quatro genes de kpcs (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Em C. elegans, dois dos quatro polipeptídeos de vitelogenina encontrados dentro dos ovócitos, YP115 e YP88, se originam a partir de um precursor polipeptídico (VIT-6) clivado pós-traducionalmente após o motivo RGKR. Nematóides transgênicos foram produzidos com construções repórteres transcricionais de GFP. Foi verificada expressão de kpc-1 tanto em neurônios quanto em células musculares e intestinais. Esses dados, aliados aos dados da literatura para os outros genes kpc de C. elegans, sugerem o envolvimento de KPC-1 no processamento de VIT-6, que é secretada por células intestinais. Ensaios de Western-blot compararam o processamento de VIT-6 em nematóides selvagens, mutantes e knock-down por RNAi para os diferentes genes kpc. A análise de nematóides mutantes e knock-down por RNAi combinado para os outros três genes de convertase de C. elegans confirmou a redundância da atividade dessas enzimas no processamento de VIT-6. / Four kpc genes are found in the Caenorhabditis elegans genome: (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Two of the four vitellogenin polypeptides, YP115 and YP88, originate from a precursor, VIT-6. VIT-6 is cleaved post-translationally after the RGKR motif. Transgenic worms carrying GFP transcription reporter constructs were produced. Expression of kpc-1 has been localized to neurons as well as muscular and intestinal cells. These data, together with the ones available from the literature for the other kpc genes, suggest the involvement of KPC-1 in the processing of VIT-6, which is secreted from intestinal cells. Western-blot analysis compared the pattern of VIT-6 processing in wild-type, mutants and RNAi-treated worms for the other kpcs. Analysis of worms treated by combined RNAi confirmed the redundancy of KPCs in VIT-6 processing.