Endocytosis is an evolutionary conserved process of internalization of cargo from the extracellular environment, be they ligands, nutritional and signaling or pathogens into cells. Following their entry, cargo is received into vesiculo-tubular network of early endosomal compartments from where they are sorted and routed to appropriate cellular destinations through transport along the endocytic network. Recycling cargo is sorted away from other cargo resident in early endosomes through tubulation resulting in fission of recycling vesicles, while those to be degraded are progressively concentrated in early endosomes to be degraded in lysosomes. Early endosomes are dynamic organelles that have been shown to move centripetally following the internalization of cargo into at the cell periphery. Their motility from the cell periphery to the juxtanuclear location of the cell involves convoluted trajectories that include directed motility, bi-directional switches, saltatory behavior and stalls. This complex motility presumably contributes toward the cargo sorting, duration of cargo residence and spatio-temporal signaling by early endosomes. How the different regimes of motility, and nature and number of molecular motors involved in early endosome motility contribute toward endosome function is not understood. The aim of this study was to probe into the regulation of endosome motility and understand how transport organizes early endosome network. Towards this end, live cell time-lapse movies of Rab5 endosomes were analyzed to derive motility properties contributing to organization of early endosomes. Consistent and significant bias toward the cell centre (minus end motility) in kinetic parameters such as speed, displacement and duration of motility contribute to centripetal flux of Rab5 early endosomes. A phenomenological property of early endosome motility is its saltatory behavior that produces saturation curves in Mean Square Displacement (MSD) plots. This phase of motility is descriptive, with no understanding of its mechanism or function. Live cell candidate RNAi screen and cytoskeletal perturbation analysis were performed to identify molecules regulating saltatory motility. To this end, cellular microtubule perturbation and RNAi knock down of several Kinesin motor candidates showed a loss in saturation behavior. Potential candidates identified have to be tested for their effect on endosome function through cargo sorting and kinetic assays to gain insights into the role of saltatory motility in endosome function. Molecular motors mediate Rab5 motility. Therefore, understanding regulation of motility requires identifying number and nature of molecular motors involved in their transport. Towards this end, a functional cargo (LDL) degradation RNAi screen targeting molecular motors was performed. The Ambion Select technology was used with 3 siRNAs targeting every gene in the library. Analysis of screen produced by lack of phenotype consistency between the multiple siRNAs targeting the same gene. Hence, a search for technology with better target specificity was initiated. Technologies tested were Ambion Select, Ambion Silencer Select, Dharmacon ON-TARGET Plus, esiRNA and Invitrogen Stealth. Invitrogen Stealth technology was found to produce the least off-targets and was most specific in terms of consistency of phenotypes produced by multiple siRNAs silencing the same target gene. Assay conditions were also found to influence the silencing specificities to a significant extent. Hence, a systematic assay optimization exercise was performed in terms of the concentration of siRNA used for transfection and time window of assay to maximize specificity of siRNA silencing. Insights obtained from methodologies developed herein not only provide invaluable guidelines in choosing RNAi commercial libraries for screens, but also underscore the importance of establishing optimal assay conditions to minimize off-targets and improve specificity of silencing target genes. The motor screen was repeated with RNAi library from Invitrogen Stealth. Several potentially interesting candidates have been identified. Also, correlation analyses of phenotypes produced in the screen have indicated toward potential regulatory motor complexes, all of which await biochemical validation.

TRIM22 ist ein intrazelluläres Protein, das ein heterogenes Aufgabenspektrum erfüllt. Bisher wurden antivirale Funktionen und Zusammenhänge mit zellulären Prozessen wie Zelldifferenzierung und Zellproliferation beschrieben. Im Rahmen dieser Arbeit wurde eine Beteiligung an der mikroRNA-Prozessierung untersucht, sowie Lokalisationsstudien des Proteins durchgeführt. Lokalisationsstudien erfolgten mittels IF-Mikroskopie, während Proteininteraktionen anhand der Co-Immunpräzipitation untersucht wurden. Die funktionellen Untersuchungen erfolgten durch Luziferaseassays. Zu Beginn wurde die subzelluläre Expression des endogenen und ektopisch exprimierten TRIM22-Proteins untersucht. TRIM22 konnte sowohl im Nukleus, als auch in der perinukleären Umgebung und am Zytoskelett lokalisiert werden. Zudem konnte eine Co-Lokalisation des endogenen TRIM22 mit dem Zentrosom bestätigt werden, was jedoch nicht für ektopisch exprimiertes TRIM22 zutraf. Des weiteren wurde der Einfluss der TRIM22-Über- bzw. Unterexpression auf die Zellvitalität überprüft. Nach TRIM22-Knockdown mittels RNAi zeigten sich vermehrt mitotisch aberrante und apoptotische Zellen. Bei Überexpression konnten vermehrt polyploide Zellen nachgewiesen werden. Zudem gab es hierbei Hinweise auf Zellvitalitätsstörungen.  Im letzten Teil der Arbeit gelang mittels IF-Mikroskopie und Co-Immunpräzipitation die Erstbeschreibung einer Interaktion zwischen TRIM22 und Komponenten der zellulären Silencing-Maschinerie. Diese Beobachtung konnte durch den Nachweis einer funktionellen Beteiligung des Proteins an der mikroRNA-Prozessierung erweitert werden. Die beschriebenen Lokalisationen des TRIM22-Proteins bestätigen die Aussagen externer Publikationen. Das divergente Bindungsverhalten des endogenen und ektopisch exprimierten TRIM22 bezüglich des Zentrosoms wurde erstmalig beschrieben und ist vermutlich auf Proteininteraktionen zurückzuführen. Das funktionelle Spektrum des TRIM22-Proteins wurde im Rahmen dieser Arbeit um eine Beteiligung in der mikroRNA-Prozessierung erweitert. Eine Funktion als Trägerprotein und ein Mitwirken in der Silencing-Maschinerie wären denkbar und sollten in zukünftigen Studien überprüft werden.

Made available in DSpace on 2015-03-26T13:42:29Z (GMT). No. of bitstreams: 1 texto completo.pdf: 546340 bytes, checksum: c6ef1899560e5249142f8d7f9943743d (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Soybean is one of the most important crops in the world extensively used as a food and feed source. However, the proteins present in soybean seeds are not considered ideal because they contain low amounts of the essential amino acids methionine and lysine. Adverse nutritional and other effects following consumption of raw soybean meal have been attributed to the presence of endogenous inhibitors of digestive enzymes and allergenic factors. Among these proteins are the P34 protein and the Bowman-Birk protease inhibitors. The absence of these factors would increase the nutritional quality of the soybean seeds, as well as the availability of this product to large number of consumers. Recent advances in plant biotechnology have enabled the silencing of specific genes. One of the techniques used to achieve this goal is RNA interference (RNAi). This method of posttranscriptional gene silencing is highly efficient, especially when the construct results in the formation of a intron-spliced hairpin RNA. It is proposed that RNAi works by enzymatic RNA degradation induced by double-stranded RNA. The main goal of this work was to construct expression cassettes for RNAi-based silencing of genes encoding antinutritional factors in soybean seeds. To achieve seed-specific expression, specific primers were designed for the amplification of the promoter region of the alpha-subunit of the beta-conglycinin gene. Appropriate restriction sites - Sac I and Xho I - were added to the primers which were used to amplify a 634 bp fragment. This product was cloned into a pGEMTEasy vector and cloning was verified by PCR, enzymatic digestion and sequencing. The sequenced clone was analyzed in silico for identification of cis-elements related to seed specific expression. The elements TATA box, CAAT box, RY LEG box and RY FLEB box were found. The fragment of interest was isolated from vector pGEM-TEasy by cleavage with enzymes Sac I and Xho I and inserted in the expression vector pKANNIBAL. This new vector was designated pBKN. For the construction of the expression cassettes, primers were designed according to published sequences deposited in the GenBank. The fragments of interest were isolated by the RT-PCR method using RNA isolated from soybean seeds. RT-PCR was also used to analyze the expression of the gene of interest in the seed and in other organs of the plant (root, stem and leaves). These analyses showed that the expression of these genes was continuous and abundant during all stages of seed development, and the corresponding transcripts were present in all organs analyzed. The fragments obtained by RT-PCR were sequenced and their identity was confirmed by BLAST analysis. To clone the sense sequence, the vectors pKANNIBAL and pBKN, and the fragments of interest were cleaved with enzymes Xho I and Kpn I. The ligation reaction was catalyzed by T4 DNA ligase and competent cells of Escherichia coli were transformed by heat shock. Cloning was verified by PCR and enzymatic digestion. The cloning of the antisense fragment was done using restriction enzymes Xba I and Cla I and vectors containing the sense fragments. The complete expression cassettes were cloned into the binary vector pCAMBIA 3301 and verified by PCR. / A soja é uma das mais importantes culturas do mundo e como apresenta um alto teor protéico, ela tem sido muito utilizada na alimentação animal e humana. Apesar disso, as proteínas encontradas na semente desta leguminosa não são consideradas ideais por apresentarem baixo teor de metionina e lisina, além de fatores alergênicos e de inibidores de proteases. Dentre estes fatores antinutricionais estão a proteína P34 e os inibidores de protease do tipo Bowman-Birk. A ausência destes fatores aumentaria a qualidade da semente de soja, bem como a tornaria disponível para uma faixa mais ampla de consumidores. Recentes avanços na biotecnologia vegetal têm possibilitado o silenciamento completo ou em altos níveis de genes específicos. Uma destas técnicas é conhecida como interferência por RNA ou RNA interference. Este método é altamente eficiente e a clivagem do mRNA alvo é induzida pela presença de pequenos RNAs dupla fita na célula. O objetivo deste trabalho foi a construção de cassetes de expressão para silenciamento gênico, via interferência por RNA, dos inibidores de protease do tipo Bowman-Birk e da proteína P34 em sementes de soja. Para a expressão semente-específica dos transgenes, primers específicos foram desenhados para a amplificação da região promotora do gene da subunidade [alfa] da [beta]-conglicinina. A estes oligonucleotídeos foram adicionados sítios de restrição Sac I e Xho I - adequados para sua clonagem em vetores de expressão em plantas. Foram clonados 634 pb no vetor pGEM-TEasy. A clonagem foi confirmada por PCR, clivagem enzimática e sequenciamento do clone isolado. A seqüência clonada foi analisada in silico para a identificação de cis-elementos relacionados à expressão semente específica de genes colocados sob controle deste promotor. Os elementos TATA box, CAAT box, RY LEG box e RY FLEB box encontrados. O fragmento de interesse foi, então, isolado do vetor pGEM-TEasy por clivagem com as enzimas Sac I e Xho I e inseridos em vetores de expressão em plantas (pKANNIBAL) originando o vetor pBKN. Para a construção dos cassetes de expressão, a escolha da região do cDNA de cada gene de interesse a ser amplificada foi determinada com o auxílio do programa BLOCK-iTTM RNAi Designer (INVITROGEN), e os primers específicos foram desenhados de acordo seqüências depositadas no banco de dados público GenBank. Inicialmente foi feita, por meio de RTPCR, uma análise da expressão dos genes de interesse durante o desenvolvimento da semente e em demais órgãos da planta (raiz, caule e folha). Essa análise evidenciou expressão contínua e abundante desses genes durante todo o desenvolvimento da semente bem como a presença de transcritos correspondentes em todos os órgãos vegetais analisados. Após sua amplificação, todos os fragmentos obtidos a partir de cDNA de semente foram sequenciados e sua identificação foi confirmada por meio de alinhamento utilizando o programa BLAST. Para a clonagem da seqüência sense, os vetores pKANNIBAL e pBKN, além dos fragmentos amplificados referentes aos genes de interesse, foram clivados com as enzimas Xho I e Kpn I. A reação de ligação foi realizada utilizando T4 DNA ligase e células de Escherichia coli ultracompetentes foram transformadas por meio de choque térmico. Os transformantes foram analisados por PCR e reação de clivagem enzimática. A clonagem do fragmento antisense foi realizada da mesma forma, utilizando as enzimas Xba I e Cla I e os clones contendo o fragmento sense como vetores. Em seguida, as construções foram transferidas para o vetor binário pCAMBIA 3301 e confirmadas por PCR.

A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas, que possui caráter aleatório, e a recombinação homóloga, que possibilita o gene targeting. Algumas ferramentas como as TAL Effector Nucleases (TALENs) e o RNA interferência (RNAi) podem ser utilizadas para aumentar a taxa de integração específica e assim melhorar a eficiência e o direcionamento da edição gênica. Nesse trabalho utilizamos o silenciamento gênico mediano por short interference RNA (siRNA) para inibição temporária dos genes ATF7IP uma metiltrasferase, EP300 uma acetiltransferase e KU70 (NHEJ) e um par de TALENs complementares a uma região do gene da distrofina canina. Células Caninas MDCK I foram transfectadas por lipofectamina 2000 (Invitrogen) com 320pmol de siRNAs para ATF7IP e Ep300; e 64 pmol do SiRNA para KU70 em diferentes grupos, 40 horas depois as células foram transfectadas com 15 μg vetor molde derivado do pEGFP-N1 (Clonatech) e com 10 μg dos RNAm das TALENs. A seleção se deu em meio DMEM high com 600μg/ mL de G418 (Lonza) por 14-16 dias. As colônias coletadas através de biópsias foram analisadas por Polimerase Chain Reaction e sequenciamento gênico. Três pares de primers foram utilizados; um controle endógeno (GAPDH), um controle interno do inserto (Neo qPCR) e um para confirmação da recombinação homóloga (DMD3). Os grupos apresentaram grande variação na taxa de mortalidade celular e consequentemente no número de colônias: Com o grupo ATF7IP+Vetor (648c) apresentando maior número de colônias e o grupo EP300+Ku70+Vetor+TALENs o menor (1c). A maior taxa de recombinação ocorreu nos grupos no grupo ATF7IP +Ku70+Vetor+TALENs com 40% das células positivas para neomicina apresentado o evento gene targeting, um aumento considerável na taxa de recombinação quando comparada a porcentagem de 3,1% do controle transfectado somente com o vetor molde. Mostrando que o uso conjunto das TALENs com siRNAs foi um sucesso para o aumento de eventos de edição gênica direcionada. / The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were transfected with 15 μg of a vector derived from pEGFP- N1 (Clontech) containing two regions homologous to the canine DMD gene (left arm length: 873 bp and right arm length: 1370 bp) and 10 μg of TALEN mRNA. The cell selection was achieved with DMEM high glucose with 600μg/ml G418 for 14-16 days. The colonies collected through biopsies were analyzed by polymerase chain reaction and gene sequencing. Three pairs of primers were used; an endogenous control (GAPDH) , an internal control of the insert (Neo qPCR) and a primer set to confirm the occurrence of homologous recombination events (DMD3). .Groups showed great variation in cell death rate and consequently in the number of colonies: ATF7IP+Vector had highest number of colonies (648c) and the group EP300+Ku70+Vetor+TALENs the lowest one (1c) The highest rate of homologous recombination was in ATF7IP +Ku70+Vetor+TALENs group that had 40% of the neomycin positives cells confirmed as gene targeting events, a considerable increase in the recombination rate compared to the 3.1% in the control group transfected only with the template vector. That shows that the combined use of siRNAs and TALENs was a success for increasing directed gene editing events.

HIPK2 (homeodomain interacting protein kinase 2) é uma proteína quinase nuclear, originalmente identificada por interagir com homeoproteínas. Sua atividade quinase contribui para a regulação de diversas vias, ativando o programa de morte celular em resposta a estímulos externos ou promovendo diferenciação celular por atuar como co-fator de homeoproteínas. A extensa similaridade estrutural com as outras proteínas da mesma família, HIPK1 e HIPK3, sugere que as três HIPKs possuem funções redundantes. Este trabalho pretendeu contribuir para a elucidação das funções de HIPK2 avaliando a expressão desta durante o desenvolvimento em camundongo. Observou-se ampla distribuição do transcrito desde o dia 9.5 pós-coito (dpc). Com o progresso do desenvolvimento, a expressão passou a se concentrar principalmente em tecidos nervosos, sugerindo uma participação de HIPK2 na morfogênese destes. Além disso, foi desenvolvido um sistema in vitro para se avaliar as funções de HIPK2 humana. A inibição de HIPK2 por RNAi em cultura celular humana levou ao aumento de expressão de genes HIPKs, indicando que possa haver um mecanismo de controle transcricional que ajuste os níveis de proteínas HIPKs na célula. O silenciamento de HIPK2 também provocou aumento de expressão de alguns genes homeobox avaliados, sugerindo que HIPK2 possa reprimir a expressão destes genes em humanos. A análise global de expressão gênica e proteômica nas células deficientes para HIPK2 poderá indicar as diferentes vias de atuação desta proteína. / HIPK2 is a nuclear protein kinase, originally identified because of its interaction with homeoproteins. Its kinase activity can regulate several pathways, triggering the apoptotic program in response to external stimuli or promoting differentiation by acting as a cofactor for homeoproteins. The extensive similarity with the other two proteins from the same family, HIPK1 and HIPK3, suggests that HIPKs have redundant functions. This work tried to contribute to the body of knowledge regarding the functions of HIPK2 addressing its expression during the mouse development. The transcript is broadly expressed in mouse embryos from day 9.5 post-coitum. As development progresses, its expression concentrates mainly in neural tissues, suggesting a role of HIPK2 in its morphogenesis. In addition, an in vitro system was developed to study HIPK2 functions in human cells. The HIPK2 silencing by RNAi in human cell culture led to a raise in expression of the other HIPKs genes, indicating that there might be a transcriptional mechanism controlling HIPKs proteins levels in the cell. HIPK2 inhibition also caused an increase in expression of some homeobox genes, suggesting that HIPK2 can negatively regulate their expression in humans. A global approach of gene expression and proteomics analysis in the HIPK2 deficient cells will help to identify different pathways in which HIPK2 participates.

Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy Maia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Desde seu descobrimento, o fenômeno de silenciamento gênico por RNA (RNAi) rapidamente se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia molecular. Uma destas possibilidades é sua aplicação no campo da entomologia agrícola, mais especificamente para o controle de insetos-praga como uma alternativa de alta eficiência, especificidade e com impacto ambiental reduzido. Por meio da geração de plantas transgênicas expressando RNAi para genes essenciais de insetos-praga específicos, a ingestão destas moléculas de RNAi pelo inseto mediante herbivoria pode resultar no silenciamento do respectivo gene, resultando em fenótipos que podem variar entre perda de apetite, infertilidade ou até a morte. Neste contexto, o presente trabalho teve como objetivo provar a viabilidade de aplicação desta técnica para a interação Tomateiro x Tuta absoluta, cultura de grande expressão econômica e social no mercado nacional e internacional e que é amplamente atacada por esta praga, com prejuizos que podem alcançar a ordem dos 100% da produção. Por meio da clonagem de genes ortólogos essenciais descritos na literatura e de genes altamente expressos nos primeiros estádios larvais, após a caracterização transcriptômica em escala do inseto, foram realizados ensaios de alimentação contendo moléculas de dsRNAs que possuíam estes genes como alvo. Também, foi realizado a transformação genética de tomateiro cultivar \"Micro-Tom\" com dois destes genes (V-ATPase A e Arginina kinase) para a realização de ensaios de herbivoria. Com os resultados obtidos nestes experimentos, foram mostradas sólidas evidências da viabilidade da técnica de RNAi para o controle de Tuta absoluta, evidenciado pelo silenciamento gênico específico observado no inseto e consequentemente os efeitos nocivos deste silenciamento. / Since their discovery, the phenomenon of gene silencing by RNA ( RNAi ) has rapidly become a widely studied and used technique in the molecular biological field. One of these possible applications is in the entomology field, more specifically for the control of insect pests, as a high efficiency, specificity and with reduced environmental impact alternative. Through the generation of transgenic plants expressing dsRNA targeting essential insect genes, their ingestion by the insect and consequently the uptake of the silencing RNA, may result in specific gene silencing, resulting in a variety of phenotypes that can range from loss of appetite, infertility to death. In this context, this study aimed to prove the feasibility of this technique to control tomato leaf miner (Tuta absoluta) in tomatoes plant, a major crop worldwide and seriously attacked by this pest, with losses that can reach 100%. For the present work, orthologous genes from successfully cases of insect gene silence described in the literature, was selected together with highly expressed genes in the early larval stages of T. absoluta, chosen after the insect molecular characterization and used in feeding assays with dsRNAs molecules to targeted these genes. Also, genetic transformation of the \"Micro-Tom\" tomato cultivar with two of these genes (V-ATPase and Arginine kinase) was conducted for testing in an herbivore assay. With these two approaches was possible to get solid evidences of the feasibility of the RNAi technique to control this insect, evidenced by specific gene silencing observed and its consequent effect on pest phenotype.

Abelhas adultas produzem vitelogenina, a principal proteína da hemolinfa. Ela está envolvida na reprodução, comportamento, imunidade, longevidade e regulação da organização social. A interferência por RNA interference é a mais promissora ferramenta para estudos de função gênica, baseada na introdução de duplex de RNA (dsRNA) que induz a degradação de transcritos alvo-específicos. Injeção de dsRNA altera a transcrição de vitelogenina, mas evidências apontam que a ativação do sistema imune em abelhas seja um efeito colateral destaa manipulação. Desenvolvemos um método para o silenciamento do gene codificador de vitelogenina no desenvolvimento pós-embrionário, que minimiza os efeitos da manipulação, onde 0,5 ?g de dsRNA de vitelogenina (dsVg) ou de GFP (controle exógeno, dsGFP) foi oferecido na dieta natural de larvas de segundo estágio, as quais foram mantidas na colônia. Nosso enfoque principal foi a compreensão dos efeitos do silenciamento pós-transcricional de rainhas e operárias de A. mellifera, em especial na fase larval. Operárias adultas reconhecem larvas tratadas e as remove. Mantemos certa distância entre as células de cria que recebiam o tratamento e a remoção de larvas tratadas diminuiu consideravelmente. A expressão de transcritos de vitelogenina em indivíduos sem tratamento e tratados foi analisada no quinto estágio larval de ambas as castas, bem como em operárias adultas de 7 dias e rainhas recémnascidas, utilizando-se PCR em tempo real e a expressão do gene codificador de actina como controle endógeno. Em adultos, controles sem tratamento e dsGFP expressaram quantidades similares de transcritos de vitelogenina. Os grupos alimentados com dsVg tiveram expressão reduzida de vitelogenina, a saber: quinto estágio larval de operárias (91%) e de rainhas (71%), operárias de 7 dias (88%) e rainhas recém-nascidas (70%). O silenciamento da vitelogenina não afetou a morfologia dos adultos, mas sim a fisiologia de larvas de ambas as castas, como nos títulos de hormônio juvenil e concentração de proteínas circulantes na hemolinfa. Concluímos que a ingestão de dsRNA é um método não-invasivo que induz silenciamento gênico e, assim, uma ferramenta eficiente para estudos funcionais pós-genoma. Os mecanismos regulatórios do gene codificador de vitelogenina e seu papel na diferenciação de castas estão em discussão. / Adult bees produce vitellogenin (Vg), the main protein in hemolymph; it is involved in honey bee (Apis mellifera) reproduction, behavior, immunity, longevity and regulation of social organization. Genetic interference mediated by injection of double-stranded RNA (dsRNA) is a powerful tool for the analysis of gene function in Apis mellifera. Injection of dsRNA effectively alters vitellogenin transcription; however, evidence has been found of immune system activation in treated bees, which could be a collateral effect of treatment. Consequently, we developed a non-invasive protocol for disruption of the A. mellifera genes exemplified by vitellogenin mRNA silencing, to understand it, mainly, in the female larval context. Second instar larvae were treated as follows: the treatment group received 0,5 ?g of double-stranded vitellogenin RNA (dsVg) mixed with larval food deposited in the worker brood cells; control group 1 was left to develop without treatment, while control group 2 received dsGFP (Green Fluorescent Protein), as an exogenous control. Treated and control larvae were maintained in the colony until adult emergence. Workers recognized dsRNAtreated larvae and frequently removed them. To circumvent this problem we increased the distance between the treatment groups. Vg gene expression were determined for fifth instar larvae of both castes and for 7 day-old workers and newly-emerged queens, evaluated by quantitative real time PCR, using actin as an endogenous control. For adults, we found that controls, dsGFP- and non-treated bees expressed similar amounts of Vg transcripts. The dsVg-fed groups had significantly reduced Vg gene expression in fifth instar larvae of workers (91%) and queens (71%) and, also, in 7 day-old workers (88%) and newly-emerged queens (70%). Disruption of the Vg gene did not affect adults morphology but physiological larval traits of both castes, as juvenile hormone titre and protein concentration. We conclude that dsRNA ingestion is an effective non-invasive method for inducing knockdown and an efficient approach for post-genome functional studies. The regulatory mechanisms of vitellogenin gene and its rules during caste differentiation are discussed.

The membrane system of a eukaryotic cell is a large and complex system handling the transport, exchange and degradation of many kinds of material. Recent research shows that double-stranded RNA (dsRNA) mediated gene silencing (RNA interference) is a membrane related process. After long dsRNA is processed to small interfering RNA (siRNA) by Dicer, the guide strand and passenger strand are separated in the RNA induced silencing complex (RISC) by Argonaute. The process of loading siRNA into RISC has been suggested to occur at the rough Endoplasmic Reticulum (rER).The components of RISC also associate with late endosomes/multivesicular bodies (MVBs). Furthermore, disturbing the balance between late endosomes/MVBs and lysosomes has been shown to affect the efficiency of silencing. We use the nematode Caenorhabditis elegans as our model organism to study two questions: how does membrane transport affect RNAi and spreading of RNAi from the recipient cells to other tissues (systemic RNAi); and how does RNA transport contribute to the multigenerational silencing induced by dsRNA (RNAi inheritance)? Using SID-5, a protein required for efficient systemic RNAi, as bait in a yeast two-hybrid (Y2H) screen, we got 32 SID-5 interacting candidate proteins. Two of these are the SNARE protein SEC-22 and the putative RNA binding protein C12D8.1. In two additional Y2H screens, we found that SID-5 interacts with multiple syntaxin SNAREs, including SYX-6, whereas SEC-22 only interacts with SYX-6. SNAREs usually function in vesicle fusion processes. We found the two SNARE proteins SEC-22 and SYX-6 to be negative regulators of RNAi and to localize to late endosomes/MVBs. In addition, loss of sid-5 leads to an endosome maturation defect. Finally, we found that the putative RNA binding protein C12D8.1 negatively regulates RNAi inheritance and that C12D8.1 mutant animals show impaired RNAi upon targeting a new gene. Taken together, the results presented in this thesis provide us with more evidence for the connection of the membrane transport system and RNAi. The identification of a putative negative regulator of RNAi inheritance further enriches this research field.

Le PTGS (post-transcriptional gene silencing) est un mécanisme de défense qui cible les acides nucléiques invasifs d’origines endogènes (transposons) ou exogènes (pathogènes, transgènes). Des mutations dans les gènes JMJ14 et NAC52 ont été isolées lors d’un crible génétique visant à identifier des mutants déficients en PTGS. JMJ14 code une histone déméthylase ciblant la lysine 4 bi- ou tri-méthylée de l’histone H3, tandis que NAC52 code un facteur de transcription. Ces deux protéines forment un complexe qui régule la transcription de centaines de gènes endogènes. Toutefois, le rôle de ce complexe chromatinien dans l’expression des transgènes et surtout dans le PTGS reste incompris. JMJ14 interagit avec NAC52 mais aussi avec une protéine de type guanine exchange factor de la famille RCC1. Des mutations dans l’un ou l’autre des membres du complexe RCC1-JMJ14-NAC52 réduisent la transcription des transgènes. JMJ14 se fixe au promoteur de façon indépendante de NAC52, tandis que NAC52 a besoin de JMJ14 pour se fixer à la région transcrite. Toutefois, JMJ14 et NAC52 ne semblent pas requis pour la transcription proprement dite. En effet, un niveau normal de transcription est restauré chez le double mutant jmj14 drm2, indiquant que le rôle du complexe RCC1-JMJ14-NAC52 semble être d’empêcher la méthylation de novo du promoteur par DRM2.L’effet des mutations jmj14 et nac52 sur la transcription des transgènes ne peut expliquer leur effet sur certaines formes de PTGS. En effet, les mutations jmj14 et nac52 n’affectent pas le PTGS induit constitutivement. Par contre, elles empêchent la systémie du PTGS induit localement. Des mutations dans le gène SPCL45 codant une Serine Carboxy Peptidase-Like qui interagit avec NAC52, mais pas JMJ14, ont le même effet. En revanche, la mutation rcc1 n’affecte pas la systémie du PTGS, suggérant que c’est au sein d’un complexe JMJ14-NAC2-SPCL45 que JMJ14 et NAC52 contrôlent le PTGS systémique. Ce complexe pourrait agir directement sur la chromatine du transgène pour permettre d’enclencher le PTGS en réponse à la perception du signal systémique, ou indirectement en contrôlant l’expression d’un gène endogène codant une protéine régulant la systémie du PTGS. Afin de mieux comprendre le rôle de JMJ14 dans la systémie du PTGS, un crible génétique visant à isoler des suppresseurs de la mutation jmj14 a été réalisé. Seize mutants correspondants à sept gènes codant des protéines ayant un rapport avec la chromatine et une action antagoniste à JMJ14 ont été caractérisés. Les mutations dans ces sept gènes pourraient supprimer l’effet de jmj14 en augmentant la transcription du transgène cible et donc la quantité du signal systémique de PTGS. Un 17ème mutant pourrait quant à lui affecter qualitativement le signal systémique de PTGS ou la perception du signal dans les cellules qui le reçoivent. Le gène correspondant reste à identifier. / Post-transcriptional gene silencing (PTGS) is a defense mechanism that targets invading nucleic acids from endogenous (transposons) or exogenous (pathogens, transgenes) origins. Mutations in JMJ14 and NAC52 have been retrieved from a genetic screen aiming to identify PTGS deficient mutants. JMJ14 encodes an histone demethylase targeting the bi- or tri-methylated lysine 4 of histone H3, while NAC52 encodes a transcription factor. Both act in a complex that regulates the transcription of hundreds endogenous genes. However, the function of this chromatin complex in transgene expression and in PTGS is not known. JMJ14 interacts with NAC52 but also with a guanine exchange factor of the RCC1 family. Mutations in any member of the RCC1-JMJ14-NAC52 complex reduce transgene transcription. JMJ14 binds to the transgene promoter independently of NAC52, whereas NAC52 requires JMJ14 to bind on the transcribed region. However, JMJ14 and NAC52 do not seem to be required for transcription itself. Indeed, a wild-type level of transcription is restored in the jmj14 drm2 double mutant, suggesting that the complex RCC1-JMJ14-NAC52 prevents de novo DNA methylation of the promoter by DRM2. The effects of jmj14 and nac52 mutations on transgene transcription cannot explain their specific effect on some forms of PTGS. Indeed, jmj14 and nac52 do not affect constitutively-induced PTGS, but prevent the systemic spreading of locally-induced PTGS. Mutations in SCPL45, encoding a Serine-Carboxy Peptidase-Like that interacts with NAC52, but not JMJ14, have the same effect. In contrast, rcc1 does not affect the systemic PTGS, suggesting that a JMJ14-NAC52-SCPL45 complex is involved in the control of systemic PTGS. This complex could act directly on transgene chromatin to trigger PTGS in response to the PTGS signal, or indirectly by controlling the expression of an endogenous gene encoding a protein regulating systemic PTGS. To better understand the function of JMJ14 in systemic PTGS, a genetic screen aiming to identify suppressors of jmj14 have been performed. Sixteen mutants corresponding to seven genes encoding proteins related to chromatin and having an antagonist function to JMJ14, have been characterized. Mutations in theses seven genes could suppress jmj14 by increasing transgene transcription and consequently the quantity of the PTGS systemic signal. A seventeenth mutant could have a qualitative effect on the PTGS systemic signal or could affect the perception of this signal in recipient cells. The corresponding gene remains to identify.