Orientador: Eliana Maria Zanotti Magalhães / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:25:44Z (GMT). No. of bitstreams: 1 Simoes_LucianaFranceschi_D.pdf: 13158668 bytes, checksum: 8fb7b4a656ae812560a18a5466806eea (MD5) Previous issue date: 2014 / Resumo: A busca de novos tratamentos para a esquistossomose é relevante devido a constatação de linhagens do Schistosoma mansoni com perda de sensibilidade aos fármacos atuais. A interferência por RNA (RNAi) é uma técnica que pode ser usada no silenciamento de um gene especifico, através de um RNA dupla fita (dsRNA). Essa ferramenta se mostra eficaz em aplicações terapêuticas em vírus, príons e patógenos intracelulares. Estudos mostram que a técnica do RNAi é bastante eficiente no verme Caenorhabditis elegans. Até o momento poucos trabalhos analisaram a técnica de RNAi no combate a esquistossomose. Este trabalho teve como objetivo aplicar a técnica da interferência por RNA em S. mansoni e avaliar sua eficiência em testes in vitro e in vivo, tendo como alvo o gene fucosiltransferase, enzima envolvida no processo de fucosilação de lipídios e proteínas essenciais ao parasita e o gene glicosilfosfatidilinositol, que atua como ancora de superfície, na qual se ligam antígenos protéicos e glicoprotéicos. A supressão gênica foi estudada em testes in vitro e in vivo, com observações da atuação na biologia, morfologia e mortalidade dos vermes. Como resultados nos testes in vitro, obtiveram-se alterações tegumentares nos adultos, extravasamento do conteúdo interno de fêmeas e alterações na oviposição, apresentando os grupos tratados maior quantidade de ovos mortos e malformados quando comparados com os grupos controles. Nos experimentos in vivo verificou-se diminuição no numero de vermes recuperados, mortalidade de vermes em alguns grupos tratados, e alteração na oviposição, quando examinados pelas técnicas de Kato e oograma. Análise histológica do fígado mostrou diferenças no número, na área e na conformação dos granulomas dos grupos tratados quando comparados com os grupos controles. A mortalidade de ovos eliminados nas fezes de camundongos após dois dias de tratamento também se mostrou significativamente maior nos grupos tratados com moléculas para silenciamento. Esses resultados sugerem o potencial uso da técnica de RNAi no estudo da esquistossomose / Abstract: The search for new schistosomiasis treatments is relevant due to the observation of resistant Schistosoma mansoni strains for current drugs. The interference by RNA (RNAi) is a technique which can be used for silencing a specific gene using a double strain RNA (dsRNA). This methodology presents efficacy in therapeutic use in virus, prions, and intracellular pathogens. Studies demonstrate that RNAi technique is very efficient in Caenorhabditis elegans worm. Up to the moment, a few studies have analyzed the RNAi technique against schistosomiasis. Thus, the aims of this study were apply the interference by RNA in S. mansoni and evaluate its efficiency in in vitro and in vivo tests, having the fucosyltransferase gene, an enzyme involved in parasite essential lipids and proteins fucosilation process, and the glicosylfosfatidilinositol gene, which acts as anchor-surface where proteic and glicoproteic antigens are bonded, as targets. The genic suppression was studied in in vitro and in vivo tests, with observation of its action in the biology, morphology, and mortality of the worms. The results for in vitro testswere integumentary alteration in adults, internal content extravasation in females, and oviposition alteration, with higher amount of dead eggs and not well formed eggs in the treated groups in comparison with the control groups. The in vivo tests presented a decrease in the number of recovered worms, worm mortality in some groups, and oviposition alteration when analyzed under the Kato and oogram techniques. Histological analysis of the liver presented differences in the number, area, and conformation of the granulomas of the treated groups in comparison with the control groups. The mortality of the eggs eliminated on the mice feces after two days of treatment were also significantly higher in the groups treated with the silencing molecules. These results suggest the potential use of the RNAi technique in the schistosomiasis treatment / Doutorado / Relações Antrópicas, Meio Ambiente e Parasitologia / Doutora em Biologia Animal

Orientador: Maria de Fátima Sonati / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T19:29:31Z (GMT). No. of bitstreams: 1 Wobeto_VaniaPerettideAlbuquerque_D.pdf: 2287567 bytes, checksum: ff964a9410a59697f79714370d17c0c7 (MD5) Previous issue date: 2012 / Resumo: As fosfatidilinositol-fosfato quinases (PIPKs) pertencem a uma família de enzimas lipídio-quinases que geram vários mensageiros lipídicos, incluindo um importante segundo mensageiro denominado fosfatidilinositol-4,5-bifosfato, que participa da regulação de diversas atividades celulares, como a modulação do citoesqueleto de actina, o transporte de vesículas, a formação de adesão focal e diversos eventos nucleares, incluindo a expressão gênica. A subfamília da PIPK está dividida, conforme a especificidade de sinalização, em tipo I (isoformas ?, ? e ?), tipo II (isoformas ?, ? e ?) e tipo III. Em estudo recentemente realizado em nosso laboratório, o gene da PIPKII? mostrou-se diferencialmente expresso em reticulócitos de dois irmãos com Doença de Hemoglobina H, sendo maior no paciente com maior nível de hemoglobina anômala, paralelamente à expressão do gene da globina ?, sugerindo uma possível relação entre a PIPKII? e a produção de globinas. Além disso, análises realizadas durante a diferenciação eritróide de células CD34+, em cultura, demonstraram que as expressões dos genes das PIPKs aumentam à medida que essas células se tornam mais diferenciadas. O papel da PIPK no processo hematopoiético, no entanto, tem sido pouco explorado. No presente trabalho, investigamos a localização celular da PIPKII? e sua expressão gênica e protéica durante a diferenciação eritroide, megacariocítica e granulocítica de células da linhagem hematopoiética e demonstramos que o silenciamento do gene da PIPKII?, em células K562, resulta em diminuição da proliferação deste tipo celular, aumento de expressão do gene da globina ? e uma tendência de elevação da expressão do gene da globina ?. Esses resultados corroboram a sugestão prévia de existência de relação entre a PIPKII? e a expressão dos genes de globinas, relação que deve continuar a ser investigada em células eritróides normais e de pacientes com hemoglobinopatias / Abstract: Phosphatidylinositol-phosphate kinases (PIPKs) belong to a family of lipid kinase enzymes that generate various lipid messengers, including an important second messenger known as phosphatidylinositol-4,5-biphosphate, which participates of the regulation of several cell activities, such as modulation of the actin cytoskeleton, vesicle transport, formation of focal adhesions and various nuclear events, including regulation of gene expression. The PIPK subfamily is divided into types I (isoforms ?, ? and ?), II (?, ? and ?) and III according to signaling specificity. In a recent study in our laboratory, the PIPKII gene was differentially expressed in reticulocytes from two siblings with hemoglobin H disease: expression of this gene, as well as that of the 'beta'-globin gene, was greater in the patient with the higher level of the abnormal hemoglobin, suggesting a possible relationship between PIPKII and the production of globins. In addition, analyses carried out using CD34+ cells from cultures undergoing erythroid differentiation showed that PIPK gene expression increased as the cells became more differentiated. However, there has been little research into the role of PIPK in the hematopoietic process. In this study we investigate the cellular location of PIPKII and the gene and protein expression of this enzyme during erythroid, megakaryocytic and granulocytic differentiation of hematopoietic lineage cells. We show that PIPKII gene silencing in K562 cells results in reduced proliferation of this cell type, increased 'gama'-globin gene expression and a tendency towards increased 'alfa'-globin gene expression. These results corroborate the previous suggestion of a relationship between PIPKII and globin gene expression, a relationship that should be the subject of further investigation in normal erythroid cells and erythroid cells from patients with hemoglobinopathies / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Traditional methods of pest control are threatened by the development of insecticide resistance, both to traditional insecticides and Bt toxins. Discovery of RNA interference (RNAi) has created opportunities to develop new insect control mechanisms. However, RNAi responses appear to be robust in coleopteran pests, but other orders, e.g. Lepidoptera and Hemiptera, present varied or ineffective RNAi responses. Current delivery strategies for double-stranded RNA (dsRNA) include microinjection, ingestion, and soaking. These approaches have benefits and problems. This study investigates the potential for porous silica nanoparticles (pSNPs) to improve the delivery of dsRNA and induce an RNAi response in Spodoptera frugiperda cells. Initially, the binding conditions of RNA onto porous and nonporous silica nanoparticles was examined, and the movement of RNA on and within pSNPs was observed. That information was then applied to in vitro studies for examining the capacity of silica nanoparticles to protect dsRNA from degradation by nucleases. This work culminated in an in vivo assay for measuring apoptosis when dsRNA is delivered to insect cells by pSNPs. Results of these studies show that silica nanoparticles bind nucleic acids and that dsRNA is mobile, pSNPs protect dsRNA from nuclease degradation, and pSNP/dsRNA complexes can induce apoptosis in lepidopteran insect cells.

Abstract Innate immunity provides the first line of defense against invading, pathogenic microorganisms in all multicellular organisms. The fruit fly Drosophila melanogaster has turned out to be an excellent model organism to elucidate mechanisms of innate immune responses because of the highly conserved intracellular signaling cascades mediating these ancient immune functions in flies and mammals. In the present study, RNA interference (RNAi) -based functional genomics were utilized to identify novel components of Drosophila’s immune reactions. Mediators of bacterial phagocytosis, nuclear factor kappa B (NF-κB) signaling and the antiviral RNAi pathway were screened in hemocyte-like S2 cells. Follow-up studies were executed in mammalian cells as well as in Drosophila larvae and adult flies to gain broader significance for the results. Seven novel components essential for efficient phagocytosis of bacteria were identified. Eater was defined as Drosophila’s most important phagocytic receptor showing novel epidermal growth factor (EGF)-repeat -based microbial recognition properties. Additionally, Abelson interacting protein (Abi), capping protein alpha (cpa), 14-3-3ζ, tousled-like kinase (tlk), CG2765 and CG15609 were determined as intracellular effectors of phagocytosis, the three former ones executing their evolutionarily conserved functions through remodeling of the actin cytoskeleton. Eater, together with Scavenger receptor class C, type I (Sr-CI), was demonstrated to be responsible for double-stranded RNA (dsRNA) uptake into S2 cells and, when ectopically expressed, into mammalian cells via clathrin-mediated endocytosis. Proteasome component Pros45 and RNA helicase Belle were established as mediators of the intracellular RNAi pathway, whereas essential roles in antimicrobial signaling via the immune deficiency (Imd) pathway were addressed for Inhibitor of apoptosis 2 (Iap2) and Tak1-associated binding protein (TAB). Iap2 and TAB were shown to affect nuclear translocation of NF-κB -like transcription factor Relish. The present study identifies several novel mediators of the Drosophila immune response and provides insight into mechanisms of fly host defense. As insects serve as vectors of human diseases (e.g. malaria), knowledge about Drosophila immune mechanisms may help to better understand the transmission and pathogenesis of these diseases and develop treatments to fight these infections. Additionally, knowledge gained from model organisms serves as valuable background information, often conducting human research into new tracks. / Tiivistelmä Synnynnäinen immuniteetti on elintärkeä puolustusjärjestelmä taudinaiheuttajia vastaan. Kodeissakin yleinen banaanikärpänen, Drosophila melanogaster, on osoittautunut erinomaiseksi synnynnäisen immuniteetin tutkimusmalliksi, erityisesti teknisesti yksinkertaisen ja eettisesti ongelmattoman geneettisen muunneltavuutensa ansiosta. On myös havaittu, että solunsisäiset, immunologisia signaaleja välittävät mekanismit ovat evoluutiossa hyvin säilyneitä. Hyvin usein samankaltaiset geenituotteet toimivat signaalinsiirtäjinä sekä kärpäsen että ihmisen soluissa. Tämän työn tarkoituksena oli RNA-häirintää (RNAi) sekä muita nykyaikaisia solu- ja molekyylibiologisia tutkimusmenetelmiä hyödyntäen tunnistaa uusia kärpäsen synnynnäiselle immuunipuolustukselle välttämättömiä geenituotteita. Bakteerien fagosytoosille, viruspuolustukselle ja tumatekijä nuclear factor kappa B:n (NF-κB) välittämälle signaloinnille välttämättömiä signalointimolekyylejä pyrittiin identifioimaan laajan mittakaavan RNA-häirintään perustuvilla seuloilla kärpäsen soluissa. Saatujen tulosten merkitystä nisäkkäiden immuunipuolustukselle tutkittiin myös hiiren soluissa. Seitsemän geenituotteen osoitettiin olevan bakteerien fagosytoosille tärkeitä kärpäsen soluissa. Aiemmin tuntematon geenituote, joka nimettiin Eateriksi, osoitettiin kärpäsen tärkeimmäksi bakteereja fagosytoivaksi reseptoriksi. Eaterin solun ulkoisen osan osoitettiin tunnistavan taudinaiheuttajia uudella epidermaalisen kasvutekijän (epidermal growth factor, EGF) kaltaisella toistosekvenssillä. Myös useiden solun tukirankaan, sytoskeletoniin, liittyvien proteiinien (Abi, cpa, 14-3-3ζ) sekä aiemmin vähemmän tunnettujen geenituotteiden (CG2765, CG15609, tlk) osoitettiin osallistuvan bakteerien fagosytoosiin. Näistä kolmen ensinmainitun immunologinen tehtävä havaittiin evoluutiossa säilyneeksi, kärpäsestä hiireen. Eaterin, yhdessä kärpäsen toisen scavenger reseptorin (Sr-CI) kanssa, havaittiin myös toimivan kaksijuosteisen RNA:n (dsRNA) reseptoreina kärpäsen soluissa, mahdollistaen helpon ja tehokkaan RNA-häirinnän. RNA-häirinnän, ja siten mahdollisesti myös viruspuolustuksen, välittäjiksi identifioitiin proteasomin alayksikkö Pros45 ja RNA-helikaasi Belle. Lisäksi Inhibitor of apoptosis 2 (Iap2) ja Tak1-associated binding protein (TAB) todettiin kärpäsen immune deficiency (Imd) signalointireitin komponenteiksi, jotka osallistuvat antimikrobisten peptidien tuotantoon välittämällä NF-κB:n kaltaisen kärpäsen transkriptiotekijän (Relish) siirtymisen tumaan aktivoimaan immuunipuolustusta välittävien geenien ilmentymistä. Tämän tutkimuksen tulokset valottavat banaanikärpäsen immuunipuolustuksen mekanismeja. Koska hyönteiset toimivat monien ihmisten infektiotautien välittäjinä, kärpäsen immuniteetin tuntemus luo mahdollisuuksia kehittää hoitoja näitä tauteja vastaan. Lisäksi malliorganismeista saatu tieto luo uusia teorioita ja näkökulmia, johtaen usein myös lääketieteellistä tutkimusta uusille raiteille.

Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...

Master of Science / Department of Entomology / Kristopher S. Silver / Kun Yan Zhu / The yellow fever mosquito, Aedes aegypti, is a significant vector of several viral diseases, including Zika, dengue fever, yellow fever, and chikungunya. Since vaccines are not currently available for these viruses, control of the disease vectors by using insecticides is the most common practice for preventing disease. As a result, Ae. aegypti has developed resistance against many of the most commonly used insecticides, including organophosphates and pyrethroids. The rise in resistance in vector mosquitoes requires the search for new control strategies, such as RNA interference (RNAi), to manage mosquito populations. Vacuolar H[sup plus]+-ATPase (V-ATPase), a multi-subunit enzyme involved in many cellular processes, including membrane energization, acidification of organelles, and entry of dengue virus into the cytoplasm, is a potential target for RNAi, though little is known about its genetic structure or expression patterns in Ae. aegypti. In this study, I performed genome-wide surveys to identify the genes encoding different subunits of the V-ATPase protein complex, partially characterized the molecular properties and expression patterns of selected V-ATPase subunit genes, and tested the feasibility of using oral-based delivery of nanoparticles formed from double-stranded RNA (dsRNA) and chitosan to suppress the expression of selected V-ATPase subunit genes in Ae. aegypti. My genome-wide surveys revealed that Ae. aegypti V-ATPase consists of 13 different subunits (A, B, C, D, E, F, G, H, a, c, c”, d, e) encoded by 14 genes. Analysis of exon-intron arrangements for each gene demonstrated that each V-ATPase subunit gene has between one (subunit c) and 12 (subunit C) exons, with most genes (11) having 3 to 6 exons. Subsequent phylogenetic analysis of the deduced amino acid sequences of each subunit showed that V-ATPase subunits A, B, C, F, G, H, and a exhibited high levels of conservation among all the examined species, but subunits D, E, c, c”, d, and e showed high conservation only among dipteran species. Analysis of the expression profiles in different tissues and developmental stages of three specific V-ATPase subunits (A, D, and H) showed that whereas the expression of these genes varied between tissues and developmental stages, the patterns of expression of subunits A, D, and H were very similar. The highest mRNA expression level was observed in Malpighian tubules in fourth-instar larvae. Interestingly, expression of subunits A, D, or H in different tissues of adults was highest in male hindgut versus Malpighian tubules in females. Feeding mosquito larvae with chitosan nanoparticles made with dsRNA complementary to subunits A, D, or H resulted in significant suppression of mRNA transcript levels of each of these subunits. Peak suppression of V-ATPase A, D, or H transcripts occurred on the fifth day, where the gene transcript level was suppressed by 66.0, 27.3, or 70.4%, respectively, as compared with those of the control. Additionally, feeding of dsRNA/chitosan nanoparticles targeting subunit D caused mortality starting on day 3, with cumulative larval mortality reaching 14.8% on the sixth day. These results suggest that oral delivery of dsRNA/chitosan nanoparticles can substantially suppress target gene expression in Ae. aegypti larvae. However, increasing RNAi efficiency in targeting V-ATPase subunit genes in mosquito larvae appears to be necessary in order to obtain higher larval mortality using oral delivery of dsRNA/chitosan nanoparticles.

Les monocytes inflammatoires Ly-6Chigh murins, et leurs homologues humains CD14+CD16-, jouent un rôle important dans l'initiation et la persistance des maladies inflammatoires chroniques. Leur action délétère dans ces pathologies a mené au développement de stratégies thérapeutiques visant à les éliminer ou empêcher leur recrutement aux sites inflammatoires. Toutefois, ces méthodes se sont avérées peu spécifiques des monocytes et surtout d'une faible efficacité compte tenu de l'aspect hautement inflammatoire des monocytes. Le besoin de développer de nouvelles stratégies est donc nécessaire. Les objectifs de ma thèse ont donc été dans un premier temps de caractériser in vivo le ciblage spécifique des monocytes inflammatoires par la formulation liposomale DMAPAP. Dans un second temps, l'utilisation de DMAPAP pour formuler des siRNA a permis d'évaluer l'efficacité thérapeutique d'une stratégie basée sur l'inhibition spécifique de gènes jouant un rôle clef dans l'inflammation dans les monocytes inflammatoires. Mon travail a permis de montrer dans un modèle préclinique d'arthrite que l'inhibition de gènes régulateurs de l'inflammation dans les monocytes Ly-6Chigh est une approche thérapeutique efficace permettant d'induire une immunomodulation des réponses pathogéniques des lymphocytes T effecteurs, aboutissant au défaut de recrutement des cellules immunitaires dans les articulations et à une amélioration des signes cliniques. J'ai également validé le transfert de cette technologie ex vivo sur cellules humaines primaires. L'inhibition de gènes clefs dans les monocytes inflammatoires représente donc une stratégie prometteuse pour le développement de futures thérapies dans la polyarthrite rhumatoïde. Par ailleurs, mes résultats confirment le rôle central des monocytes inflammatoires dans les pathologies inflammatoires chroniques. / Inflammatory mouse Ly6Chigh monocyte subset and its human counterpart, defined as CD14+ CD16-, play key roles in the initiation and chronicization of immune-mediated inflammatory disorders (IMID). Deleterious effects of monocytes led to the development of therapeutic strategies aiming at depleting them or preventing their recruitment to inflamed tissues. However, these methods are poorly specific with weak efficacy considering the high number of inflammatory monocytes and their marked level of activation. The need for developing new therapeutic approaches is obvious. The aim of my thesis was to characterize selective delivery of a siRNA-containing lipid formulation to the Ly-6Chigh monocyte population and at evaluating the therapeutic potential of this targeted strategy. Using the cationic lipid-based DMAPAP vehicle for in vivo RNAi-mediated gene silencing, my work allowed demonstrating, in a preclinical mouse model of arthritis, the efficacy to inhibit master genes of inflammation specifically within Ly-6Chigh monocytes upon systemic injection. Reduced disease severity in mice was associated with an overall systemic immunomodulation of the pathogenic T cell populations and led to defective mobilization of immune cells to arthritic joints. Importantly, the formulation was successfully optimized in a perspective of clinical application and the targeting of human CD14+CD16- inflammatory monocytes was validated ex vivo. Overall, my findings demonstrate that the silencing of a key gene within Ly-6Chigh monocytes is a promising strategy for future therapeutic intervention in the context of IMID and reinforces the pivotal role of Ly-6Chigh monocytes in inflammatory processes.

RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA polyplexes are equally as effective as naked dsRNA for gene knockdown on a concentration basis, these materials are assimilated into cells in a manner independent of dsRNA specific transport proteins. The mechanism of uptake is likely clathrin-mediated endocytosis. In addition, I identify a significant and yet unreported side-effect associated with chitosan exposure, the dysregulation of a major myosin isoform. Next, I have determined the efficacy of chitosan/dsRNA polyplex nanoparticles under different environmental conditions. The presence of inorganic ions (phosphate and nitrate) at realistic environmental concentrations does not alter the efficacy of the nanoparticles for gene knockdown, nor do they inhibit knockdown by naked dsRNA. These conditions did not cause any significant changes to the hydrodynamic diameter or zeta potential of the particles themselves between treatments. By contrast, a pH higher than six and the presence of natural organic matter significantly reduce the efficacy of the nanomaterials at gene knockdown but leave knockdown by naked dsRNA unaffected. Though some changes in polyplex size are observed in the pH treatments, these changes are comparatively small, and particles remain well within the size that can be ingested by C. elegans. At pH 8, the charge of the particles is effectively neutral. Similarly, concentrations of natural organic matter >2.5 mg/L cause a charge reversal of the particles, from strongly cationic to strongly anionic. Large aggregates are also visible in each of these treatments. Lastly, I characterize the efficacy of a suite of different polymer and solid core nanomaterials for dsRNA delivery, similar to the above. Poly-L-lysine, poly-L-arginine, Ge-doped imogolite, and poly-L-arginine-citrate coated Au nanoparticles all fail to cause any appreciable knockdown in the same C. elegans reporter system. Uptake of the polymers was exceedingly poor, and though the Au particles appear to have been ingested, there is no evidence of significant gene knockdown. Furthermore, poly-L-arginine caused significant injury to the mouthparts of C. elegans exposed to these materials. Layered double-hydroxide nanoparticles were successful at gene knockdown, and appear to function slightly better than naked dsRNA alone, and were translocated in C. elegans in a similar fashion to naked dsRNA. Taken together, these findings aid in the development of safe and effective RNAi biological control agents.

No description available.

Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins. Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance. Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway. Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.