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Period, which is broadly conserved in metazoans, regulates circadian timing of neurophysiology as well as cell fate specification. Studies in mouse and humans indicate that period functions as a tumor suppressor and controls adult stem cell differentiation. However, regulation of period function in developmental pathways has not been characterized and appears to be different from its regulation and function in circadian pathways. lin-42 is the Caenorhabditis elegans ortholog of period and has both circadian and developmental timing functions. During post-embryonic larval development, cyclic expression and function of lin-42 controls stage-specific and reiterative cell fate choices of a subset of epidermal stem cells called seam cells. We are studying lin-42 regulation of seam cell fate during C. elegans larval development as a model for understanding the mechanisms of period regulation of adult stem cell fate in mammals. This dissertation describes the research undertaken to characterize the cis-regulatory elements and the trans-regulatory factors that control lin-42 expression. We used direct molecular interaction assays (Electrophoretic Mobility Shift Assay, EMSA) (Chapter 2) followed by an RNA interference (RNAi)-based genetic screen (Chapter 3) to identify lin-42 transcriptional regulators. Using the EMSA, we identified three 50 to 100 base pair regions (binding regions, BR1-3) in the lin-42 5â noncoding sequences that were bound with specificity by C. elegans nuclear proteins. These binding regions represent putative cis-regulatory elements that may serve as transcription factor binding sites (TFBSs). We attempted to identify by mass spectrometry the proteins that bind to the BR sequences. We also used Phylogenetic Footprinting and bioinformatics screens to identify candidate C. elegans transcription factors (TFs) that may bind to putative TFBSs within the BR sequences. Using an RNAi-based screen, we tested the candidate TF genes for potential genetic interactions with lin-42. We identified ZTF-16, a member of the Hunchback/Ikaros zinc-finger transcription factor family, as a potential lin-42 activator and, using quantitative real-time PCR, confirmed that ztf-16 mutation results in down-regulation and loss of cycling expression of lin-42. We further determined that loss of ztf-16 results in seam cell development defects that phenocopy lin-42 loss-of-function, thus validating ZTF-16 as a transcriptional activator of lin-42. / Ph. D.

Yes / Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed.

Wireworms are the subterranean larval stage of click beetles (Coleoptera: Elateridae), generalist herbivores that feed on a wide range of crops and cause serious damage. Wireworm management is challenging due to their long life cycle, subterranean habitat, wide range of host plants, and lack of effective chemical approach in most crops. Thus, developing an effective alternative control approach is an urgent need. The overall aim of this research study was to evaluate alternative [to insecticides] tactics that could be used as components of effective IPM strategies against wireworms. In Chapter 1 we provide an overview of wireworm's biology and management practices. In Chapter 2 we focused on biological control to evaluate the efficacy of entomopathogenic nematodes and fungi as single and mixed application against Limonius californicus and L. infuscatus in organic vegetable farms. The results showed the higher efficacy of commercial EPN, Steinernema feltiae, against Limonius species than entomopathogenic fungi. In Chapter 3, we focused on cultural control as a component of integrated pest management for wireworm control. We evaluated the efficacy of brown and yellow mustard and their byproducts, including defatted seed meal and seed meal concentrated extract against Limonius spp. Our finding indicated the effectiveness of brown mustard concentrated extract on reducing the wireworm population. However, it has a negative impact on the field-residing entomopathogenic nematodes. In Chapter 4, in the component of cultural control, we evaluated the wireworm, L. californicus, preference to three different crops, pea, bean, and wheat, for identifying an effective trap crop. We also quantified CO2 and organic volatile compounds released from each plant's root and evaluated the wireworm response toward the synthetic volatiles. Detailed results are provided in chapter 4. Finally, we looked at the novel new technology of RNA interference to find the effective target genes in wireworms for application as bioinsecticides and/or transgenic plants to control wireworms. In the concluding chapter, Chapter 6, we summarized our findings on the efficacy of different management tactics, biological, cultural, and molecular, to control Limonius species in cereals and organic production systems. / Doctor of Philosophy / Wireworms, the larval stage of click beetles (Coleoptera: Elateridae), are belowground pests that feed on a wide range of crops such as potatoes, cereals, vegetables, and sugar beet and can cause serious economic damage. Wireworm management is challenging because they live for long periods in the soil and feed on almost all crops. Moreover, most insecticides available for wireworm control, such as neonicotinoids, are not effective in reducing wireworm populations and just serve as feeding deterrents. Thus, developing an effective alternative control approach is an urgent need. The overall aim of this research study was to evaluate alternative [to insecticides] tactics that could be used as components of effective IPM strategies against wireworms. In Chapter 1 we provide an overview of wireworm's biology and management practices. In Chapter 2 we focused on biological control to evaluate the efficacy of entomopathogenic nematodes and fungi when applied individually and in combination with each other against sugar beet wireworm in organic vegetable farms. The results showed the commercial nematode, Steinernema feltiae was more effective in reducing sugar beet wireworm than fungi. In Chapter 3, we focused on cultural control as a component of integrated pest management for wireworm control. We evaluated the efficacy of brown and yellow mustard and their byproducts including defatted seed meal and seed meal concentrated extract against sugar beet wireworms in spring wheat. Our finding indicated the effectiveness of brown mustard concentrated extract in reducing the wireworm population. In Chapter 4, in the component of cultural control, we evaluated the sugar beet wireworm preference for three different crops, pea, bean, and wheat, for identifying an effective trap crop. We also quantified CO2 and organic volaille compounds released from each plant's root and evaluated the wireworm response toward the synthetic volatiles. Detailed results are provided in chapter 4. Finally, we looked at the novel new technology of RNA interference to find the effective target genes in wireworms for application as bioinsecticides and/or transgenic plants to control wireworms. In the concluding chapter, Chapter 6, we summarized our findings on the efficacy of different management tactics, biological, cultural, and molecular, to control wireworm species in cereals and organic production systems.

Les pucerons sont parmi les principaux ravageurs des cultures dans les régions tempérées. Leur succès comme parasites de plantes repose sur leur fort potentiel reproductif dû à la parthénogénèse durant le printemps et l'été ainsi qu’à la symbiose avec Buchnera aphidicola. Cette bactérie symbiotique obligatoire fournit aux pucerons les acides aminés essentiels qui sont déficients dans leur alimentation déséquilibrée (la sève élaborée des plantes), et contribue ainsi à leur développement et reproduction. Le premier volet de ce travail de thèse a consisté à déterminer les besoins en acides aminés des différents stades embryonnaires, afin d’identifier des facteurs clé de l’association symbiotique au cours du développement du puceron du pois Acyrthosiphon pisum. Cette étude, conduite sur des embryons prélevés in vivo ou mis en culture in vitro, a révélé i) des exigences métaboliques évoluant au cours de développement du puceron, ii) une dépendance au compartiment maternel pour l’approvisionnement des embryons en acides aminés, et iii) de forts besoins en acides aminés aromatiques, notamment en tyrosine, pour les stades embryonnaires tardifs et le premier stade larvaire précoce. Le deuxième volet de cette thèse a alors eu pour objectif l’identification de gènes cibles à l’intérieur des voies révélées par l’approche métabolique. A l’aide d’une puce à ADN dédiée au génome du d’A. pisum, les profils d’expression des gènes du puceron ont été analysés au cours de son développement embryonnaire. L’analyse fonctionnelle des différents groupes de gènes montre que ceux liés au métabolisme des acides aminés présentent de hauts niveaux d’expression et des variations significatives entre les différents stades. La voie métabolique des acides aminés aromatiques et tout particulièrement les gènes menant à la synthèse de la tyrosine, ainsi que les gènes/voies liés à la formation et à la maturation de la cuticule, sont parmi les plus sollicités chez les embryons tardifs. L’ensemble des résultats obtenus par les approches métabolique et transcriptomique suggère une synthèse et une accumulation de tyrosine au cours du développement embryonnaire, en vue de son utilisation comme précurseur pour la sclérotisation et le tannage cuticulaire après la ponte. Le dernier volet de ce travail de thèse a consisté en une analyse fonctionnelle du rôle du gène ACYPI007803, codant l’enzyme catalysant la synthèse de la tyrosine à partir de la phénylalanine, par la technique d’ARN interférence (RNAi). Une augmentation de la mortalité des larves pondues par les femelles traitées est corrélée à la diminution de l’expression du gène cible dans les compartiments symbiotiques (les chaines embryonnaires et les bactériocytes maternels) et confirme le rôle clé du gène ACYPI007803 dans le développement des embryons chez le puceron du pois. / Aphids are among the main crop pests in temperate regions. Their success as parasites of plants is based on their strong reproductive output due to parthenogenetic reproduction during spring and summer and to their symbiosis with Buchnera aphidicola. This obligatory symbiotic bacterium supplies aphids with essential amino acids poorly available in their unbalanced food (the phloem sap of plants), and so contributes to their development and reproduction. The first part of this work consisted in determining amino acid needs of different embryonic stages, in order to identify key factors of the symbiotic association during the pea aphid development. This study, led on embryos taken in vivo or cultivated in vitro in culture media, allowed us to identify: i) the evolution of metabolic requirements of embryos during development, ii) a dependence of embryos from the maternal compartment for their supply in amino acids, and iii) strong needs in aromatic amino acids, particularly in tyrosine, of the late embryonic stages and the early first larval stage of the pea aphid. The second part of this thesis had for objective the identification of key genes inside pathways revealed by the metabolic approach. Using a dedicated oligonucleotides microarray, the gene expression profiles of the aphid were analysed during the development of the insect. The functional analysis of different gene groups showed that genes involved in amino acids metabolism are globally over-expressed, but they also showed significant transcriptional regulations in the switches between the different stages studied here. The metabolic pathway of aromatic amino acids and particularly the genes involved in the biosynthesis of tyrosine, as well as genes / pathways involved in the formation and the maturation of the cuticle, were among the most solicited in the late embryos. These transcriptomic results, taken together with those obtained by the metabolic approach, suggest that the amino acid tyrosine is synthesized and accumulated by the pea aphid during its embryonic development, in order to later be used as precursor for the sclerotization and the cuticular tanning, processes that occur after insect laying. The last part of this work consisted in a functional analysis of the gene ACYPI007803, coding the enzyme catalysing the tyrosine synthesis from the phenylalanine, by using the RNA interference (RNAi) technique. An increase of the mortality of larvae laid by the treated females was correlated with the decrease of the expression of the target gene in the symbiotic compartments (the embryonic fraction and the maternal bacteriocytes) so confirming the key role of the ACYPI007803 gene in the development of the pea aphid embryos.

P-Glykoprotein als Produkt des MDR1-Gens stellt einen gut untersuchten Mediator der Multidrugresistenz (MDR) in humanen Malignomen dar. Die Überexpression dieses ABC-Transporters steht in Korrelation zu einer erniedrigten Tumorremission und einer kürzeren Überlebensrate der Patienten. Bisherige Versuche, das Protein über niedermolekulare Substanzen (MDR-Modulatoren) zu inhibieren, vermochten in allen bisherigen klinischen Studien nicht zu überzeugen, so daß diese bis heute keinen Eingang in Standardtherapieschemata gefunden haben. Ziel dieser Arbeit war es, mittels RNA-Interferenz Strategien die Expression von MDR1 zu hemmen und eine Reversion der zellulären Chemoresistenz sowohl im Zellkultur- als auch im Tiermodell zu erreichen. Für die in vitro Untersuchungen an drei humanen multidrug-resistenten Karzinomzellinien wurden verschiedene siRNA (short interfering) Duplexe und shRNA (short hairpin)-exprimierende Vektoren gegen die MDR1 mRNA entwickelt. Die Behandlung der Zellen mit siRNAs führte zu einer bis zu 91 %igen Inhibition der MDR1 mRNA-Expression und zu einer Sensitivierung der Zellen gegenüber dem Anthrazyklin um 89 %. Diese Effekte konnte über einen Zeitraum von drei bis fünf Tagen aufrechterhalten werden. Die stabile Expression von anti-MDR1 shRNAs führte in zwei der untersuchten Zellmodelle zu einer dauerhaften und kompletten Überwindung des MDR1-abhängigen Resistenzphänotyps. Im Mausmodell konnte durch intratumorale Applikation des anti-MDR1 shRNA-kodierenden Vektors mittels low-volume Jet-Injektion eine komplette Reversion der MDR1-Überexpression sowie eine Wiederherstellung der Chemosensitivität gegenüber Doxorubicin in dem resistenten Tumormodell erreicht werden. Die Effizienz der kombinierten Gen- und Chemotherapie wird durch die Verminderung des in vivo Tumorwachstums auf das Volumen des von der sensiblen Zellinien-abgeleiteten Tumors reflektiert. / Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR in three human cancer cell lines by RNA interference (RNAi) technology, two small interfering RNA (siRNA) constructs and four H1-RNA gene promoter-driven expression vectors encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules were constructed. In all cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to a maximum of 89%. The introduction of anti-MDR1/P-gp shRNA expression vectors leads in two of the three human cancer cell lines to a complete reversion of the MDR phenotype. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. In a mouse xenograft model a complete in vivo restoration of MDR1 overexpression and chemosensitivity to doxorubicin could be obtained by intratumorally jet-injected anti-MDR1 shRNA in a multidrug resistant human cancer tumor model.

Parasitische Würmer gehören mit über 3,5 Milliarden Betroffenen zu den weltweit verbreitetesten Infektionskrankheiten. Der Erfolg dieser Parasiten beruht auf ihren ausgefeilten Mechanismen mit denen sie das Immunsystem ihrer Wirte manipulieren. Interessanter Weise gehen Wurminfektionen mit einer geringeren Wahrscheinlichkeit an Allergien zu erkranken einher. Wie genau die Parasiten das Immunsystem manipulieren ist weitgehend unbekannt. Um diese Mechanismen besser studieren zu können, wurde im Rahmen dieser Arbeit versucht RNA interference (RNAi), anhand des Modellmoleküls Tropomyosin zu etablieren. Wie sich am Beispiel des Strongyliden Heligmosomoides polygyrus bakeri zeigte, ist RNAi als Manipulationsmethode für Nematoden nicht oder nur in geringem Maße geeignet. Dies lässt sich auf das Fehlen von Aufnahme- und Verbreitungsmechanismen für Doppelstrang-RNA zurückführen. Desweiteren wurden die Auswirkungen von rekombinantem Tropomyosin der Filarie Acanthocheilonema viteae (rAv-TMY) auf die Entstehung allergischer Atemwegserkrankungen im Mausmodell untersucht. Eine viermalige Behandlung mit rAv-TMY in einem Zeitraum von vier Wochen führte zu verringerten entzündlichen Reaktionen in den Atemwegen. Die Analyse immunologischer Parameter ergab, dass rAv-TMY signifikant den Einstrom von Entzündungszellen in die Atemwege reduziert, allem voran den Einstrom von Eosinophilen. Dies lässt sich durch die verringerte Ausschüttung an IL-5, Eotaxin und MCP-5 zurückführen. Zudem wurde die Bildung von antigenspezifischen IgE verringert während sich die Produktion blockierender IgG1 Antikörper erhöhte. Diese Arbeit belegt somit die anti-allergischen Eigenschaften von rAv-TMY. Damit stellt rAv-TMY ein interessantes Kandidatenmolekül zur Behandlung allergischer Reaktionen dar. Desweiteren kann der Vergleich von allergenem, nicht allergenem und modulatorischem Tropomyosin wichtige Informationen über die allgemeinen Eigenschaften von Allergenen und ihrer molekularen Struktur geben. / Parasitic worms are among the world''s most prevalent infectious diseases with more than 3.5 billion. The success of these parasites is based on their sophisticated ways to manipulate the immune system of their hosts. Interestingly, worm infections abate the risk to develop allergic disorders. How exactly parasitic worms modulate the immune system is so far largely unknown. In order to be able to investigate parasite induced modulation, this work aimed to establish RNA interference (RNAi), a method of genetic manipulation, using tropomyosin as target gene. As shown for the example of Heligmosomoides polygyrus RNAi is not or only to a small extent useful as method to genetically manipulate nematodes. This can be explained with the lack of uptake and spreading mechanisms for double stranded RNA. Furthermore, this work examined the impact of the recombinant muscle protein tropomyosin of Acanthocheilonema viteae (rAv-TMY) on the course of a rodent model of allergic airway inflammation. A four-time treatment with rAv-TMY over a period of four weeks resulted in decreased inflammatory responses in the airways. The analysis of immunological parameters showed that rAv-TMY significantly reduces the influx of inflammatory cells into the airways, especially eosinophils. The reduced eosinophil influx can be attributed to the decreased expression of IL-5, eotaxin and MCP-5 in the airways. In addition, the formation of antigen-specific IgE was impaired whereas the production of the blocking antibody IgG1 was increased. These results demonstrate the anti-allergic properties of rAv-TMY. For this reason rAv-TMY becomes an interesting model molecule for the treatment of allergic diseases. Furthermore, the comparison of allergenic, non-allergenic and modulatory tropomyosin might put some light on the nature of allergens and their molecular patterns.