Einleitung: Die Hände des medizinischen Personals gelten als wichtigste Übertragungsquelle von Krankheitserregern. Methicillin- und multiresistente Erreger stellen die Tiermedizin vor besondere Herausforderungen und limitieren therapeutische Optionen. Obwohl die chirurgische Händedesinfektion einen wichtigen und alltäglichen Bestandteil der Infektionsprävention darstellt, scheinen die Grundkenntnisse hierüber selbst bei chirurgischen Fachtierärzten gering zu sein. Studien haben gezeigt, dass 66 % der Chirurgen sich nicht an etablierte Standardprotokolle halten. Neben der Händedesinfektion stellen sterile OP-Handschuhe eine zusätzliche Barriere für die Übertragung von Bakterien dar. Allerdings sind perforierte Handschuhe mit einem höheren Risiko für postoperative Infektionen (SSI) verbunden, wobei die SSI-Rate in der Pferdechirurgie bis über 60 % reicht. Ziele der Untersuchungen: Die Hauptziele dieser Arbeit lagen in der Erhebung der individuellen Gewohnheiten bei der Durchführung der chirurgischen Händedesinfektion in einer Pferdeklinik (Phase 1) und dem Vergleich der Keimreduktion mit einem Standardprotokoll (Phase 2). Ferner wurden die Proben auf Bakterienspezies gescreent, die SSI induzieren können. Darüber hinaus wurde die Rate von Handschuhperforationen bestimmt. Material und Methoden: Die Observation der individuellen Gewohnheiten (Phase 1) umfasste die Dauer der Händewaschung und -desinfektion, die genutzte Desinfektionsmittelmenge und Zusammenfassung 62 die Verwendung von Bürsten. Das Standardprotokoll in Phase 2 beinhaltete eine 1-minütige Händewaschung mit flüssiger, pH-neutraler Seife ohne Bürsten und die Händedesinfektion über 3 Minuten. Alle Teilnehmer (2 Chirurgen, 8 Klinikmitarbeiter, 32 Studenten) verwendeten Sterillium® für die Händedesinfektion. Die Gesamtkeimzahlen wurden jeweils vor und nach dem Händewaschen, nach der Desinfektion und nach der Operation bestimmt. Zur Probennahme wurden die Hände für 1 Minute in 100 ml sterile phosphatgepufferte Lösung getaucht und die Bakterienkulturen auf Columbia-Schafblutagar angezüchtet. Die Bakterienkolonien wurden manuell ausgezählt und die Bakterienspezies mittels MALDI-TOF identifiziert. Die Handschuhe wurden postoperativ mit einem modifizierten Wasser-Leck-Test auf Perforationen untersucht. Ergebnisse: In Phase 1 und Phase 2 wurden 46 bzw. 41 Händedesinfektionen durchgeführt. Die individuellen Gewohnheiten unterschieden sich deutlich zwischen den Teilnehmern hinsichtlich der Dauer des Händewaschens (bis zu 8 min) und der Desinfektion, sowie der Menge des verwendeten Desinfektionsmittels (bis zu 48 ml). Die Dauer des Händewaschens in Phase 1 und 2 zeigte keinen statistisch signifikanten Effekt auf die Bakterienreduktion. Bei Verwendung des Standardprotokolls war die Reduktion der Keimzahlen nach der Desinfektion im Vergleich zur täglichen Routine signifikant höher (p < 0.001). Die mittlere Reduktion in Phase 1 betrug 90,72 % (LR = 3,23; rechte Hand) und 89,97 % (LR = 3,28; linke Hand) im Vergleich zu 98,85 % (LR = 3,29; rechte Hand) und 98,92 % (LR = 3,47; linke Hand) in Phase 2. Bei acht Teilnehmern (19 %) wurde MRSA (spa Typ t011, CC398) nachgewiesen. Die MRSA-Isolate konnten ferner einer Subpopulation zugeordnet werden, die besonders mit Pferdekliniken assoziiert wurde (hauptsächlich t011, ST398, Gentamicin-resistent). Handschuhperforationen traten bei 54 % (Chirurgen) bzw. 17 % (Assistenten) der Handschuhe auf, wobei eine höhere Prävalenz bei invasiven Eingriffen und Operationen mit einer Dauer von > 60 Minuten vorlag. Die Mehrheit (85 %) der Perforationen blieben vom Operationsteam unbemerkt, wobei Zeigefinger und Daumen die am häufigsten punktierten Stellen waren. Insgesamt nahmen die Bakterienzahlen an den Händen im Laufe der Zeit erneut zu, insbesondere wenn eine Handschuhperforation auftrat. Schlussfolgerung: Die Einhaltung eines Standardprotokolls nach neuestem Stand der Wissenschaft trägt zu einer quantitativ höheren und gleichmäßigeren Keimreduktion beider Hände bei. Die Implementierung eines standardisierten Händedesinfektionsplans sichert die Qualität der aseptischen Maßnahme und ist besonders für die Ausbildung und Schulung von Studenten mit geringer chirurgischer Erfahrung unerlässlich.:1 Einleitung ............................................................................................................................ 1 2 Literaturübersicht ............................................................................................................... 3 2.1 Residente und transiente Hautflora ............................................................................. 3 2.2 Asepsis und Antisepsis ............................................................................................... 4 2.3 Grundlagen und allgemeine Voraussetzungen für eine effektive Händehygiene ......... 4 2.4 Händewaschung .......................................................................................................... 5 2.4.1 Limitationen der Händewaschung ........................................................................ 5 2.5 Händedesinfektion ....................................................................................................... 6 2.5.1 Historie der Händedesinfektion ............................................................................ 6 2.5.2 Hygienische Händedesinfektion ........................................................................... 8 2.5.3 Chirurgische Händedesinfektion .......................................................................... 8 2.5.4 Aliphatische Alkohole ......................................................................................... 10 2.5.5 Dauer und Wirksamkeit der chirurgischen Händedesinfektion .......................... 12 2.5.6 Compliance ........................................................................................................ 13 2.5.7 Zulassung und Prüfung von Händedesinfektionsmitteln .................................... 15 2.6 Nosokomiale Infektionen, Surgical Site Infections (SSI) ........................................... 16 2.6.1 Staphylococcus aureus ...................................................................................... 18 2.6.1.1 Methicillin-resistente Staphylococcus aureus (MRSA) ................................... 18 2.7 Handschuhe .............................................................................................................. 21 2.7.1 Nutzen und Limitationen von Handschuhen ...................................................... 21 3 Veröffentlichung ............................................................................................................... 24 3.1 Eigenanteil zur Veröffentlichung ................................................................................ 24 3.1.1 Publikation ......................................................................................................... 26 4 Diskussion ........................................................................................................................ 50 5 Zusammenfassung ........................................................................................................... 61 6 Summary .......................................................................................................................... 63 7 Literaturverzeichnis .......................................................................................................... 65 Danksagung .............................................................................................................................. 79 / Introduction: Hands of medical personnel are considered the most important source of pathogen transmission. Methicillin- and multiresistant strains of pathogens provide particular challenges to veterinary medicine and limit therapeutic options. Surgical hand disinfection is a major aspect of infection prevention, but basic knowledge seems to be low, even among specialized veterinary surgeons. Studies revealed that 66 % of surgeons do not adhere to established standard protocols. Besides hand disinfection, sterile surgical gloves provide an additional barrier to the transmission of bacteria. However, perforated gloves are associated with a higher risk of surgical site infections (SSI), with an SSI rate in equine surgery exceeding 60 %. Objective: The major objectives were to assess current habits for presurgical hand preparation (phase 1) among personnel in a veterinary equine hospital and to compare the effectiveness in reducing bacteria from hands with a standardized protocol (phase 2). Moreover, samples were screened for bacteria known to cause surgical site infection. The rate of glove perforation was determined, additionally. Material and methods: Individual habits were recorded with regards to the time taken for washing and disinfecting hands, the amount of disinfectant used, as well as the usage of brushes (Phase 1). In contrary to the personal habits, the applied standardized protocol (Phase 2) defined washing hands for 1 minute with liquid neutral soap without brushing and disinfection for Summary 64 3 minutes. All participants (2 surgeons, 8 clinic members, 32 students) used Sterillium® for disinfection. Total bacterial counts were determined before and after hand washing, after disinfection and after surgery. In brief, hands were immersed in 100 ml sterile phosphate-buffered saline for 1 minute and cultures were inoculated onto Columbia sheep blood agar using the spread-plate method. Bacterial colonies were manually counted. Surgical gloves were investigated for perforations after surgery using a modified water leak test. Results: Fourty-six and 41 hand disinfection preparations were carried out during phase 1 and phase 2, respectively. Individual habits differed distinctly between participants regarding the duration of handwashing (up to 8 min) and disinfection as well as the amount of disinfectant used (up to 48 ml). The duration of hand washing in phase 1 and 2 revealed no statistically significant effect on reducing bacteria. In contrary, using the standardized protocol in phase 2, reduction in bacterial numbers after disinfection was significantly higher (p < 0.001) compared to current habits. The mean reduction in phase 1 was 90.72 % (LR = 3.23; right hand) and 89.97 % (LR = 3.28; left hand) compared to 98.85 % (LR = 3.29; right hand) and 98.92 % (LR = 3.47; left hand) in phase 2. Eight participants (19 %) carried MRSA (spa type t011, CC398) which is well established as a nosocomial pathogen in veterinary clinics. The isolates were further assigned to a subpopulation which is particularly associated with equine clinics (mainly t011, ST398, gentamicin-resistant). Glove perforation occurred in 54 % (surgeons) and 17 % (assistants) of gloves, respectively, with a higher number in invasive procedures and operations lasting > 60 minutes. The majority (85 %) of perforations was unnoticed by the surgical team, with index fingers and thumbs most frequently affected. Overall, bacterial numbers on hands mainly increased over time during surgery, especially when glove perforation occurred. Conclusion: Adherence to state-of-the-art standardized protocols contributes to a quantitatively higher and constant germ reduction on both hands. The implementation of a standardized hand disinfection protocol ensures a high quality of aseptic measures and is essential for the education and training of students with little surgical experience.:1 Einleitung ............................................................................................................................ 1 2 Literaturübersicht ............................................................................................................... 3 2.1 Residente und transiente Hautflora ............................................................................. 3 2.2 Asepsis und Antisepsis ............................................................................................... 4 2.3 Grundlagen und allgemeine Voraussetzungen für eine effektive Händehygiene ......... 4 2.4 Händewaschung .......................................................................................................... 5 2.4.1 Limitationen der Händewaschung ........................................................................ 5 2.5 Händedesinfektion ....................................................................................................... 6 2.5.1 Historie der Händedesinfektion ............................................................................ 6 2.5.2 Hygienische Händedesinfektion ........................................................................... 8 2.5.3 Chirurgische Händedesinfektion .......................................................................... 8 2.5.4 Aliphatische Alkohole ......................................................................................... 10 2.5.5 Dauer und Wirksamkeit der chirurgischen Händedesinfektion .......................... 12 2.5.6 Compliance ........................................................................................................ 13 2.5.7 Zulassung und Prüfung von Händedesinfektionsmitteln .................................... 15 2.6 Nosokomiale Infektionen, Surgical Site Infections (SSI) ........................................... 16 2.6.1 Staphylococcus aureus ...................................................................................... 18 2.6.1.1 Methicillin-resistente Staphylococcus aureus (MRSA) ................................... 18 2.7 Handschuhe .............................................................................................................. 21 2.7.1 Nutzen und Limitationen von Handschuhen ...................................................... 21 3 Veröffentlichung ............................................................................................................... 24 3.1 Eigenanteil zur Veröffentlichung ................................................................................ 24 3.1.1 Publikation ......................................................................................................... 26 4 Diskussion ........................................................................................................................ 50 5 Zusammenfassung ........................................................................................................... 61 6 Summary .......................................................................................................................... 63 7 Literaturverzeichnis .......................................................................................................... 65 Danksagung .............................................................................................................................. 79

Depuis plusieurs années, un nombre conséquent d’études décrivent une augmentation de l’incidence de pathologies liées à la fonction de reproduction masculine. Ces anomalies ont été regroupées sous le terme de « syndrome de dysgénésie testiculaire ». Ce syndrome aurait pour origine les effets délétères de polluants environnementaux sur le développement du testicule en période fœtale. Parmi ces polluants environnementaux, les phtalates et le bisphénol A (BPA) sont les plastifiants les plus produits et les plus répandus dans les objets de consommation courante. De nombreuses études leur sont consacrées et ont permis de les classer au rang de perturbateurs endocriniens en mettant notamment en cause leurs effets reprotoxiques. Mon travail de thèse est une étude des effets de ces deux perturbateurs endocriniens sur le développement du testicule fœtal.Nous avons réalisé une première étude concernant les effets du BPA sur le développement du testicule fœtal. Grâce au modèle de culture organotypique, nous avons développé notre étude dans trois espèces : le rat, la souris et l’Homme. Nous démontrons que le BPA diminue la sécrétion de testostérone dans le testicule fœtal humain à partir d’une concentration de 10-8M, alors que chez le rat et la souris, la sécrétion de testostérone n’est affectée qu’à partir de 10-5M de BPA. Nous avons également démontré une diminution de l’expression du gène de l’Insl-3, dans ces mêmes conditions. Ceci nous a permis de mettre en évidence une différence de sensibilité entre les espèces. Pour tenter de comprendre les mécanismes par lesquels le BPA exerce son effet toxique, nous avons comparé ses effets à ceux du DES, autre perturbateur endocrinien œstrogénomimétique. Contrairement au BPA, le DES diminue la sécrétion de testostérone fœtale chez les rongeurs, et non chez l’Homme. Ce résultat suggère l’implication de deux voies de signalisation différentes pour ces deux xéno-œstrogènes. Cette hypothèse est d’ailleurs renforcée par l’étude que nous avons SourceMécanismes d’action des perturbateurs endocriniens bisphénol A et phtalates sur le développement du testicule fœtal / par Thierry N’Tumba-Byn ; sous la direction de Virginie Rouiller-Fabre, Université Paris Sud, 2013 [Thèse de Biologie de la Reproduction et du Développement]réalisée sur des souris invalidées pour le récepteur des œstrogènes ERα, dans lesquelles l’effet anti-androgénique du BPA persiste, contrairement à celui du DES.Parallèlement, nous avons recherché les mécanismes d’action des phtalates et de leur métabolite actif le plus répandu, le MEHP (mono-2-éthyl-hexyl phtalate). Dans la continuité de plusieurs travaux réalisés dans notre laboratoire sur les effets du MEHP, nous avons tenté de comprendre les mécanismes par lesquels le MEHP induit un effet pro-apoptotique dans les cellules germinales mâles. Nous avons mis en évidence une augmentation de l’expression du gène Stra8 dans les cellules germinales traitées au MEHP. Ce résultat nous suggère que le MEHP pourrait induire une différenciation erronée des cellules germinales mâles. De plus, nous avons recherché les récepteurs et la voie de signalisation activée par le MEHP. Nous observons que les agonistes des récepteurs PPARα et de PPARγ entrainent dans les cellules germinales les mêmes phénotypes que le MEHP, à savoir une augmentation du taux d’apoptose et de l’expression du gène Stra8. / For several years, an increase in the incidence of pathologies connected to the male reproductive functions has been described in numerous studies. These anomalies are classified under the term “testicular dysgenesis syndrome”. This syndrome might find its origins in the deleterious effects of environmental pollutants on the testis development in fetal period. Among theses environmental pollutants, phthalates and bisphenol A (BPA) are the most produced plasticizers found in products of common use. Many studies were performed in order to determine their effects, and allowed to classify them as endocrine disruptors because of their reprotoxic effects. My thesis work is a study of the effects of these two endocrine disruptors on the fetal testis development.Our first study focuses on the effects of BPA on the fetal testis development. Using the organotypic culture model, we developed our study in three species: rat, mouse and human. We demonstrated that BPA decreases the testosterone secretion in the human fetal testis from a 10-8M concentration, while in rat and mouse the testosterone secretion is only affected by 10-5M BPA. We also demonstrated a decreased Insl-3 gene expression, in the same conditions. These results allowed us to evidence a difference of sensibility between species. To understand the mechanisms involved in the BPA toxic effect, we compared it with the effect of DES, another endocrine disruptor with estrogenic activity. Unlike BPA, DES decreases the fetal testosterone secretion in rodents and not in human. This result suggests the involvement of two different signalisation pathways for these two xenoestrogens. This hypothesis is reinforced by the study that we performed in mice invalidated for the estrogen receptor ERα. In those mice, the anti-androgenic effect of BPA is maintained, unlike DES effect.In parallel, we investigated the mechanisms of action of phtalates and particularly of their most prevalent active metabolite, the MEHP (mono-2-ethyl-hexyl phthalate). Following previous studies performed in our laboratory concerning the effects of MEHP, we intended to understand the mechanisms by which MEHP induces the apoptosis in male germ cells. We evidenced an increase in Stra8 gene expression in MEHP treated germ cells. This result suggests that MEHP might induce a wrong differentiation in male germ cells. Furthermore, we investigated the receptors and the signalisation pathway activated by MEHP. We observe that PPARα and PPARγ receptors agonists induce the same phenotypes as MEHP, namely an increase in the apoptosis and in Stra8 gene expression in germ cells.

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...

Testicular germinal tumors (TGCT) are relatively rare solid tumors in adults. Even so, they affect more than 700 men a year in the Czech Republic, mostly young patients aged 18- 45 years. A large number of patients are curable by a combination of surgery and chemotherapy, yet about 50 men a year in the Czech Republic succumb to this tumor, in the vast majority of cases due to the development of resistance to chemotherapy containing cisplatin. The rare occurrence and high curability are probably the cause of infrequent molecular and clinical studies carried out in these tumors, and our understanding of the biological processes leading to primary tumor development and the development of cisplatin resistance (CDDP) is still limited. At present, no specific molecular markers that could be used as prognostic or predictive factors and improve patient stratification or treatment tailoring are available in TGCT management. In this work, we studied the molecular-genetic background of TGCT development and CDDP resistance at several levels. To comprehensively study the development of cisplatin resistance, we prepared and analyzed CDDP-exposed TGCT cell lines. Long-term exposure to CDDP increased resistance 10-fold in the NCCIT cell line, while no significant resistance was achieved with Tera-2. The...

The aim of this work was to gain new knowledge about mechanisms of autoimmune uveitis and to test new therapeutic possibilities that have not yet been studied in uveitis or whose effect is questionable. The main emphasis was placed on the role of microorganisms in the process of uveitis. A mouse model of experimental autoimmune uveitis including a germ-free model was used to achieve the aims and samples of patients' intraocular fluids were analyzed. In the experimental model, the intensity of inflammation was evaluated in vivo clinically and post mortem histologically. The effect of immunomodulatory treatment was evaluated. The intensity of inflammation was compared between groups of germ-free and conventional mice. The therapeutic effect of antibiotics administered to affect microbiome was investigated in conventional mice. In intraocular fluid samples of patients with autoimmune uveitis signs of infection were monitored and levels of cytokines and other factors were evaluated. Evaluation of the effect of immunomodulatory therapy has demonstrated the efficacy of mycophenolate mofetil, which supports its wider use in the treatment of autoimmune posterior uveitis in human medicine. The decrease in bacterial load has led to a decrease in the intensity of inflammation, thereby confirming the importance of...

During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type. Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways. Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.