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.

Die subakute sklerosierende Panenzephalitis (SSPE), eine durch das Masernvirus (MV) verursachte sogenannte „ slow virus “ Infektion des zentralen Nervensystems, ist eine progrediente chronische Erkrankung, die zum Tod führt und bisher medikamentös nicht heilbar ist. Da die RNAi-Strategien grundsätzlich zur Inhibition von Viren in Säugetierzellen geeignet sind, stellt die RNAi eine Möglichkeit dar, die Infektion auf molekularer Ebene anzugreifen. Dafür wurden verschiedene miRNA-Expressionskassetten, welche gegen zwei Sequenzen im MV- Hämagglutinin-Gen (H) und sechs Sequenzen im MV-Nukleokapsid-Gen (N) gerichtet sind, konstruiert und in MV infizierte Zellen eingebracht. Diese miRNAExpressionskassetten wurden auf zwei verschiedenen Wegen in die Zelle eingebracht: Zum einen wurden sie über ein miRNA-Expressionsplasmid (pmiR), welches in die Zellen transfiziert wird, transient exprimiert; zum anderen wurden sie durch virale Vektoren (HIV, SIV und MoMLV) stabil in die Zellen transduziert. Dies ermöglicht die Integration der miRNAExpressionskassette in das Genom der Zelle und dadurch die Expression der miRNAs für einige Wochen. In erster Linie zielt die Wirkung der RNA-Interferenz auf die Degradierung der spezifischen MV-mRNAs. Diese Degradierung konnte mit Hilfe quantitative Reverse Transkription real time-PCR (qRT-PCR) nachgewiesen werden. Die transiente Expression der verschiedenen miRNAs gegen das MV-N-Gen bzw. MV-H-Gen führte in jedem Fall zu einer Reduktion der viralen genspezifischen mRNAs. Die Reduktion der MV spezifischen mRNA betrug 99,8% für das MV-N-Gen und 91,2% für das MV-H-Gen. Die Wirkung der RNA-Interferenz zielt am Ende auf die Reduktion der neu gebildeten infektiösen Viruspartikel und ihrer Verbreitung in der Zellkultur, welche die spezifische miRNA gegen MV-N oder MV-H exprimiert. Dieser Effekt konnte nur durch Plaque-Assay überprüft werden. Die Plaque-Assays, die mit den Überständen der miRNA-behandelten Zelllinien durchgeführt wurden, zeigten ebenfalls eine Reduktion der neu gebildeten infektiösen MV-Partikeln von 97,6% für die miRNA gegen das MV-H-Gen und 99,0 % für die miRNA gegen das MV-N-Gen. Die intrazelluläre Expression der miRNAs führte zu einer Hemmung der Virus-Ausbreitung in MV-infizierten Zellen. Die Reduktion betrug hier durch die Expression der miRNA-N10 98,8% und durch die Expression der miRNA-H2 80,0%. Hier zeigte sich, dass die Inhibition der viralen Proteinsynthese durch den RNAi-Mechanismus auch die Verbreitung der MVInfektion durch Zell-Zell-Fusion behindern kann. Dies zeigte sich durch die verringerte Bildung von Plaques bzw. Synzytien in miRNA-behandelten Zelllinien. Die vorliegende Arbeit zeigte, dass RNAi effektive gegen MV-Infektion in Zellkultur eingesetzt werden kann. Als nächster Schritt sollte daher dieser RNAi-Effekt im etablierten Tiermodell ausgetestet werden.

Oncogenes drive cancer by hijacking normal cellular functions involved in proliferation and survival. Suppression of the driving oncogene is highly effective for promoting tumor regression, a phenomenon termed "oncogenic addiction." By using unbiased genetic tools to functionally probe oncogenic addiction, we can identify cancer dependencies and characterize aspects of oncogenic signaling.

The bloodstream form of Trypanosoma brucei differentiates into the stumpy form in the mammalian bloodstream, completing differentiation into the procyclic form on uptake by the tsetse fly. The underlying genetic events occurring during this differentiation process in pleomorphic cell lines were investigated through whole-genome microarray studies of key time points during differentiation from stumpy form cells to the procyclic form found in the insect midgut. The microarray was extensively validated and bioinformatic experiments conducted to detect motifs over represented in stumpy form or slender form cells. A positional-dependent motif was identified that was over represented in stumpy form cells, possibly representing a regulatory domain. The transcripts found to be enriched in stumpy form cells included a chloride channel, although RNAi directed against this gene showed no phenotype, suggesting the protein is redundant, as three other homologous proteins exist in the genome and showed similar mRNA profiles on the microarray. Stumpy form cells are G0 arrested and two proteins implicated in G0/G1 regulation in other organisms, Target of Rapamycin (Tor) and Cdh1, were investigated in T. brucei to determine whether these proteins were involved in differentiation. The result of depletion of either protein was rapid cell death in bloodstream form cells, although treatment with the drug rapamycin did not have any effect on the cells in contrast to other eukaryotes where this drug causes G1 arrest. A method for synchronisation of bloodstream form cells was also designed using a supravital dye and flow cytometry to allow investigation into cell cycle-dependent processes. This method was particularly suitable for harvesting populations enriched in G0/G1 stage cells, however differentiation of the isolated G0/G1 and G2/M populations did not show significantly different differentiation kinetics.

No description available.

Hepatitis C virus (HCV) has an internal ribosomal binding site (IRES) located near the 5ʹ end of its genome. The HCV IRES is capable of direct binding to the 40S small ribosomal unit and eukaryotic initiation factor eIF3, and can initiate translation after the assembly of the whole 80S ribosome. Various molecular types can act as IRES inhibitors. Small molecule compounds seem to be the most promising agent for use in the clinic. The main objective of the thesis was to develop a system for searching for small molecule compound inhibitors of HCV IRES in a library of chemical compounds. Several variants of vector carrying bicistronic cassettes were prepared. After validating their functionality by transient transfection of mammalian cell cultures, mammalian stable cell lines were established. These stable cell lines will allow for automatization of the search for small molecule compound inhibitors of HCV IRES. Our second objective was to study the variability of HCV IRES sequences in patient samples. The samples were analysed by temperature gradient gel electrophoresis (TGGE). Select specimen were sequenced, cloned into a vector with bicistronic cassette and analysed by flow cytometry. In this was we evaluated the effect of specific mutations in the HCV IRES sequence on the level of IRES dependent...

Master of Science / Department of Biochemistry / Gerald Reeck / Aphids mount a remarkable salivary secretion to overcome plant host defenses. Our group has previously reported a gene unique to aphids enriched in the salivary glands of the pea aphid A. pisum, C002, which is required for successful feeding on its host plant Vicia fava. Here I present an analysis of genetic variation within the available EST data for C002 in pea aphids. From 596 total ESTs, 332 are full-length, and segregate into 8 validated haplotypes based on the criteria I set in place to access the quality of EST data. Additionally, Armet, is a putative multi-functional gene implicated as a neurotrophic factor during development, and as a part of the unfolded protein response during stress. I employ RNA interference in the model organism T. castaneum to determine the effect of transcript knockdown during development from early in-star larval stages, through pupation, and its effect on adult emergence. I report that knockdown of Armet transcript significantly hinders the ability for beetles to emerge from the pupae.

L’analyse par spectrométrie de masse des protéines qu’interagies avec Dicer-2, R2D2 a identifié six sur huit composés du complexe chaperon TRiC / CCT. L’élimination de l'un des six composants du complexe TRiC / CCT identifiés par spectrométrie de masse a conduit à une réplication virale accrue d'au moins un des trois virus testés. L’ensemble des mes résultats suggèrent un rôle nouveau pour le complexe TRiC / CCT dans l'ARNi antiviral. Mes résultats soulèvent la question de savoir pourquoi le GRR d’AGO2 semble être nécessaire dans le contexte d'une réponse antivirale. Mes résultats indiquent que le complexe TRiC / CCT participe à l’étape de dissociation et à la relocalisation dynamique d'AGO2-L pendant l’infection virale. / The mass spectrometry analysis of the complexes associating with Dicer-2, R2D2, and AGO2 identified six out of the eight subunits forming the TRiC/CCT complex. Knockdown of one of the six subunits identified is sufficient to increase the replication of DCV (DrosophilaC Virus). My results identify an interaction between the TRiC/CCT complex and the antiviral RNA interference. This interaction raises the question of how the GRR region of AGO2 is necessary for the antiviral response. My results suggest that the TRiC/CCT complex is involved in the dynamic dissociation and relocalization of AGO2 during viral infection.

Heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) requires conserved components that mediate RNA interference (RNAi) directed methylation of histone H3 on lysine 9 (H3K9). Fission yeast heterochromatin is mainly found at centromeres, telomeres, and the mating-type locus. At centromeres, transcripts from repetitive elements are processed to siRNAs and RNAi promotes chromatin modification by recruiting the Clr4 methyltransferase. RNAi is not required to maintain silent chromatin at the mating-type locus. This RNAi-directed form of centromeric heterochromatin provides an ideal system for in vivo screening to allow the identification of compounds that inhibit the activity of proteins involved in RNA silencing, chromatin modification and heterochromatin assembly in fission yeast and may inhibit conserved proteins in other organisms. A dominant selectable marker gene system at fission yeast centromeres that reports loss of heterochromatin integrity by increased resistance to G418 in 96-well plate format liquid cultures was developed. The resulting strain was used to screen a nontargeted chemically diverse compound library in vivo to identify compounds that disrupt the integrity of RNAi-directed heterochromatin. Two compounds, Emi1 and Emi14, were identified and found to cause a significant decrease in the level of H3K9 methylation on the outer repeats at fission yeast centromeres. Growth in the presence of Emi1 or Emi14 also caused a reduction in H3K9 methylation levels at the mating-type locus, suggesting that they do not act through RNAi. Consistent with this, Emi1 and Emi14 did not cause a decrease in centromeric siRNA levels. Analyses therefore suggest that Emi1 and Emi14 do not disrupt RNAi but that they inhibit downstream events in chromatin modification and heterochromatin assembly. Cells lacking RNAi due to loss of Dicer (dcr1Δ) or cells lacking the histone deacetylase (HDAC) Sir2 (sir2Δ) retain significant but lower levels of H3K9 methylation on the centromeric outer repeats. When dcr1Δ or sir2Δ cells were grown in the presence of Emi1 or Emi14 a further reduction in H3K9 methylation levels was observed on the outer repeats. This mimics the effect of combining clr3Δ with dcr1Δ or sir2Δ and suggests that Emi1 and Emi14 may interfere with SHREC function. SHREC is a chromatin remodelling complex that includes the HDAC Clr3 and the chromatin remodeler Mit1 and is known to contribute to heterochromatin integrity. Expression profiling performed on Emi1 and Emi14 treated cells confirmed the previous results. The changes in gene expression following Emi1 and Emi14 treatment were compared to known mutants defective in heterochromatin integrity. The profile of expression changes following Emi14 treatment was found to correlate with alterations in the expression pattern observed in cells with SHREC components deleted. No correlation with mutants lacking other HDACs or RNAi components was detected. Emi1 had a weaker correlation with defective SHREC function and thus may also partially inhibit the SHREC complex. Murine erythroleukemia (MEL) cells harbouring a silenced eGFP reporter transgene were used to assess whether Emi1 and Emi14 also affect silencing in mammalian cells. Emi1 was found to disrupt silencing at the eGFP reporter and this correlated with a decrease in H3K9 methylation. Structurally related analogues of Emi1 and Emi14 were selected and tested in the fission yeast assay. Interpretation of the obtained structure-activity relationships allowed identification of the chemical moieties key to Emi1 and Emi14 activity. Overall, an approach was developed to identify two novel small molecule inhibitors of a well-characterized chromatin modification pathway. The SHREC complex was identified as the putative target of these two compounds and structurally related active analogues were identified for them. Importantly, one of the compounds was also active in mammalian cells, highlighting the usefulness of this approach in identifying compounds that affect higher organisms.

"Adhesion Molecules have been associated with a number of neurological and psychological disorders (humans), and implicated in various developmental processes (animals). Better understanding the development of the nervous system and the roles of adhesion molecules in it may be crucial to better understanding these disorders. LIGs, Leucine Rich Repeat and ImmunoGlobulin containing transmembrane proteins, represent a novel class of such adhesion molecules and have been implicated in various neuronal processes, including neurite outgrowth, axonal pathfinding, neuronal regeneration and survival. Two such LIGs are Kek1 and Kek2, members of a Drosophila LIG family, which have been reported to function in axonal pathfinding and synaptic plasticity, respectively. It is unclear what their roles in these processes are, as well as if other members of the Drosophila LIG family have similar roles. Current studies aim to survey the Kekkon family function in the nervous system, looking to identify new phenotypes and/or to elucidate the mechanisms underlying previously identified phenotypes. To achieve this goal, tissue specific inducible RNAi technique was employed. Validating of a number of transgenic RNAi stocks obtained was necessary and showed that all stocks obtained promoted specific and efficient knock down of target gene. Next an assessment of RNAi knockdown efficacy in developing nervous system was carried out and knockdown was shown to be weak if not in the presence of Dicer-2 co-misexpression. A number of screens for general behavioral phenotypes were performed including ubiquitous, neural, and imaginal discs knockdown. These uncovered possible effects of kek1 neural knockdown, as well as possible interaction of Kek1 with neurotactin, neuroglian and kek2. NMJ analysis of Kek5 and Kek6 was also carried out and preliminary results indicate possible interaction of kek5 in NMJ, although no statistical significance was detected."