MA thesis - Daniela Cvachová - 2011 Abstract: This thesis compares two Czech translations of the drama The Importance of Being Earnest by Oscar Wilde. The comparison is based on findings of currently recognised theoreticians of translation (Levý, Knittlová, Králová, Reiss). The translation of Jiří Zdeněk Novák is half a century older than the translation by a team of young beginning translators led by Stanislav Rubáš and Zuzana Šťastná. The thesis focuses on the comparison of those aspects that are characteristic for Wilde's works and difficult for translators: lexicological problems like puns, bon mots, allusions to realia. In spite of the fact that the drama is not of a large extent, it offers a wide range of "tough nuts to crack" like that.

Le polyomavirus humain BK (virus BK) établit une infection persistante asymptomatique dans les voies rénales de 80% de la population humaine. Chez les patients transplantés, la réactivation du virus BK est à l'origine de néphropathies et de cystites hémorragiques. L'augmentation des pathologies associées au virus BK en même temps que l'utilisation de traitements immunosuppresseurs de plus en plus puissants souligne un lien étroit entre la réponse immunitaire de l'hôte et la réactivation virale. Cependant la réponse immune à l'infection par le virus BK, en particulier le rôle des cytokines antivirales dans le contrôle de l'infection est peu documentée. Ici, nous avons étudié l'efficacité antivirale des interférons (IFN) sur la multiplication du virus BK. Nous avons testé les IFN-alpha, lambda et gamma sur la souche Dunlop du virus BK dans les cellules Véro et MRC 5. L'IFN-gamma inhibe de façon dose-dépendante la transcription virale de la région précoce et de la région tardive ainsi que l'expression de la protéine virale VP1. Un moindre effet antiviral a été observé avec l'IFN-alpha et l'IFN lambda. Ces résultats sont associés à une phosphorylation prolongée de STAT 1 avec l'IFN-gamma, non retrouvée avec l'IFN-alpha et lambda. La différence d'efficacité entre ces trois types d'IFN suggère que certaines protéines induites seulement par l'IFN-gamma ont un effet antiviral dans l'infection par le virus BK. L'analyse transcriptionnelle révèle neuf protéines qui pourraient être impliquées dans cet effet antiviral spécifique. Parmi elles, nous avons étudié l'effet antiviral de l'indoleamine 2,3-dioxygénase (IDO) et les protéines de liaison au guanylate (GBP ou guanylate binding protéines), GBP1 et GBP2, sur le virus BK. Nos résultats montrent que GBP1 et GBP2 mais pas IDO contribuent à l'activité antivirale de l'IFN-gamma sur le virus BK. Trouver le mécanisme d'action de ces protéines antivirales induites par l'IFN pourrait nous aider à développer une stratégie thérapeutique / The human polyomavirus BK (BK virus) establishes an asymptomatic persistent infection in the urinary tract of 80% of the human population. In transplant recipients, reactivation of the BK virus infection is the cause of nephropathy and hemorrhagic cystitis. Diseases associated with BK virus infections are increasing at the same time as potent immunosuppressive therapies are developing. This highlights the importance of components of the immune system in controlling viral reactivation. However, the immune response to the BK virus, particularly the role of antiviral cytokines in infection control, is poorly documented. Here, we investigated the antiviral efficacy of interferons (IFN) on the BK virus multiplication. We tested IFN-alpha, lambda and gamma on the Dunlop strain of BK virus in Vero cells and MRC 5 cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose dependent manner and decreased the expression of the early and late viral transcripts. A weaker antiviral effect was observed with IFN-alpha and IFN-lambda. These results are associated with a prolonged STAT1 phosphorylation with IFN-gamma but not with IFN-alpha and lambda. The difference of efficacy between these three types of interferon suggests that some interferon induced proteins only produced by IFN-gamma had an antiviral effect on BK virus infection. Transcriptomic analysis reveals that nine proteins could be involved in this specific antiviral effect. Among them, we selected and investigated the antiviral effect of indoleamine 2,3-dioxygenase (IDO) and guanylate binding protein 1 and 2 (GBP1 and GBP2) on the BK virus. Our results suggest that GBP1 and GBP2 but not IDO contribute to the antiviral activity of IFN-gamma on the BK virus. Finding the action mechanism of these IFN gamma induced antiviral proteins could help to develop a therapeutic strategy

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Cell signaling is central to integration of internal and external cues that regulate development and homeostasis. Most development is thought of as pre-adult, but limited developmental processes occur in adults. Gametogenesis incorporates elements of both these facets, with a distinct developmental plan for gamete synthesis which is regulated by integration of homeostatic inputs such as nutrient status, and environmental cues. Signaling pathways integrate and transduce information from these cues to evoke a response. A decline in homeostasis and subsequent cues occurs over time, in the case of reproductive tissues leading to a progressive loss of fertility. The Janus Kinase and Signal Transducer and Activator of Transcription or Jak/Stat signaling pathway is conserved between vertebrates and invertebrates and is necessary for numerous functions needed to maintain organism and reproductive homeostasis, as well as contributing to various developmental events. The pathway in the fruit fly Drosophila melanogaster, is composed of a single receptor, Domeless, one Janus kinase, Hopscotch, one known effector, Stat92E, and the Unpaired family of ligands consisting of Upd, Upd2, and Upd3. Jak/Stat signaling is highly pleiotropic in both sexes with involvement in homeostasis and reproduction, making it an ideal model for studying the role of signaling in reproductive aging. Reduction of pathway activity in females results in a higher proportion of unfertilized eggs, which increases with age, and in males leads to a premature onset of infertility. Central to both is integration through cell signaling to evoke an appropriate response. This dissertation explores two of the requirements for Jak/Stat signaling: the pleiotropic requirement for Jak/Stat activity during oogenesis and male reproductive maintenance. Jak/Stat functions from the beginning of oogenesis, in the stem cell niche. From there it participates in multiple functions including specification of a subset of somatic cells called the border cells through the polar cells, a pair of cells at either pole of the egg. Pathway stimulation in the border cells drives their migration with the polar cells to the oocyte boundary, where the polar cells each form an extension in a coordinated manner into the micropyle, the means for sperm entrance during fertilization. Loss of Jak/Stat activity in the border cells prevents border cell migration. While border cell migration has been well studied, polar cell involvement after completion of border cell migration is less well known. To investigate the requirements for polar cell activity and Jak/Stat activity after the completion of border cell migration, we reduced Jak/Stat signaling in the polar cells which, while having no effect on border cell migration, results in blocked micropyles due to loss of coordination of extensions during their outgrowth. Reduced function in the polar cells did not significantly affect expression of adhesion molecules. But, the loss of Stat92E is phenocopied by loss of DE-cadherin. Hence, these results indicate a previously unknown autocrine requirement for Jak/Stat activity in the polar cells. The testes also have a continuous requirement for Jak/Stat activity for stem cell maintenance and differentiation of the germline into mature sperm. Reproductive maintenance not only requires sustained production of gametes, but reproductive tissues are also subject to deterioration of homeostatic functions that contribute to organismal aging. Males from thirty-nine lines of the Drosophila Genetic Reference Panel (DGRP), a panel of inbred, fully sequenced lines, were screened for age at infertility. Data were used to perform a genome-wide association study (GWAS) to identify the genetic architecture of reproductive aging. Candidate variants associated with cell signaling regulators, genes with functions in maintaining cell homeostasis, and organism behavior were uncovered. Notably, several SNPs fell in and near Ptp61F, a negative regulator of Jak/Stat activity. While variants in the primary components of the Jak/Stat pathway were not identified, the general classes of candidate loci functions reflect the requirements for homeostasis, metabolism, and development that have been shown by other studies examining the genetics of aging and fecundity. Thus, we show that Jak/Stat has an amazing amount of pleiotropy that encompasses both the real-time functions of fertility and the time related process of aging.

Les interférons (IFNs) de type I sont des cytokines qui jouent un rôle capital dans les défenses immunes, antivirales et antiprolifératives de l'organisme. En se liant à leur récepteur de surface, composé des deux sous-unités IFNAR1 et IFNAR2, ils induisent la cascade de signalisation JAK/STAT qui aboutit à leur effets biologiques. L'objectif de ma thèse était d'identifier des mécanismes de régulation de la signalisation des IFNs. Dans ce contexte, nous nous sommes intéressés à la palmitoylation du récepteur de l'IFN de type I, une modification lipidique souvent impliquée dans le trafic et la signalisation des protéines. Par marquage métabolique au palmitate tritié, nous avons montré qu'IFNAR1 et IFNAR2 sont palmitoylées. Le domaine cytoplasmique d'IFNAR1 contient deux cystéines, Cys463 et Cys502, qui sont des sites potentiels de palmitoylation. A l'aide de mutants sur chacune de ces cystéines, nous avons montré qu'IFNAR1 est palmitoylée uniquement sur sa cystéine la plus proche de la membrane plasmique, la Cys463. Un mutant non palmitoylé dans lequel cette cystéine a été remplacée par une alanine nous a permis de constater que la palmitoylation d'IFNAR1 n'est pas impliquée dans son trafic intracellulaire, dans son endocytose ni dans sa stabilité, mais qu'en revanche elle joue un rôle crucial dans l'activation de la voie de signalisation JAK/STAT. De façon concordante, la palmitoylation d'IFNAR1 est requise pour l'activation transcriptionnelle des gènes induits spécifiquement par l'IFN-a. Par contre, un défaut de palmitoylation n'influence nullement l'activité antiproliférative de l'IFN-a, en dépit du rôle de cette modification dans la signalisation.

Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5. TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5. We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms Lori Nicole Griner Abstract Myeloproliferative neoplasms (MPNs) are diseases of hematopoietic stem cell origin and are characterized by uncontrolled growth of cells of the myeloid compartment. The Philadelphia chromosome negative classical MPNs, including polycythemia vera, essential thrombocythemia, and myelofibrosis, are diseases of dysregulated JAK2 signaling. In fact, the majority of MPN patients have activating mutations in JAK2 (e.g JAK2-V617F), a tyrosine kinase that contributes to the growth and survival of myeloid cells. While MPNs were first described over sixty years ago, a significant need remains to develop therapeutic strategies for them. Inhibitors of JAK2 are currently being developed, and one inhibitor, ruxolitinib, was recently approved for certain MPN patients. Ruxolitinib has made profound impacts on improving splenomegaly and constitutional symptoms in MPN patients, but it and other JAK2 inhibitors have not significantly reduced the JAK2 mutant allele burden, and thus such inhibitors have not induced remission in these patients. The current consensus in the MPN field supports JAK inhibition for the treatment of patients, but a further understanding of MPNs and JAK2 signaling, as well as improved JAK2 inhibitors, may be necessary for treating MPN patients. The work described in this dissertation has uncovered novel requirements for JAK2-V617F-driven signaling and transformation. We demonstrate that JAK2-V617F co-localizes with lipid rafts, cholesterol-rich microdomains within the plasma membrane that function to serve as platforms for signaling complex formation. Signaling complex formation is a necessary component for dysregulated signaling induced by JAK2-V617F. We provide evidence that cholesterol altering-lipid raft disrupting agents attenuate JAK2-V617F-driven signaling. We also show that cholesterol-lowering statins are effective at downregulating JAK2 signaling and inducing apoptosis in JAK2-V617F-driven cell lines. Importantly, we show that statins, inhibitors of the mevalonate pathway, inhibit the growth of primary MPN cells, while the same statin doses have no effect on healthy controls. Impressively, we demonstrate that statins cooperate with multiple JAK inhibitors, including ruxolitinib, to inhibit cell growth and induce apoptosis of JAK2-V617F-driven cells. This report establishes statin-mediated inhibition of the mevalonate pathway as a potential approach to improve MPN therapeutics. We propose future studies with statins and JAK2 inhibitors in the treatment of MPNs.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The JAK/STAT pathway is but one of the signal transduction cascades responsible for proper development and homeostasis. Gain-of-function mutations of pathway components are causative agents of several leukemias, highlighting the necessity for proper regulation of signal transduction. Drosophila presents an attractive model to study JAK/STAT signaling because mutations in the pathway behave in an analogous manner. Furthermore, the Drosophila cascade is much simpler as only one of each component required for activation has been characterized; whereas in mammals, there are many ligands, receptors, 4 JAKs and 7 STATs.Suppressors of Cytokine Signaling (SOCS) are one family of molecules which regulate JAK/STAT signaling via a negative feedback loop. All SOCS share a distinct modular domain architecture, which we exploited to locate three putative SOCS homologues within the Drosophila genome. I present the identification and initial characterization of one of these homologues, Socs44A. I show that Socs44A is not responsive to or dependent on JAK activity. However, I demonstrate that Socs44A is capable of downregulating JAK/STAT signaling in the developing wing but not inoogenesis, indicating that its ability to regulate the pathway is tissue specific, a phenomenon observed in the mammalian model.Signal transduction pathways are integrated at multiple levels. This interplay allows for combinatorial signaling, resulting in a higher order of complexity in the signals that can be received and interpreted by a cell. Well documented are the interactions between the JAK/STAT and the EGFR/MAPK pathways. In this work, I show that Socs44A can genetically interact with, and upregulate, the EGFR/MAPK pathway, analogous to a recent report involving SOCS-3.Starting with the Drosophila genome sequence, I initiated a reverse genetic approach to studying the function of the Socs44A locus. During the course of this investigation, I designed and implemented a novel post-processor of the BLAST algorithm, called Multi-BLAST, which facilitates retrieval of multiple domain sequences from public databases. In what would have been the ultimate achievement of this study, I attempted two mutagenesis screens designed to isolate Socs44A loss-of-function alleles. Progress on these screens is reported.

Equine herpesvirus-1 (EHV-1) is one of the major viral pathogens causing respiratory disease, abortion, perinatal mortality and neurologic disease among horses resulting in significant economic losses to the equine industry. The virus can also remain latent in the horses and recrudesce at any time. Type-I interferons (IFNs) act as a first line of defense against many viral infections. In this study we investigated the type-I IFN response against the neuropathogenic T953 strain of EHV-1 in equine endothelial cells (EECs). The results showed that after a transient induction of IFN-β mRNA as well as protein at an early time (3h) post infection (p.i.), T953 strain of EHV-1 suppressed further induction of IFN-β at later times (12h onwards). Studies were done to confirm that the suppression of type-I IFN induction at later time points was not due to the normal IFN-β induction kinetics, it was rather because of the active interference by the virus. Investigation of the mechanisms by which T953 interferes with IFN-β production revealed that the virus degraded the endogenous level of the transcription factor, interferon regulatory factor 3 (IRF-3) and also down-regulated the activation of IRF-3 followed by its accumulation in the nucleus. However, T953 infection caused degradation of nuclear factor κB (NF-κB) inhibitory protein IκBα and also induced p50 subunit to translocate into nucleus from cytoplasm suggesting activation of NF-κB signaling. This also indicated that inhibition in the type-I IFN production was probably not due to the inhibition of NF-κB. The results of these studies also indicated that T953 virus was resistant to the biological effect of the recombinant equine IFN-α in vitro. Investigation of the reason of this resistance showed that T953 virus interfered with the cellular JAK-STAT signaling mechanism by which type-I IFN exerts its antiviral effect. Moreover, the studies revealed that downstream of the JAK-STAT signaling, T953 virus also inhibited the expression of cellular antiviral proteins including interferon stimulated gene 56 (ISG56) and viperin. Altogether, these data indicate that the T953 strain of EHV-1 interfered with the host cell innate immune responses by modulating type-I IFN mediated immune responses at multiple levels in vitro.