The highly unstructured interferon regulated transcription factor IRF-1 is a tumour suppressor protein that plays vital roles in the antiviral and DNA damage response pathways. To identify interacting factors that regulate IRF-1 function and expand on the available limited information on its interactome, an in vitro screen was developed using peptide-aptamer affinity chromatography coupled with mass spectrometry. Discrete identified which bind to a number of potential transcriptional regulators including NPM1, YB-1 and TRIM28. The screen also proved useful in identifying binding proteins to the C-terminal Mf1 domain, which is vital for IRF-1-mediated growth suppression and Cdk2 repression, and additionally regulates IRF-1 stability. Thus, an LXXLL motif in the MF1 domain was found to be required for the binding of Hsp70 family members and cooperation with Hsp90 to regulated IRF-1 turnover and activity. These conclusions were supproted by the finding the Hsp90 inhibitors suppressed IRF-1-dependent transcription shortly after treatment, whilst at later time points inhibition of Hsp90 led to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 was increase by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear, but not cytoplasmic, IRF-1. Additionally, a stress specific interaction between IRF-1 and the Hsp70-associated ubiquitin E3 ligase CHIP, that targets Hsp70/Hsp90 clients for proteasomal degradation, was demonstrated. Consequently, decreases in IRF-1 protein levels in cells exposed to heat stress or heavy metal ions were accompanied by the formation of IRF-1:CHIP complexes. Based on observations that CHIP ubiquitination of IRF-1 occurred both in the presence and absence of Hsp70, a model was proposed wherein Hsp70 serves as a factor that recruits CHIP to its substrates and its dissociation from the complex allosterically activates CHIP-dependent substrate ubiquitination. In support of this model, in vitro and biophysical evidence is presented, showing that CHIP in complex with Hsp70 is less flexible and less effective as an E3 ligase that CHIP alone. Thus, in agreement with recent studies, the work done in this thesis highlights the importance of conformational flexibility and of direct binding or 'docking' of CHIP to its substrate(s) in its mechanism of action.

Introduction: Interferon regulatory factor-7 (IRF-7), the “master regulator” of type 1 interferon, has shown to orchestrate anti-viral immune responses via fine-tuning expression of interferons and interferon-stimulated genes. Methods: IRF-7 levels were examined using multi-parametric flow-cytometry in HIV-uninfected Manitoban donors and in HIV-infected and HIV-uninfected Kenyan volunteers from a well-characterized Kenyan sex worker cohort. IRF-7 expression level was reduced by IRF-7 specific siRNA or shRNA encoded in lentivirus and administered into ex-vivo CD4+ T cells by transfection or transduction. Results: In unstimulated PBMC, IRF-7 was constitutively expressed at low levels in every defined subset we examined. We observed less HIV-infected cells (~10%) with IRF-7 knockdown, suggesting that IRF-7 may play a role in HIV infection. Conclusions: Unexpectedly, it was found that even though IRF-7 had been implicated in orchestrating antiviral events, reducing IRF-7 expression in ex vivo CD4+ T cells did not increase the cellular susceptibility to productive HIV infection. / February 2017

Interferon regulatory factor 1 (IRF-1) is a tumour suppressor protein and transcription factor. It has been shown to modulate target gene expression in response to stimuli, which include viral infection and DNA damage, and to be down-regulated in several forms of cancer. This thesis details the development of an intrabody, an intracellular antibody, that binds specifically to endogenous IRF-1. The binding of the intrabody to IRF-1 enhanced transcription from IRF-1-responsive reporter gene constructs and endogenous promoters, thus it was shown to activate IRF-1. Intrabody binding also increased the rate at which IRF-1 was degraded, suggesting that the intrabody epitope may be regulating both IRF-1 activity and turnover. These results were supported point mutation within the intrabody epitope (P325 to A) as the resultant mutant also displayed both a higher transcriptional activity and increased rate of degradation. In an effort to understand the mechanisms which regulate IRF-1 activity a search for novel IRF-1-interacting proteins was carried out using phage peptide display. This in vitro technique enables the identification of peptides able to bind a specific target protein. The sequence of these peptides can then be used to search protein databases for homologous, full-length proteins that could also bind the target protein. This led to the identification of an IRF-1-binding peptide that held sequence similar to a region of Zinc Finger 350 (ZNF350), a transcription factor involved in regulating the DNA damage response. Subsequently, endogenous ZNF350 and IRF-1 were co-immunoprecipitated from a human cancer cell line. The extreme C-terminus of IRF-1 was shown to be sufficient for an interaction with ZNF350, although a second, more N-terminal site was also shown to be essential for a stable intracellular interaction. This data sheds new light on the role of the extreme C-terminus of IRF-1 in modulating the protein‟s activity. This study also provides new and IRF-1-specific molecular tools, in the form of intrabodies and IRF-1-binding peptides, which could be used in the future to further characterise the activity and regulation of this tumour suppressor protein.

Interferon regulatory factor 1 (IRF-1), the founding member of IRF family, is a nuclear transcription factor first described as a transcription factor that binds to the upstream region of interferon induced genes following viral infection. In addition, IRF-1 has been reported to be involved in cell growth regulation, induction of apoptosis, immune responses, post-transcriptional modification, and cell transformation by oncogenes. Thus, IRF-1 shows accumulative evidence supporting the theory that IRF-1 functions as a tumour suppressor. However, we still lack the knowledge in the regulation and function behind IRF-1 and many other tumour suppressors due to the lack of synthetic tools that can aid in understanding the mechanism of cancer biology. Here we described the creation of synthetic tools that can be applied to study the role of a transcription factor(s) in cancer biology. Firstly, we described the creation, using recombineering technology, of universal bacterial artificial chromosome (BAC) targeting vector. This targeting vector, carry a cre-conditioned STOP cassette that can be targeted at a desired specific area. The resulted targeting vector can aid the generation of mice models with a conditioned knock-in subtle mutation(s). The resulted cre-conditioned mice models are an essential tool for any outstanding research project in cancer biology. Secondly, we described the development of Flp-In System™ from Invitrogen; the system can ease the generation of isogenic stable mammalian expression cell lines. Using this system, we created two isogenic stable cell lines expressing wild-type IRF-1 and a mutant that abolish IRF-1 DNA binding ability (W11R). Both cell lines were investigated using microarray analysis revealing new IRF-1 target genes. We reported the up-regulation of expected standard interferon regulatory genes such as, interleukin-24 (IL-24) and interferon regulatory factor-2 binding protein-2 (IRF2BP2) and the up-regulation of standard apoptotic genes such as, early growth response-1 (EGR-1) and prostate transmembrane protein, androgen induced-1 (PMEPA1) confirming the role of IRF-1 as a tumour suppressor. However, we also reported the up-regulation of secreted phosphoprotein-1 (SPP1) and SH3 and PX domains-2A (SH3PXD2A) which are matricellular protein produced by cancer cells playing a role in cellular adhesion, invasion, tumour growth progression and metastasis. Thus, we proposed a new biological role of IRF-1 in cellular movement. Thirdly, we described the development of a synthetic stable reporter cell line which can report IRF-1 transcriptional activity; such reporter cell line can be used once large scale screening is needed. The created stable reporter cell line was used to screen a kinase inhibitor library which has revealed C3 as an IRF-1 modifier. The newly identified IRF-1 modifier regulates IRF-1 transcriptional activity by inhibiting platelet-derived growth factor receptor (PDGFR) and/or vascular endothelial growth factor receptor (VEGFR) tyrosine kinase. Finally, we validated the synthetic Flp-In System™ by testing the system using a novel oncoprotein model. We have developed a stable cell line that overexpresses an oncoprotein named Anterior Gradient 2 (AGR-2). We have found that AGR-2 can attenuate IRF-1 protein levels dependent of p53. In addition, AGR-2 has been identified as a cellular survivor factor during unfolding protein response. In conclusion, this study descried the creation and the validation of synthetic tools: synthetic cassette for cre-conditioned mice creation, the Flp-In System™ for isogenic stable cell line creation, and IRF-1 reporter cell line for high throughput screening. All synthetic tools were validated and used to investigate IRF-1, a transcription factor that plays a role in cancer and immune system.

By modelling and simulating the ESA spacecraft Rosetta in a plasma with solar wind parameters, and simultaneously simulating a particle detection experiment of Langmuir probe voltage sweep type using the ESA open source software SPIS Science, we investigate the features of Rosetta’s envi- ronment in the solar wind and the e↵ect of photoemission from the space- craft on the measurements made by the Langmuir Probe instrument on board Rosetta. For a 10 V positively charged spacecraft and Maxwellian distributed photoelectron emission with photoelectron temperature, Tf = 2 eV in a plasma of typical 1 AU solar wind parameters: ne = 5 ⇥ 106 m3, vSW = 4 ⇥ 105 m/s, Te = 12 eV, Tion = 5 eV, we detect a floating potential of 6.4 (± 0.2) V at Langmuir probe 1. Two models used in literature on photoemission was used and compared and we report a clear preference to the Maxwellian energy distribution of photoelectrons from a point source model with our simulation result.

Syftet med detta examensarbete är att undersöka hur läraren i praktiken använder samtalet i undervisningen och vår utgångspunkt läggs i de klassrumssamtal som initieras av läraren. För att uppnå syftet har vi valt att ställa oss frågor där vi belyser hur samtalsstrukturen ser ut,vilken roll läraren tar i samtalet samt om det ställs autentiska eller icke-autentiska frågor och hur elevernas svar används för att skapa vidare samtal. För att kunna svara på frågeställningarna har vi använt oss av observationer där vi observerat klassrumssamtalet. Resultatet visar på att läraren tar en tydlig ledarroll där denne innehar rätten att initiera ämnen och att leda samtalet och därmed också fördela ordet mellan eleverna. Vi har även funnit att de frågor läraren ställer till största del är av icke-autentisk karaktär.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

Interferon γ is an important T-cell helper type 1 (Th1) cytokine involves in antimicrobial immunity. It is a part of the inflammatory immune response in the site of infection. However, for its proper function, the regulation of immunity is necessary to avoid injury of the tissue caused by long-term inflammation. While interferon γ triggers expression of proinflammatory genes, it also regulates genes which inactivate immune response. The B7-H1 molecule belongs among these inhibitory regulators. Furthermore, antitumour effect of interferon γ is well-known as well. After extensive experiments, interferon γ was tested as an immunotherapeutic drug against melanomas in clinical trials. However, the trials had to be terminated prematurely because of unsuccessful results. It started to be clear that interferon γ could have also a protumour effect. Interferon γ upregulates the expression of B7-H1 molecule which aids tumour in escape from immunity. The B7-H1 molecule possesses a binding site for interferon regulatory factor 1 (IRF-1) in its promoter region. This IRF-1 is induced by interferon γ - JAK/STAT signalling pathway. In our previous research, we observed interferon γ induced DNA demethylation of promoters in genes that are involved in antigen presenting machinery. Additionally, DNA methylation of...

Under de senaste årtiondena har den svenska skolan gått från att vara tämligenenspråkig och monokulturell till dagens mångkulturella och flerspråkliga skolmiljö(Skolverket 2016). Denna förändrande elevsammansättning medför andraförutsättningar för lärarna och då bland annat gällande att fördela ordet i klassrummettill en elevgrupp bestående av både elever med svenska som förstaspråk och elever medsvenska som andraspråk. Uppsatsens syfte är att studera hur en gymnasielärare i ensådan elevgrupp ställer frågor till eleverna och sedan följer upp deras svar. För att undersöka detta genomfördes två lektionsbesök som både observerades med stödav ett observationsschema och som spelades in både med diktafon och medvideokamera. Utifrån observationsschemat och inspelningarna analyserades sedanlektionerna efter en analysmetod där både lärarens sätt att ställa frågor och sätt att följaupp frågorna kategoriserades. Resultatet visade att läraren i viss utsträckning fördelar ordet i klassrummet baserat påom eleverna har svenska som förstaspråk (SVE) eller som andraspråk (SVA). De frågorsom ställde högst krav på den eleven som skulle svara, gick som regel till en elev medsvenska som förstaspråk. Dessutom valde läraren ofta att ställa sina frågor rakt ut iklassen vilket gjorde att några få elever, två SVE-elever och en SVA-elev, besvarade enstor del av den totala mängden frågor. Samtidigt var läraren mån om att ge positivåterkoppling på eleverna svar och såg till att ta sig tid att hjälpa eleverna fram till rättsvar på frågan både i fall där elevens svar var svårt att förstå rent språkligt eller därsvaret var felaktigt.

Hepatitis C virus (HCV) is a serious global health problem with an estimate of 170 million carriers worldwide. Most individuals exposed to this blood-borne pathogen develop chronic infection, which may result in severe liver complications as well as end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options are suboptimal with no effective vaccines available to date. Development of a readily accessible mouse model that is permissive to natural HCV infection is important to facilitate drug and vaccine discovery, and also to better understand the viral pathogenesis. The inherent difficulty is that HCV displays very limited tropism, infecting only livers from humans or chimpanzees. An attempt was made to elucidate the key determinants in rendering the murine intracellular environment permissive to HCV replication. The results revealed that deletion of the interferon regulatory factor-3 and overexpression of microRNA-122 can independently enhance viral subgenomic replication in murine fibroblasts, with microRNA-122 being the stronger determinant. Interestingly, the phenotype established by these genetic manipulations was insufficient to support full-length HCV genome replication. Murine hepatic cell lines, with or without microRNA-122 expression, were also non-permissive to genomic HCV replication, despite the fact that translation of viral RNA was observed. These results suggest that additional host-specific factor(s) are required to support replication of full-length HCV RNA. These studies provide insight on the essential factors capable of influencing permissiveness of rodent cells to HCV replication, and also suggest genetic modifications to be considered when modeling the complete viral life cycle in a rodent animal model.