Les maladies inflammatoires de l'intestin (MII) sont caractérisées par des réponses immunitaires incontrôlées dans l'intestin. Des études génétiques ont associé un polymorphisme dans le gène de l'IL23R à la résistance aux MII. IL23R code pour la protéine de l’IL-23r, une sous-unité du récepteur à l’IL-23 (IL-23R). Ce récepteur appartient à la famille de l’IL-12R, contenant plusieurs récepteurs hétérodimériques. D’ailleurs, IL-12R et IL-23R partagent la sous-unité IL12Rb1. Néanmoins, ces deux récepteurs favorisent des réponses immunitaires distinctes (Th1 vs Th17). Ce mémoire caractérise les dynamiques d’expression cellulaires de l’IL-23R et l’IL-12R, afin d’élucider leurs rôles dans l’inflammation. Nous avons établi qu’IL-23R et IL-12R ne sont jamais co-exprimés, malgré qu’ils partagent la sous-unité IL-12Rβ1. Parmi les cellules de rates de souris, la protéine IL-23r est trouvée dans certaines cellules T TCRγδ ou T CD4+, quelques cellules B et des cellules Lti-like. La protéine IL-12Rβ2 est exprimée par quelques cellules B. L’analyse de l’expression de l’IL-23R et l’IL-12R dans différents organes révéla que la plus grande proportion de cellules exprimant l’IL-23R se retrouve dans la lamina propria de l'intestin grêle, alors que les cellules exprimant l’IL-12Rβ2 ont été retrouvées en proportion équivalente dans tous les organes lymphoïdes. Ces observations appuient les études génétiques suggérant un rôle prédominant de l’IL23R dans les intestins. Finalement, des cultures in vitro suggèrent que l’IL-23R ou l’IL-12R avaient des réactions croisées à l’IL-12 ou l’IL-23. L’étude de l’IL-23R dans les MII devrait donc être complémentée par l’étude de l’IL-12R, car les deux récepteurs pourraient avoir des rôles complémentaires. / Inflammatory bowel diseases (IBD) are characterised by uncontrolled immune responses in the gut. Genome-wide association studies (GWAS) have identified a protective polymorphism for IBD in the IL23R gene. IL23R codes for the IL-23r protein, one of the two subunits of IL-23R. IL-23R belongs to the IL-12R family, which contains many heterodimeric receptors. For example, both IL-12R and IL-23R share the IL-12Rβ1 subunit. Nevertheless, IL-12R and IL-23R are associated with different immune processes (Th1 vs. Th17). This thesis characterizes the cellular patterns of expression of both IL-23R and IL-12R, to further elucidate their roles in inflammation. We established that IL-23R and IL-12R were never co-expressed together, even though they share the IL-12Rβ2 subunit. Analysis of murine splenocytes revealed that IL-23R is expressed by some TCRγδ T-cells, a few B-cells, CD4+ T-cells and several Lti-like cells. IL-12R protein was found in a few B-cells. The analysis of IL-23R and IL-12R expression in different organs revealed that the lamina propria of the small intestine was the organ containing the largest proportion of IL-23r+ cells. IL-12R+ cells were found in constant numbers throughout the organs. Finally, in vitro cultures showed that IL-23R and IL-12R had crossed reaction to IL-12 and IL-23. Study of IL-23R in IBD should always be accompanied by IL-12R analysis, because both receptors could have complementary roles.

Hormone-dependent cancers (HDCs), such as those of the prostate, ovary, breast and endometrium, share characteristics that indicate similar underlying mechanisms of carcinogenesis. Through steroid hormone signalling on "down-stream" target genes, the growth, development and progression of HDCs are regulated. One such family of target genes, highly expressed in HDCs and regulated by steroid hormones, are the tissue kallikreins (KLKs). The KLKs are a multigene family of serine proteases involved in physiological processes such as blood pressure regulation, inflammation, and tumour development and progression via the hydrolysis of specific substrates. Although the KLK gene family is clearly implicated in tumourigenesis, the precise roles played by these genes are largely unknown. Additionally, except for the androgen-responsive genes, KLK2 and KLK3, the mechanisms underlying their hormonal regulation in HDCs are yet to be identified. The initial focus of this thesis was to examine the regulation of the kallikreins, KLK1 and KLK4, by estradiol and progesterone in endometrial and breast cancer cell lines. From these studies, progesterone clearly regulated KLK4 expression in T47D cells and therefore, the focus of the remaining studies was to further examine this regulation at the transcriptional level. An overview of the results obtained is detailed below. Human K1 and hK4 protein levels were increased by 10 nmol/L estradiol benzoate, progesterone, or a combination of the two, over 48 hours in the endometrial cancer cell line, KLE. However, these same treatments resulted in no change in KLK1 gene or hK1 protein levels in the endometrial cancer cell lines, HEC1A or HEC1B (only hK1 analysed). Progesterone treatment (0-100 nmol/L) over 24 hours resulted in a clear increase in KLK4 mRNA at the 10 nmol/L dose in the breast cancer cell line, T47D. Additionally, treatment of T47D cells with 10 nmol/L progesterone over 0-48 hr, resulted in the rapid expression of the hK4 protein at 2 hr which was sustained for 24 hr. Further analysis of this latter progesterone regulation with the antiprogesterone, RU486, over 24 hours, resulted in an observable decrease in hK4 levels at 1 µmol/L RU486. Although the estrogen and progesterone regulation of the hK1 protein was not further analysed, the data obtained for hK4 regulation in T47D cell lines, supported the premise that this gene was progesterone-responsive. The rapid expression of hK4 protein by progesterone at two hours suggests that KLK4 transcription is directly coupled to progesterone regulation, perhaps through progesterone receptor (PR) binding to progesterone-responsive regions within the KLK4 promoter or far "up-stream" regions. Thus, the following further studies were performed. To test this hypothesis, the transcription initiation site (TIS) and 5' flanking regions of the KLK4 gene in T47D cells were interrogated. Primer extension and 5' RACE identified the TIS 78 bp 5' of the putative ATG site for translation as identified by Korkmaz et al. (2001). This KLK4 gene transcript consists of only four exons, and thus excludes the pre/pro signal peptide. Although a TATA-box is not present within -25 to -30 bp 5' of the identified TIS, a number of consensus binding motifs for Sp1 and estrogen receptor half-sites were identified. It is possible that the Sp1 sites are involved in the basal levels of transcription for this gene. Additionally, a putative progesterone response element (PRE) was identified in the far "up-stream" regions of the KLK4 gene. Basal levels of transcription were observed within the KLK4 proximal promoter region when coupled to a luciferase reporter gene and transfected into T47D cell lines. Additionally, the KLK4 proximal promoter region did not induce the luciferase reporter gene expression when progesterone was added to the system, however, estradiol was inhibitory for luciferase gene expression. This suggests that the proximal promoter region of the KLK4 gene could contain functional EREs but not PREs. In keeping with this hypothesis, some ER half-sites were identified, but PR sites were not obvious within this region. The identified PRE in the far "up-stream" region of the KLK4 gene assembled the progesterone receptor in vitro, and in vivo, as assessed by electromobility shift assays and chromatin immunoprecipitation assays (EMSAs and ChIPs), respectively. The binding of the PR to the KLK4 PRE was successfully competed out by a PR antibody and not by an androgen receptor antibody, and thus confirms the specificity of the KLK4 PRE-PR complex. Additionally, the PR was recruited and assembled onto and off the progesterone-responsive KLK4 region in a cyclic fashion. Thus, these data strongly suggest that the PR represents one of the core components of a transcription complex for the KLK4 gene, and presumably also contributes to the expression of this gene. Moreover, these data suggest a functional coordination between the PR and the KLK4 progesterone-responsive region in T47D cells, and thus, provide a model system to further study these events in vivo.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.

All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.

The thesis deals with the employment of direct forms of presentation in the genre of hard news. The texts for the analysis were excerpted from the main British broadsheet newspapers. The classification of forms of presentation is based on Semino and Short (2004) and focuses on their deictic and syntactic properties, interpreted in terms of the pragmatic concepts of perspective, faithfulness claims and the role of the reported and reporting speaker. Attention is paid to various direct forms, including direct speech, free direct speech and combined forms, i.e. non-direct forms appearing with a partial direct quote. The thesis draws heavily on the work by White (1998), whose approach to genre is informed by the ideas proposed by the Sydney School and Systemic Functional Linguistics. Hard news is characterised by the orbital generic structure, consisting of the nucleus and a number of specifying satellites (White 1998). The occurrence of direct and combined forms of presentation is explained by their deictic, syntactic and pragmatic properties, the generic role and characteristic features of the nucleus and individual satellites, and the overall function of hard news. The function of hard news and forms of presentation is also discussed in terms of the more general concepts of heteroglossia, dialogue...

No description available.

The conflict between the First Amendment and the Sixth Amendment is not new nor is it easily decipherable. Both amendments appear to have absolute priority, yet they appear to conflict (Erickson, 1977). The First Amendment declares unequivocally, "Congress shall make no law...abridging the freedom of speech, or of the press[,]" while the Sixth Amendment states with equal force, "In all criminal prosecutions, the accused shall enjoy the right to a speedy public trial, by an impartial jury of the state and district wherein the crime shall have been committed..." (U.S. Constitution, Amendment I, Amendment VI). Free speech and an unrestricted press can lead to a partial jury, but a jury unbiased by the media may mean restricted speech. In the judicial system the debate about how to balance these two competing constitutional rights has raged for decades, but one critical area—the nature and characteristics of requests for judicial "gag" orders—has been largely ignored. This thesis analyzed 103 cases from the Media Law Reporter volumes 19 through 33 (approximately 1991-2005) where gag orders were requested because of pretrial publicity. Those 103 cases were evaluated for the type of case, the reason for the case, when the gag order was requested, who requested the gag order, why they requested the gag order, who opposed the gag order, why they opposed the gag order, and why the gag order was granted or denied. It was found that although the issue of gag orders and their use in trials is not settled there is a general pattern to how they tend to be used. This study found that gag orders are most commonly used by judges in serious criminal trials, particularly at the federal level. Further, these cases usually involved juries, and the targets of the gag order were the parties involved in the trial, not the press.

Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap-abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt. Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt. Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden. / Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E‑BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap-independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.

No / Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.

This study aims to draw on some of the ethical guidelines enshrined in the South African Press Code (SAPC 2007:10). This SAPC states that “the press shall be obliged to report news truthfully, accurately, fairly and in a balanced manner, without any intentional or negligent departure from the facts”. This insight is used in order to analyse the way in which the 2008 xenophobic attacks were reported in South Africa by the Sowetan and Daily Sun newspapers. Overall, the findings show that the two newspapers adhered to the South African Press Code (2007), and were ethical in their 2008 news reports. This specifically contradicts the dominant perception of most mainstream newspaper readers, who thought that, the Daily Sun is just a tabloid newspaper which “represents the lowest standard of journalism” (Wasserman 2012:1), because of its sensational crime and sex stories. / Communication Science / M.A. (Communication)