Texte remanié de: Diss.--Philosophie--Paderborn--Universität-Gesamthochschule Paderborn, 1995. / Bibliogr. p. 135-144.

The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.

Nous avons montré un rôle d’un canal K+, le canal SK3, dans la migration des cellules cancéreuses de sein MDA-MB-435s et le développement de métastases ostéolytiques du cancer du sein. Lors de l’ostéolyse, la [Ca²+]ext augmente dans le microenvironnement osseux. Nous avons voulu déterminer si cette élévation de [Ca²+]ext, pouvait moduler l’expression et l’activité du canal SK3. Nous avons montré que l’augmentation de la [Ca²+]ext: i) favorise l’expression du canal SK3. Cet effet fait intervenir le récepteur au calcium (CaSR), qui en diminuant la [AMPc]int réduit l’activité de la PKA et lève ainsi son inhibition de la transcription du gène KCNN3 (codant pour SK3) ; ii) favorise la migration cellulaire dépendante du canal SK3, mécanisme impliquant également le CaSR ; iii) active le canal SK3 qui, par ailleurs, voit son activité réduite par l’élévation d’AMPc intracellulaire. De plus, l’augmentation d’AMPc délocalise un canal calcique partenaire de SK3, le canal Orai1, et diminue l’entrée constitutive de Ca²+ et la migration dépendantes du canal SK3. En conclusion, nos résultats montrent que l’expression et l’activité de SK3 sont régulées par l’AMPc et le Ca2+ extracellulaire. Ceci permet d’envisager une nouvelle stratégie thérapeutique ciblant l’AMPc pour le traitement des métastases osseuses du cancer du sein. / We showed that a K+ channel, SK3 channel, is a mediator of MDA-MB-435s breast cancer cells migration and of osteolytic bone metastasis development of breast cancer. Since [Ca²+]out rises during osteolysis, in bone microenvironment, we study if this [Ca²+]out elevation could modulate SK3 expression and activity. We show that [Ca²+]out elevation: i) increases SK3 expression threw CaSR activation which, in turn, decreases [cAMP]int and PKA activation, leading to loss of its inhibitory effect on KCNN3 transcription; ii) increases SK3-dependent migration threw CaSR activation; iii) increases SK3 channel activity that is in addition, decreased by [cAMP]int elevation. Furthermore, cAMP elevation moves the Ca2+ channel Orai1 (SK3 partner) outside of lipid rafts and reduces the SK3 dependent-constitutive Ca²+ entry and cell migration. Our results show that both SK3 expression and activity are regulated by cAMP and extracellular Ca²+. These results underscore an innovative opportunity to use therapeutic approaches targeting cAMP for the treatment of breast cancer bone metastasis.

L'adénosine monophosphate cyclique (AMPc) est une molécule très largement conservée dans le monde du vivant. Chez les bactéries, cette molécule signal est impliquée, entre autres, dans l'adaptation aux changements de milieu, au développement de la virulence, à la sporulation et la compétence. Bien qu'il fut démontré dans les années 1970 que cette molécule nécessitait un transporteur afin de traverser la membrane cellulaire, l'identité de celui-ci restait jusqu'à très récemment inconnue. Ce n'est qu'en 2011 que Hantke et ses collègues démontrèrent l'implication de la protéine TolC dans l'export de l'AMPc. Au cours de ma thèse, j'ai démontré le rôle prépondérant du complexe Opp dans l'import d'AMPc chez Escherichia coli. Ce complexe, formé des protéines OppABCD et F, est connu pour transporter des oligopeptides et se trouve dans la membrane interne de nombreuses bactéries. Pour arriver à la conclusion de son implication dans le transport d'AMPc, une banque de mutants a été créée dans une souche ne produisant pas d'AMPc (cyaA-). Afin d'identifier le transporteur responsable de l'import de l'AMPc, différents cribles qui testent la présence de cette molécule dans la cellule, ont ensuite été réalisée sur ces clones. Après analyse des résultats obtenus, l'opéron opp a été pressenti comme étant nécessaire à l'entrée de l'AMPc dans la cellule. Le double mutant cyaA-oppA- a été créé et de nouvelles expériences sont venues confirmer notre hypothèse. La protéine OppA a ensuite était surexprimée et purifiée pour nous permettre de faire des tests biochimiques d'interaction entre AMPc et OppA, basés sur la fluorescence émise par la protéine elle-même ou un homologue de l'AMPc, l'ε-AMPc. Ces expériences nous ont permis de confirmer l'interaction entre la protéine OppA et l'AMPc. Le système Opp est donc le principal importateur d'AMPc chez Escherichia coli, mais il ne semble pas être l'unique transporteur impliqué. Un autre candidat très intéressant est le complexe Dpp, connu pour transporter les dipeptides. En effet, les expériences préliminaires effectuées démontrent une diminution de la concentration intracellulaire d'AMPc dans une cellule cyaA-dppA- comparée à celle observée dans une souche cyaA-. Les travaux réalisés pendant ces trois dernières années permettent de conclure que le complexe Opp est le principal importateur d'AMPc, très probablement secondé par le complexe Dpp. / Cyclic adenosine monophosphate (cAMP) is a signalling molecule conserved in all reigns of life. In bacteria, cAMP plays an important role in processes as diverse as the adaptation to a changing environment, the control of virulence, sporulation and competence. Although it has been proven in the 1970s that this molecule needs an active transporter to traverse the plasma membrane, the first one of these transporters was discovered only a few years ago. In 2011, Hantke et al have shown that the TolC protein is involved in the efflux of cAMP. During my PhD work I have identified the Opp complex as a major player of cAMP import into Escherichia coli. This complex, composed of proteins called OppABCD and F, is known to transport oligopeptides across the inner membrane of numerous bacterial species. To prove the involvement of the Opp complex in cAMP transport, I have used transposon mutagenesis to generate a collection of random mutants in a strain that does not produce cAMP (cyaA-). Different screens were used to detect mutants with impaired transport of extracellular cAMP into the cell. The opp operon emerged as the most promising candidate from this screen. The double mutant cyaA-oppA- was constructed and experiments designed to test the function of OppA confirmed our hypothesis. Subsequently, I overexpressed and purified OppA in order to perform biochemical experiments destined to measure the physical interaction between cAMP and OppA. I show that the Opp system is the major importer of cAMP in Escherichia coli. However, it seems that Opp is not the unique importer of cAMP. The other, very interesting candidate is the complex Dpp, known to transport dipeptides. Preliminary experiments revealed a decreased amount of cAMP in strain cyaA-dppA- compared to strain cyaA-. The experiments carried out during the last three years allow us to conclude that the Opp complex is the major importer of cAMP into E. coli and that the Dpp complex is probably a secondary transporter.

This thesis posits that the scout camp can fruitfully be studied as a technological environment, more specifically as an infrastructure of the simple life. Mainly based upon periodicals, handbooks, still and moving pictures (from the Swedish boy scouts 1910–1930, but with international outlooks) it draws upon the Foucauldian notion of the apparatus (dispositif) as well as theory on modern infrastructure. Particular attention is payed to the role of work, leisure and community building at and around the camp site.       The simplification of the camp site – socially as well as technologically – was strategic in the sense that it enabled other goals and functions on a larger, societal level. To some extent it can also be seen as a critique of a growing complexity in modern life. A shared infrastructural sensibility finally grew out of the camp life, connecting scouts on a scale that stretched from the individual to the world.

This research aims to find architectural and urban design related solutions to enhance the living conditions of refugees who are trapped in protracted refugee situations. One key issue that could be considered the main problem-generating factor in protracted refugee situations, is the lack of a clear definition for the appropriate deign life-span of refugee camps in general. As an example of that refuge situation, focus within this research is upon the two refugee camp models found in Jordan; the Zaatari camp and the Azraq camp. Both were established in response of the recent civil war in Syria, started in 2011. A comparison between the two refugee camps resulted in identifying the strong points and the shortcomings of the current models. In addition, an assessment of the refugees’ needs is made based on the refugee camp’s design guidelines and the official reports published by the different humanitarian organizations. The above mentioned researched information is utilized to develop and apply an adaptable design solution that aims to overcome the unpredictable future of the refugee situation in Azraq refugee camp in Jordan. The proposal includes a long-term plan that may develop over time and which is divided to three stages depending on the longevity of the refugee camp. Each stage takes into consideration the changing requirements and needs that the refugees develop over time.

Rational agent behaviour is frequently achieved through the use of plans, particularly within the widely used BDI (Belief-Desire-Intention) model for intelligent agents. As a consequence, preventing or handling failure of planned activity is a vital component in building robust multiagent systems; this is especially true in realistic environments, where unpredictable exogenous change during plan execution may threaten intended activities. Although reactive approaches can be employed to respond to activity failure through replanning or plan-repair, failure may have debilitative effects that act to stymie recovery and, potentially, hinder subsequent activity. A further factor is that BDI agents typically employ deterministic world and plan models, as probabilistic planning methods are typical intractable in realistically complex environments. However, deterministic operator preconditions may fail to represent world states which increase the risk of activity failure. The primary contribution of this thesis is the algorithmic design of the CAMP-BDI (Capability Aware, Maintaining Plans) approach; a modification of the BDI reasoning cycle which provides agents with beliefs and introspective reasoning to anticipate increased risk of failure and pro-actively modify intended plans in response. We define a capability meta-knowledge model, providing information to identify and address threats to activity success using precondition modelling and quantitative quality estimation. This also facilitates semantic-independent communication of capability information for general advertisement and of dependency information - we define use of the latter, within a structured messaging approach, to extend local agent algorithms towards decentralized, distributed robustness. Finally, we define a policy based approach for dynamic modification of maintenance behaviour, allowing response to observations made during runtime and with potential to improve re-usability of agents in alternate environments. An implementation of CAMP-BDI is compared against an equivalent reactive system through experimentation in multiple perturbation configurations, using a logistics domain. Our empirical evaluation indicates CAMP-BDI has significant benefit if activity failure carries a strong risk of debilitative consequence.

No description available.

The diploma thesis is The Theme of the War in Czech Prose Published in 1948-56. Following significant changes after the 1948 war, several thematic forms of Czech prose that are analyzed. After the 1948 war began to be interpreted as a precursor to a future socialist revolution, which had to be the "correct" stories - there were used books M. Pujmanové - Život proti smrti (1952) and B. Březovského - Lidé v květnu (1954). They were compares with dilogií A. Brandalda, Severní nádraží (1949) and Lazaretní vlak (1950). Another section is devoted to prose since 1954, when the artistic treatment of the subject of the war reflected their own experiences of war, concentration camps and life in the Protectorate - K. Ptáčník - Ročník jedenadvacet (1954), F. Burian - Osm odtamtud (1954), N . Frýd - Krabice živých (1956), E. Valenta - Jdi za zeleným světlem (1956). The aim of this thesis is to analyze war themes in the works of these authors in the designated time.

This diploma thesis deals with the interpretation of fiction with the theme of the Holocaust. An integral part of the work is a methodological chapter, which is focused on the issues of hermeneutics and other issues that needed to be taken into account when handling the chosen theme. This thesis further focuses on the description of the historical context, which ultimately played a significant role. The main part of the work consists of an independent analysis of selected works of Czech and foreign authors and their subsequent comparison.