Im Fokus dieser Diplomarbeit steht ein korreliertes Anderson Modell. Unser Modell beschreibt kurzreichweitige Einzelplatzpotentiale, wobei negative Korrelationen zugelassen werden. Für dieses korrelierte Modell wird mittels der fraktionalen Momentenmethode im Falle genügend großer Unordnung exponentieller Abfall der Greenschen Funktion bewiesen. Anschließend wird daraus für den nicht korrelierten Spezialfall Anderson Lokalisierung bewiesen. / This thesis (diploma) is devoted to a correlated Anderson model. Our model describes short range single site potentials, whereby negative correlations become certified. For this correlated model exponential decay of the Greens' function is proven in the case sufficient large disorder according to the fractional moment method. Subsequently, we prove Anderson localization for the not correlated special case.

This thesis operates, unashamedly, from the premise that every act of criticism involves a self-reflexive gesture of one's own concerns and ideological imprintings. For this reason Chapter One establishes the writer's own involvement - both autobiographical and theoretical - in notions of male rage and the 'working through' of these concerns. Chapter Two conducts an overview of male rage and the extant systemic literature on the subject. It sets out the various positions on the subject and posits the importance of gender (over generation) in the praxis of therapy. Furthermore, it explores the possibility that the male is equally, but differently, troubled by the hegemonic forces of patriarchy as is the woman. Without diminishing the legitimacy of the woman's experience in the face of male rage, the argument is forwarded that the male is caught in a similar struggle but without the feminine articulatory resources. This chapter details the lack of male power in the face of his supposed muscular omnipotence. Seminal analytic approaches to the question of gender are raised in Chapter Three. Working through Freud, Klein, Lacan and Masters and Johnson an attempt is made to plot the 'evolution' of the feminine and the masculine. Central to this debate is the bi-polarization of gender relations within the same sex (biology/construction) and without (phallic/vaginal, clitoral, passive/active). What emerges is that femininity is bi-focal and that the woman has more resources at her disposal that hitherto acknowledged. While the woman is always double - as both clitoral and vaginal, as lover and mother- it appears that male sexuality is far more precarious than generally perceived. It is this dis-ease on the part of the male that translates itself into envy and, with it, the need to denigrate and belittle woman as the object of that envy. In Chapter 4 an attempt is made to overlap the seemingly divergent fields of analytic and systemic methodologies via the involvement of the therapist in the eco-system of analysis. The substantial role of the therapist -- and the coercive forces placed on him/her by the couple -- is used to modify Elkaim's model and to introduce the need for a telling of the particular stories that concentrate on the unique narratives of the warring couple rather than the patriarchal regime under which these stories are constrained. Before encountering these narratives an essay is made at establishing a methodology of sorts. Newton's scientific formulations are used in order to question the binary opposition that has been, historically, established between quantitative (male) and qualitative (female) methodologies. In the process of questioning this binary opposition it becomes clear that any form of objectifying approach constitutes a refuge from the messiness that is intrinsic to the therapeutic process. The experimental methodology that is posited is precisely one that engages in the narratives of male violence - four extracts are considered, each exposing different articulations of male violence. The question of female subjectivity (and the attendant power of the sorority) is returned to in light of these stories. Central to this section is the notion that male subjectivity is far more convoluted - perhaps more that the feminine counterpart - than initially conceived. The original identification with the (m)other forever displaces him in that the later identification with the father remains distant and contrived. For the purposes of maintaining the dialogic nature of this work, a feminist appraisal of the rage narratives concludes the thesis. Don Quixote is used, by way of an Epilogue, to offer three representations of male subjectivity and to look towards alternative subject positions for the male under patriarchy. / Psychology / D.Litt. et Phil. (Psychology)

“The relationship between the criminal and victim is much more complicated than the law would care to acknowledge. The criminal and his victim work on each other unconsciously. We can say that as the criminal shapes the victim, the victim also shapes the criminal. The law differentiates distinctly between the attacker and the victim. But their relationship may be, and often is, quite close, so that their roles are reversed and the victim becomes the determining person, while the [victimiser] in the end becomes his own victim.” (Abrahamsen, 1973:35). This research was directed at establishing whether narcissistic individuals will go to extreme levels of violence, specifically murder, if their self-image is threatened. The aim was to determine the extent of pre-existing narcissistic personality disorder (NPD) in these individuals and how this contributed to the murderous action they committed. Emphasis was placed on the psychological motivation of the perpetrator, as well as the relationship that existed between the perpetrator and the victim prior to the event. As the subject of the research was a relatively unknown phenomenon, a qualitative research approach was used. The research focused on analysing specific cases of murder, more particularly cases where rage-type murders were committed. It endeavoured to identify the underlying personality dynamics to determine whether an association between rage-type murder and NPD exists. Case studies illustrating rage-type murderers who had been admitted to Weskoppies Psychiatric Hospital for a 30-day observation period were identified and analysed. These cases were selected through reviewing the case history of each individual to determine whether the murder fitted the outlined definition of a rage-type murder. The cases that met the outlined requirements were deemed suitable for the purpose of the research, where after the Minnesota Multiphasic Personality Inventory (MMPI-2) results of the selected cases were examined to determine the personality organisation of the individuals. This information was then used to determine the possible association between NPD and rage-type murder. The MMPI-2 was selected as the assessment tool as it is the most widely used personality assessment available. For the purposes of this research a two-point code type was used to indicate the presence of narcissistic personality traits. A two-point code type implies an elevation of two scales, for the purposes of this research specifically the Pd (Psychopathic deviance) scale and the Pa (Paranoia) scale, also referred to as the 4-6/6-4 code type. As interpretation based only on a two-scale elevation was considered to be overly simplistic, all the MMPI-2 clinical scales were interpreted independently, and a clinical interpretation provided in the context of each individual’s background. The 4-6/6-4 code type individual was used to indicate whether the individuals did have narcissistic personality traits, and thus were classified as having NPD. Nine cases were identified of individuals thought to be rage-type murderers, who were admitted for a 30-day period of psychiatric observation to Weskoppies Psychiatric Hospital in Pretoria. Only five cases were acknowledged as rage-type murders. All the cases selected were referred to Weskoppies Psychiatric Hospital by order of the court and involved males over the age of 20 years. The individuals involved were admitted to the Forensic Unit of the hospital and were subjected to standard psychiatric hospital observations, which included psychiatric interviews, psychological interviews, psychological testing, as well as general behavioural observations in the ward. All the information obtained during the standard psychiatric hospital observations is held in the clinical case files in the archives at the hospital. All the standard psychiatric hospital observation evaluations were completed prior to the initiation of the research, and the case records had been closed. Although more research is necessary, this research has established an association between the selected cases of rage-type murder and NPD and there is historic documented evidence suggesting that individuals with NPD will most likely react in a similar manner in similar circumstances, as a result of their underlying personality disorder. This suggests that incarceration in a correctional facility is not the most appropriate place to rehabilitate individuals. It also serves as support to why a person with NPD who commits a rage-type murder should be acquitted because of their personality disorder and subsequently be committed to a psychiatric facility as a patient of the state president. / Dissertation (MA)--University of Pretoria, 2010. / Social Work and Criminology / unrestricted

La présente thèse vise à interroger les rapports qu’entretiennent le factuel et le fictionnel dans "Ardor guerrero", "Sefarad" et "Ventanas de Manhattan" d’Antonio Muñoz Molina. En effet, si ces œuvres se présentent respectivement comme trois modalités d’une écriture factuelle, à savoir des mémoires, des témoignages et un récit de voyage, elles ne peuvent néanmoins être exemptes de fictionnalité. L’hybridation des faits et de la fiction au sein de ce corpus apparaît non seulement comme inéluctable mais aussi porteuse de sens dans la mesure où elle permet d’approcher d’une part la vision personnelle de l’auteur sur le monde et d’autre part les histoires de l’Autre dans toute sa vérité et son intimité. / This thesis aims to examine the relationships between the factual and the fictional in "Warrior’s Rage", "Sepharad" and "Windows on Manhattan" by Antonio Muñoz Molina. Indeed, if these works are presented respectively as three modalities of factual writing, that is to say memories, testimonies and a travelogue, they can’t nevertheless be free of fictionality. Hybridization of facts and fiction in this corpus is not only inevitable but also meaningful in that it allows approaching on the one hand the author's personal vision of the world and on the other hand the stories of the Other in all its truth and intimacy. / Esta tesis tiene como objetivo examinar las relaciones entre lo fáctico y lo ficticio en "Ardor guerrero", "Sefarad" y "Ventanas de Manhattan" de Antonio Muñoz Molina. De hecho, si estas obras se presentan respectivamente como tres modalidades de escritura fáctica, o sea como las memorias militares del autor, unos testimonios de perseguidos y un relato de viaje por Nueva York, no pueden sin embargo estar libres de ficcionalidad. La hibridación de los hechos y de la ficción en este corpus no sólo es inevitable, sino que permite también acercarse por un lado a la visión personal del autor sobre el mundo y por otro lado a las historias de los demás en toda su verdad y su intimidad.

We chose to base our literature study on the meme phenomena. The meme is a portion of culture that is spread among us from mind to mind. Every time we learn something by copying others we use information that has been passed on from a previous person, that information can be seen as a meme. The theoretical frame for our methodology in our exam paper is founded on Forsberg och Wengström research on literature studies. Our aim is to describe to the reader in depth what a meme is and how they could be helpful in an academic and pedagogical setting.Our study will show how memes are seen predominantly as graphic designs and as artifacts for cultural representation parallel to their imagery. This means that the cultural reference is only clear to the beholder if they have corresponding prior knowledge to the reference.Our study will also show via concrete examples how this could be used in school settings and as pedagogical tools in the classroom.

Dans l'Arctique, le risque de transmission de maladies zoonotiques comme la rage est encore une préoccupation pour la santé publique, avec plusieurs cas rapportés chaque année chez différentes espèces animales. L’interface entre la faune sauvage, les animaux domestiques et les humains pour la transmission de maladies sera en augmentation en raison des pressions grandissantes du développement anthropique et du réchauffement climatique. Les interactions directes entre les chiens domestiques des villages nordiques et les renards sauvages, les principales sources d’exposition à la rage, sont des évènements critiques pour l’exposition des humains au virus, mais très peu d’études se sont attardées à ce sujet. Les objectifs de ce projet sont 1- de décrire et de quantifier l’activité spatiale et temporelle des renards et des chiens libres et d’identifier les facteurs anthropiques et environnementaux qui influencent leur présence à proximité et dans les villages nordiques, 2- de quantifier les contacts directs et le potentiel d’interactions entre les renards et les chiens libres afin de mieux identifier les périodes et les zones avec un risque de transmission de la rage, 3- de comparer et discuter des résultats de l’activité des renards et des chiens libres et leur potentiel d’interactions entre les deux villages nordiques à l’étude et 4- d’identifier des méthodes de gestion pour diminuer les opportunités d’interactions entre les renards et les chiens libres. Afin de détecter la présence des deux carnivores, un réseau de caméras automatiques a été installé sur une grille spatiale autour et à l’intérieur de deux villages typiques du Nord, Kuujjuaq (près de la limite des arbres) et Inukjuak (à > 100 km de la limite des arbres). Pour chaque station caméra, différents facteurs anthropiques et environnementaux ont été évalués (distance au dépotoir, distance à l’aéroport, densité d’habitations, densité de chiens de traîneau, distance aux rues, distance à l’eau, indice de végétation). Les contacts directs et les indices d’interaction ont été calculés pour évaluer le potentiel d’interactions entre les renards et les chiens libres. Les opportunités d’interactions entre les renards et les chiens sont plus élevées à l’aube et au crépuscule pendant les mois d'octobre et de novembre en périphérie des deux villages, particulièrement près des chiens de traîneau et légèrement près du dépotoir municipal (KU) et de l’aéroport (IN). Les renards roux et les renards arctiques ont été observés à proximité et dans les deux villages, mais les renards roux sont plus souvent observés à Kuujjuaq (87% des observations) qu’à Inukjuak (renards arctiques : 74% des observations), ce qui reflète également des particularités biotiques et abiotiques uniques à chaque village. Les résultats suggèrent que les opportunités d’interactions entre les renards et les chiens représentent un risque d’exposition à la rage pour les chiens et les humains, et possiblement pour d’autres pathogènes nordiques transmissibles entre les chiens et la faune. Cependant, les évènements de contacts directs interspécifiques renard-chien sont rares et les opportunités d’interactions sont concentrées dans le temps et dans des zones restreintes, ce qui peut aider à cibler des mesures préventives visant à limiter les évènements de transmission. Cette étude fournit la première documentation sur l'activité des renards et leurs interactions avec les chiens libres dans les villages de l'Arctique. L’application d’une approche « Une seule Santé » devrait être utilisée pour prévenir ou diminuer le risque de transmission de la rage entre les renards et les chiens. / In the Arctic, the risk of transmission of zoonotic diseases like rabies is still a public health concern, with several cases reported each year in different animal species. The interface between wildlife, domestic animals and humans for disease transmission will be increasing due to pressures from anthropogenic development and global warming. Direct interactions between domestic dogs in northern communities and wild foxes, the main source of rabies exposure, are critical of human exposure to the virus, but very few studies have focused on this. The objectives of this project are 1- to describe and quantify the spatial and temporal activity of free-ranging dogs and foxes, and identify the anthropogenic and environmental factors that influence their presence near and within northern villages, 2- to quantify direct contacts and the potential for interactions between free-ranging dogs and foxes in order to better identify periods and areas of higher risk of rabies transmission, 3- to compare and contrast dog-fox activity and interactions between the two northern villages under study, and 4- to identify management methods to reduce opportunities for fox-dog interactions. To detect the presence of the two carnivores, we set up a network of automatic cameras near and within two typical northern villages, i.e., Kuujjuaq (near the tree line) and Inukjuak (> 100 km from the tree line). For each camera, different anthropogenic and environmental factors were evaluated (distance to the landfill, distance to the airport, density of dwellings, density of sled dogs, distance to streets, distance to water, vegetation index). Direct contacts and interaction index were calculated to assess the potential for interactions between foxes and free-roaming dogs. Interaction opportunities between foxes and dogs are more likely to occur at dawn and dusk during the months of October and November, and on the outskirts of both villages, particularly near sled dogs and slightly nearer to the landfill (KU) and the airport (IN). Red and Arctic foxes were observed in and around both villages, but red foxes were more frequently observed in Kuujjuaq (87% of observations) while Arctic foxes were more frequent in Inukjuak (74% of observations), which also reflects the biotic and abiotic particularities specific to each village. These results suggest that fox-dog contacts around and within the village are likely, posing a real risk of peri-domestic rabies transmission to dogs and humans, and possibly for other northern pathogens transmissible between wildlife and dogs. Nevertheless, interspecific direct contact events are rare and interaction opportunities are concentrated in time and space, which may help target preventive measures aimed at limiting transmission events. This study provides the first documentation on the activity of foxes and their interactions with dogs and humans in Arctic communities. Considering the potential risk-by-proximity described in our study, a “One Health” approach could be applied to prevent or lower fox-dog rabies transmission.

No description available.

Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (> 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad > 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen. / Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 > S100A1 > S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments.

Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (> 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad > 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen. / Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 > S100A1 > S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments.

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des lésions alvéolaires diffuses menant à un œdème alvéolaire lésionnel et une insuffisance respiratoire aiguë hypoxémique. Malgré les progrès récents dans la prise en charge des patients de réanimation, le SDRA reste un syndrome fréquent et associé à une morbimortalité importante. Deux mécanismes principaux du SDRA semblent associés à une mortalité plus élevée et à des réponses thérapeutiques différentes : la déficience de la clairance liquidienne alvéolaire (AFC, pour alveolar fluid clearance), l’incapacité pour l’épithélium alvéolaire de résorber l’œdème alvéolaire, et la présence d’un phénotype « hyper-inflammatoire ». Les approches pharmacologiques du traitement du SDRA restent limitées et il est nécessaire de poursuivre l’étude des voies biologiques impliquées dans la pathogénie du SDRA et dans sa résolution afin de développer des approches innovantes des prises en charge diagnostique et thérapeutique du SDRA. RAGE, le récepteur des produits de glycation avancée, est un récepteur multi-ligands, exprimé abondamment par les cellules épithéliales alvéolaires du poumon (pneumocytes), qui module de nombreuses voies de signalisation intracellulaire. De nombreuses études récentes suggèrent que sRAGE, la forme soluble principale de RAGE, pourrait servir de marqueur lésionnel du pneumocyte de type I, et que RAGE pourrait jouer un rôle-pivot dans la pathophysiologie du SDRA, en initiant et en entretenant la réponse inflammatoire alvéolaire. Nos objectifs étaient de caractériser les rôles de RAGE au cours du SDRA, grâce à une approche translationnelle combinant études cliniques et précliniques. D’abord, des études cliniques observationnelles et interventionnelles ont été conduites afin de caractériser sRAGE comme un véritable biomarqueur dans le SDRA. Ensuite, des cultures in vitro de cellules épithéliales et de macrophages, ainsi qu’un modèle expérimental in vivo de SDRA murin par instillation trachéale d’acide chlorhydrique ont été utilisés pour décrire les effets de la voie RAGE sur les mécanismes d’AFC et l’inflammation macrophagique médiée par l’inflammasome « Nod-Like Receptor family, Pyrin domain containing 3 » (NLRP3). Enfin, l’effet d’une inhibition de RAGE, par sRAGE recombinant ou par anticorps monoclonal anti-RAGE, était testée en modèle murin. Nos résultats issus des études cliniques suggèrent que sRAGE présente toutes les caractéristiques d’un biomarqueur au cours du SDRA, avec un intérêt dans le diagnostic, le pronostic et la prédiction du risque de développer un SDRA dans une population à risque. Pris ensemble, notre travail suggère que la voie RAGE joue un rôle important dans la régulation de l’atteinte pulmonaire, de l’AFC et de l’activation macrophagique au cours du SDRA. Toutefois, les mécanismes précis de cette régulation restent incertains. La forme soluble de RAGE (sRAGE), lorsqu’elle est dosée dans le plasma, présente toutes les caractéristiques d’un biomarqueur pouvant être utile en pratique clinique, mais son intérêt dans la sélection de sous-groupes (ou « phénotypes ») de patients pouvant bénéficier de traitements ciblés reste à étudier. La voie RAGE pourrait enfin représenter une cible thérapeutique prometteuse. Bien que des études de validation restent nécessaires, ces résultats pourraient ouvrir de nouvelles perspectives dans la prise en charge des patients atteints de SDRA. / The acute respiratory distress syndrome (ARDS) is associated with diffuse alveolarinjury leading to increased permeability pulmonary edema and hypoxemic respiratory failure. Despite recent improvements in intensive care, ARDS is still frequent and associated with high mortality and morbidity. Two major features of ARDS may contribute to mortality and response to treatment: impaired alveolar fluid clearance (AFC), i.e. altered capacity of the alveolar epithelium to remove edema fluid from distal lung airspaces, and phenotypes of severe inflammation. Pharmacological approaches of ARDS treatment are limited and further mechanistic explorations are needed to develop innovative diagnostic and therapeutic approaches. The receptor for advanced glycation endproducts (RAGE) is a multiligand pattern recognition receptor that is abundantly expressed by lung alveolar epithelial cells andmodulates several cellular signaling pathways. There is growing evidence supporting sRAGE (the main soluble isoform of RAGE) as a marker of epithelial cell injury, and RAGE may be pivotal in ARDS pathophysiology through the initiation and perpetuation of inflammatory responses. Our objectives were to characterize the roles of RAGE in ARDS through a translational approach combining preclinical and clinical studies. First, observational and interventional clinical studies were conducted to test sRAGE as a biomarker during ARDS.Then, cultures of epithelial cells, macrophages and a mouse model of acidinduced lung injury were used to describe the effects of RAGE pathway on AFC and inflammation, with special emphasis on a macrophage activation through NodLikeReceptor family, Pyrindomain containing 3 (NLRP3) inflammasome. Acidinjured mice were treated with an antiRAGE monoclonal antibody or recombinant sRAGE to test the impact of RAGE inhibition on criteria of experimental ARDS. Results from clinical studies support a role of sRAGE as a biomarker of ARDS, withdiagnostic, prognostic and predictive values. In addition, plasma sRAGE is correlated with a lung imaging phenotype of nonfocal ARDS and could inform on therapeutic response. Herein, we also describe in vivo and in vitro effects of RAGE activation on transepithelial fluid transport and expression levels of epithelial channels (aquaporin 5, αNa,KATPaseandαENaC) and on macrophage activation through NLRP3 inflammasome. Finally, RAGE inhibition improves AFC and decreases lung injury in vivo. Taken together, our findings support a role of RAGE pathway in the regulation of lung injury, AFC and macrophage activation during ARDS, albeit precise regulatory mechanisms remain uncertain. sRAGE has most features of a validated biomarker that could be used in clinical medicine, but whether it may help to identify subgroups (or phenotypes) of patients that would benefit from tailored therapy remains underinvestigated. Modulation ofRAGE pathway may be a promising therapeutic target, and though validation studies are warranted, such findings may ultimately open novel diagnostic and therapeutic perspectivesin patients with ARDS.