This thesis deals with a specific group of historical sites called World Heritage, which are registered on the UNESCO World Heritage List. It characterizes the basic elements of the UNESCO World Heritage idea as a cultural and social process, which engages in acts of remembering that work to create ways to understand, present and interpret the past within the present social frameworks. The thesis brings to the attention a range of viewpoints about the nature of heritage in general and the UNESCO World Heritage in specific that have emerged in the relatively new area of academic interdisciplinary heritage studies. For the purposes of exploring different aspects of the World Heritage phenomenon, following themes of heritage discourse have been developed: remembering and making public use of the past, the UNESCO approach and the ways of presenting and interpreting the past. In order to give a sense of what World Heritage looks like on the ground this thesis details some aspects of three different World Heritage sites. To study such matters in the physical world a strategy of undertaking case studies of three specific UNESCO sites and employing the methods of direct observation and a fieldwork were chosen. Closer examinations of the Czech medieval town of Kutná Hora, the Hiroshima Peace Memorial Park...

Because of the lasting impact that traumatic family events can have on psychological well-being, students who present for services at college counseling centers may be experiencing problems and symptoms associated with earlier trauma. Many college counseling centers utilize the Counseling Concerns Survey developed by the Research Consortium of Counseling and Psychological Services in Higher Education (1991) and the Outcome Questionnaire-45 (OQ-45; Lambert et al., 1996) as intake instruments to assess students who present for counseling. The major components of the Counseling Concerns Survey are the 18-item Family Experiences Questionnaire, which identifies history of family trauma, and the 42-item Presenting Problems List, which assesses students' major areas of distress. The OQ-45 measures symptom severity. While it is generally assumed that family trauma during childhood and adolescence can negatively impact future mental health and well-being, it has been unclear how specific traumatic family experiences reported on the Family Experiences Questionnaire are related to specific presenting problems as listed on the Presenting Problems List or symptom severity as measured by the OQ-45. The purpose of this study was to examine this relationship and to ascertain discernible patterns. Data from the intake instruments of 20,495 students who sought counseling services at a large western U.S. university from 1997 to 2007 was analyzed. Logistic regression of each of the 18 traumatic family history experiences was performed, using the initial OQ-45 score, the 42 Presenting Problems List items, and five Presenting Problems List factors (Draper, Jennings, & Baron, 2003) as "predictors" of the types of trauma the students might have experienced. Results showed that although family trauma of a variety of types was associated with symptom severity and various presenting problems, there did not seem to be an overall discernible pattern. The results suggest that trauma seems to have a diffuse association with presenting problems and symptom severity. However, some family traumas are associated with a greater number of presenting problems, and these traumas were identified.

Clinical and experimental studies have shown that estradiol (E2) can enhance protection against sexually transmitted infections such as HSV-2 and HIV-1. Antigen presenting cells (APCs) such as Dendritic cells (DCs) are critical for generating immune responses against these infections, and it is unclear whether unique factors present in the genital mucosa can influence immune responses by directly modulating the phenotype and function of local APCs. To address this, I hypothesized that sex hormones, such as E2 and innate factors in the local microenvironment can regulate the phenotype and function of vaginal APCs. The work summarized in this thesis addressed this central hypothesis. In the first section of the thesis, I examined whether vaginal APCs were distinct in their phenotype and function compared to those in other mucosal tissues or spleen. The results show that the vagina was enriched in CD11c+ CD11b+ MHCII− DCs. Functionally, vaginal tissue cells (TC) and CD11c+ DCs were more potent inducers of Th17 responses in co-cultures with CD4+ T cells, compared to lung, small intestine or spleen APCs. E2 was critical for the conditioning of vaginal DCs to prime these Th17 responses through an IL-1-dependent pathway, indicating that sex hormones such as E2 can directly influence the function of vaginal APCs. In the next section, I determined whether other co-factors in the genital microenvironment such as microflora and innate lymphocytes could also influence vaginal APC functions. We found that while microflora was not essential, IL-17 produced by innate lymphocytes was critical for the induction of IL-1 from DCs, and consequently for potentiating Th17 responses. Finally, I attempted to develop an in vivo mouse model where the effect of E2 on vaginal APCs could be examined in the context of genital HSV-2 infection. I tested a 7-day injectable E2 and a 21-day E2 pellet delivery model, and found that both regimes had limitations for examining E2-effects on anti-viral responses. Yet, subsequent to the work done in this thesis, we were able to confirm our observations of E2-conditioned Th17 responses in vivo in an intranasal immunization model utilizing E2 pellet delivery, and thereby addressed the mechanism underlying enhanced anti-viral protection following E2-treatment. In conclusion, this is the first study to show the effect of E2 on genital tract APCs and their ability to prime Th17 responses. It provides future avenues to examine whether modulation of this microenvironment can help optimize vaccine-induced immune responses against STIs. On a more fundamental level, it highlights the need to consider the inherent distinctions in APC populations among different mucosal tissues. / Dissertation / Doctor of Philosophy (PhD)

CD8+ T cells are critical to the adaptive immune response and are a target for vaccine development. However, the complex dynamics of cell proliferation can vary response success, providing uncertainty when designing vaccines. Computer models can provide clarity by simulating these dynamics, tracking millions of cell-cell interactions, a feat that is impractical experimentally. Our group created the STORE.1 model, a probabilistic simulation of the CD8+ T cell response to vaccination. While able to accurately simulate in vivo mouse T cell clonal expansion, intracellular dynamics are absent. Furthermore, there is no mechanism by which cell division ceases. This work builds upon the STORE.1 model by systematically explaining the division dynamics of CD8+ T cells and providing measures of the extracellular environment. The new STORE.2 model has demonstrated an ability to accurately simulate differences in CD8+ T cell expansion in WT mice and mice lacking type I conventional dendritic cells up to 170 hours after vaccination. It is the first model to simulate individual cell cycle regulator protein counts for millions of cells, and the resulting impact on pH for the extracellular microenvironment. Finally, it provides a partial mechanism behind division cessation, an important element for future models seeking to further simulate the end of the T-cell response. / Thesis / Master of Applied Science (MASc) / T-cells are an important component of the human immune system, but currently, there are no vaccines in clinical use that are designed to target them. This is because there are many different dynamics that underpin how T-cells activate, and to what degree they can replicate into a substantial pool of pathogen-clearing cells. Learning which candidate vaccines can properly elicit a strong T-cell response is time and resource consuming. Mathematical models can therefore speed development of candidate vaccines by virtually testing their T-cell responsiveness. This thesis works to improve on an existing mathematical model by introducing immunological mechanisms that determine how T-cells undergo cell division, change the acidity of their immediate surroundings, and respond to their own growing population. By doing so, this new model can be more representative of the immunological reality and begin to probe new dynamics of the T-cell response.

<h4> </h4> / <p>Asthma is an inflammatory disorder of the airways, and there has been growing insight into the cellular and molecular mechanisms underlying the inflammatory basis of this disease. Research into the inflammatory mechanisms of asthma has progressively shifted focus from downstream effectors, such as mast cells and eosinophils, up to Th2 lymphocytes and their proallergic cytokines. Even more upstream in the allergic cascade are dendritic cells (DCs), potent APCs that orchestrate immune responses. Evidence supporting a role of DCs in regulating airway allergic inflammation is derived mainly from animal studies. In animal models of asthma, myeloid DCs (mDCs) induce and maintain airway inflammation, while plasmacytoid DCs (pDCs) mediate tolerance and lung homeostasis. It remains uncertain, however, whether this concept of pro-allergic mDCs and anti-allergic pDCs translates from animal to human models. The overall objective of this thesis was to investigate the biology of DC subsets in allergen-induced asthma in asthmatic subjects. Initially, we demonstrate that both mDCs and pDCs increase in the airways of subjects with mild asthma after allergen inhalation. Next, we describe a distinct subpopulation of mDCs, called mDC2s, and demonstrate their association with allergy and asthma severity. Expanding on these findings, we show that mDC2s increase in the airways of mild asthmatics after allergen challenge. Lastly, we explore the potential of pharmacological therapies, anti-OX40L MAb and anti-TSLP MAb, to affect DCs in subjects with mild asthma, and demonstrate no effect of either drug on circulating DC subsets. The studies presented here provide evidence for multiple DC subtypes being involved in the regulation of allergen-induced inflammatory responses, and support continued investigations into the biology of different DC subsets in allergen-induced asthma.</p> / Doctor of Philosophy (Medical Science)

In zahlreichen Studien konnte an Mausmodellen gezeigt werden, dass dendritische Zellen eine wichtige Rolle im Rahmen der mukosalen Immunabwehr spielen. Eine unkontrollierte Aktivierung immunologischer Effektorzellen durch antigenpräsentierende Zellen ist die Folge, welche die Antigene der luminalen Flora folglich falsch erkennen und damit zu einer Schädigung des Gewebes führen. In der Arbeit wurden humane CD1c+CD11c+CD14-CD19- myeloide dendritische Zellen (mDCs) aus dem peripheren Blut und der intestinalen Mukosa von CED Patienten sowie von gesunden Probanden phänotypisch und funktionell näher charakterisiert. mDCs von Patienten reagieren auf LPS im Gegensatz zu DCs von Gesunden mit der Ausbildung eines aktivierten Phänotyps und der Sekretion pro-inflammatorischer Zytokine. Die Daten lassen vermuten, dass ihre tolerogene Rolle gestört ist und die Zellen so möglicherweise aktiv zum Entzündungsgeschehen durch eine Fehlreaktion auf die kommensale Flora beitragen. Es konnte gezeigt werden, dass zirkulierende mDCs von Erkrankten mehr LPS aufnehmen. Des Weiteren ist die Häufigkeit von mukosalen und aktivierten mDCs bei CED Patienten signifikant erhöht. Die vermehrte Häufigkeit von aktivierten mDCs in der entzündeten Mukosa ist ein Hinweis auf intestinales „homing“, also ein Wiedereinwandern der gereiften Lymphozyten in die Darmwand. Es ist bekannt, dass die Hefe Saccharomyces boulardii (Sb) eine Wirksamkeit bei entzündlichen sowie infektiösen Erkrankungen des Gastrointestinaltraktes hat. Kulturexperimente von mDCs mit Zellkulturüberständen von Sb (SbS) und LPS zeigten eine deutliche Reduzierung in der Expression von CD40 und CD80 sowie des Reifemarkers CD197. SbS reduzierte die Sekretion von TNF- und IL-6. Während es die Sekretion von IL-10 bei gesunden Probanden erhöhte, konnte bei CED Patienten eine leichte Abnahme verzeichnet werden. SbS vermindert die Proliferation von naïven T-Zellen in einer gemischten Lymphozytenreaktion mit gesunden mDCs signifikant. / Various animal studies have provided insights that mucosal dendritic cells play a key role in this process. However, the specific function of certain dendritic cells in IBD is still unknown. Primary CD1c+CD11c+CD14-CD19- myeloid blood (mDCs) and mucosal DCs from IBD patients and healthy controls were compared. More mDCs from IBD patients exhibited an activated phenotype shown by expression of co-stimulatory molecules. mDCs from patients secrete higher levels of pro- and anti-inflammatory cytokines. Circulating mDCs from IBD patients take up more LPS and the frequency of mucosal mDCs and the number of activated, i.e. CD40 and CD80 expressing mucosal mDCs, is significantly greater in CED. The increased frequency of activated mDCs in the inflamed mucosa suggests intestinal homing of mDCs in acute stages of IBD. Further, the data suggests an aberrant LPS response of mDCs in patients suffering from IBD which results in an inflammatory phenotype. The most widely accepted hypothesis for the cause of IBD is a disturbed interaction of the host immune system with commensal microflora and other luminal antigens. The well controlled balance of the intestinal immune system is disturbed and luminal antigens like LPS gain access to the underlying mucosal tissue via the leaky barrier. It was investigated whether the yeast preparation Saccharomyces boulardii (Sb) modulates dendritic cell function which has shown efficacy in inflammatory and infectious disorders of the gastrointestinal tract. Culture experiments of mDCs in the presence of Sb culture supernatant (SbS) significantly reduced the expression of CD40 and CD80 as well as the DC maturation marker CD197 (CCR7) induced by the prototypical microbial antigen LPS. SbS reduced secretion of TNF- and IL-6, while the secretion of anti-inflammatory IL-10 increased. IBD patients showed also a reduction in their secretion level of IL-10. SbS inhibited proliferation of naïve T cells in a mixed lymphocyte reaction with healthy mDCs.

Background Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. Methodology/Principal Findings Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. Conclusions/Significance In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.

Le respect de l’équilibre entre lymphocytes T effecteurs auto-réactifs et lymphocytes T régulateurs (LTreg) est primordial dans le maintien de la tolérance aux antigènes du soi. Les partenaires cellulaires et les mécanismes moléculaires impliqués dans la rupture de l’équilibre de cette balance ne sont pas ou peu connus dans les maladies auto-immunes. Ainsi, les travaux décrits dans cette thèse portent sur le dérèglement de la balance T effecteurs/ Treg dans deux modèles de maladies auto-immunes chez l’homme: le lupus érythémateux systémique et l’anémie hémolytique auto-immune (AHAI). Nous montrons une augmentation de l’expression de la molécule de costimulation OX40L (CD252, TNFSF4) à la surface des cellules présentatrices d’antigène circulantes et infiltrant les tissus chez les patients lupiques. Cette augmentation est corrélée à l’activité de la maladie chez l’adulte comme chez l’enfant. Elle a pour conséquence l’induction de lymphocytes T effecteurs de type Tfh (T follicular helper) et le blocage des fonctions suppressives des Treg, deux acteurs majeurs dans la physiopathologie du lupus. Dans le second projet, nous montrons une augmentation de la proportion de T8reg circulants chez les patients affectés d’une AHAI à anticorps chauds en phase de rémission. Ces Treg expriment le CD25, le FoxP3 et exercent leur fonction suppressive par un mécanisme faisant intervenir l’IL10. De faibles doses d’IL-2 permettent l’expansion de cette population cellulaire in vitro. Ces résultats apportent de nouvelles connaissances dans la physiopathologie de ces deux maladies et offrent des perspectives thérapeutiques potentielles. / Respect of the balance between autoreactive T cells and regulatory T cells (LTreg) is important to maintain tolerance to self-antigens. Cellular partners and molecular mechanisms involved in the disruption of this balance are not or little known in autoimmune diseases.Thus, the work described in this thesis focuses on the disruption of the T effector/ Treg balance in two models of human autoimmune diseases: systemic lupus erythematosus and autoimmune hemolytic anemia (AIHA). We show an increased expression of the OX40L (CD252, TNFSF4) costimulatory molecule at the surface of both circulating and tissues-infiltrating antigen presenting cells in SLE patients. OX40L expression is correlated with disease activity in adults and in children and results in Tfh (follicular helper T) effector cells induction and Treg suppressive functions inhibition, two key mechanisms in the pathogenesis of lupus. In the second project, we show an increase of the circulating T8reg proportion in patients with a warm AIHA in a non-active state. These Treg express CD25, FoxP3 and exert their suppressive function by a mechanism involving IL-10. Low-dose IL-2 allows the expansion of this cell population in vitro. These results provide new insights into the pathophysiology of these diseases and offer potential therapeutic perspectives.

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

L'utilisation des nanobiotechnologies dans le domaine de la santé est en plein essor. Les nanoparticules de poly(acide lactique) (PLA) représentent un nanosystème biocompatible capable d'accroître la spécificité et l'efficacité de traitements thérapeutiques et vaccinaux administrables par voie muqueuse et intraveineuse. Toutefois, l'optimisation de ces nanosystèmes se heurte à une caractérisation incomplète de leur biodistribution in vivo, en particulier à l'échelle cellulaire.L'objectif de ce travail de thèse est d'enrichir les connaissances sur la biodistribution des nanoparticules de PLA in vivo après administration muqueuse ou intraveineuse, dans le but d'élargir les perspectives d'optimisation et d'utilisation. Animal complexe et adapté pour les études sur organisme-entier, le modèle du poisson-zèbre (Danio rerio) a été utilisé. Pour mener à bien ce projet, une méthodologie rigoureuse d'analyse de la biodistribution des nanoparticules de PLA a été développée. Ce qui permit, après administration par balnéation, d'en révéler le fort tropisme inné envers les cellules dendritiques muqueuses. Ces données ont servi à élaborer une stratégie de ciblage, utilisant la lectine agglutinine de cacahuète, capable d'augmenter la prise en charge des nanoparticules de PLA par les branchies et la peau. Enfin, l'étude du devenir de ces nanoparticules après injection intraveineuse, a révélé de nombreuses interactions avec le système circulatoire. Ce travail a permis d'approfondir la connaissance des interactions des nanoparticules de PLA avec le vivant, soulignant le potentiel prometteur de ces nanoparticules pour la vaccination muqueuse / Medecine shows a growing interest regarding nanobiotechnologies. Among them are poly(lactic acid) (PLA) nanoparticles, which represent a biocompatible and competent nanosystem to heighten the specificity and efficacy of diverse therapeutic and vaccine treatments, following mucosal and intravenous administration. However, the further optimization of such nanosystem is poised by the lack of informations regarding their in vivo biodistribution, especially at the cellular level.The main objective of this PhD is to increment the knowledge about PLA nanoparticles biodistribution in vivo, after muquous and intravenous administration, to further expand their optimisation and use perspectives. The zebrafish model has been utilized to perform this research because of his conserved complexity as well as his suitability for whole-organism studies.To fulfill this project, a precise methodology has been developed to analyze the PLA nanoparticles biodistribution. Which allowed, after bathing administriation, to unveil their robust innate tropism toward mucous dendritic cells. From these data has been established a targeting strategy, utilizing the peanut agglutinin lectin, which has been proved to enhance nanoparticle uptakes by both gills and skin mucosae. Finally, the study of PLA nanoparticles behavior and destiny after intravenous injection, revealed numerous elaborated interactions with the circulatory system.Overall, this work has been able to strengthen our understandings of PLA nanoparticles among living organisms, furthermore highlighting their promizing potential as nanovehicles for mucosal vaccines