RAGE (receptor for advanved glycation endproducts) was identified on endothelial cells as binding partner for AGE-modified molecules. The term "Advanced glycation endproducts" involves a number of structurally diverse molecules, which derive from multiple complex rearrangements of reducing sugars with free amino-groups of proteins. They evolve during food production and also in vivo during ageing and to an accelerated degree in diabetes, where AGEs cause receptor-mediated cellular perturbations. Due to the pathological relevance the aim of this thesis was to generate a "biosensor" for AGEs. To this end, the membrane-expressed receptor (flRAGE) as well as soluble RAGE (sRAGE) were expressed in mammalian cells and investigated in numerous binding studies. These did not reveal a specific interaction of AGE-modified ligands with RAGE. In addition, the expression of RAGE on endothelial cells, as described in the literature, could not be followed neither with the help of newly generated monoclonal anti-RAGE antibodies, nor in quantitative "real time" RT-PCR analysis. These results cast doubts on the meaning of RAGE as a proinflammatory receptor in AGE-mediated pathologies and on the adequacy of RAGE for the "biosensor". At the same time the question concerning a physiological role of the receptor arose. RAGE-expression was analysed in different healthy human tissues by "real time" RT-PCR, which revealed an almost selective expression in lung tissue. An important indication for a possible physiological function of RAGE in lung provided the selective localization of RAGE on alveolar epithelial type I cells as demonstrated in frozen lung sections as well as in in vitro cultivated lung cells. RAGE could be identified as a novel, highly specific marker for the thin, expanded AT I cell, which form part of the air-blood-barrier. In the following, RAGE was found to be an interaction partner for collagen IV, a major component of the alveolar basal lamina. Membrane-expressed RAGE did not only strengthened adherence of cells but also induced cell spreading on collagen IV-coated surfaces. This preferential interaction of RAGE with collagen IV could substantially contribute to the functional morphology of AT I cells in vivo, thereby ensuring an effective bidirectional gas-exchange. The results of this thesis expose a novel, so far unnoticed aspect of the biology of RAGE, which presumably contributes to the phenotypic characteristic und function of normal human lung tissue. / RAGE (receptor for advanced glycation endproducts) wurde als Interaktionspartner auf Endothelzellen für AGE-modifizierte Moleküle identifiziert. Unter den "Advanced glycation endproducts" werden eine Vielzahl strukturell unterschiedlicher Moleküle zusammengefasst, die durch mehrstufige komplexe Umlagerungen zwischen reduzierenden Zuckern und freien Aminogruppen von Proteinen entstehen. Sie entstehen sowohl bei der Herstellung von Lebensmitteln, als auch in vivo während des Alterns und in erhöhtem Maß bei Diabetes, wobei sie Rezeptor-vermittelt Zellstörungen hervorrufen. In der vorliegenden Arbeit wurde zunächst aufgrund der pathologischen Relevanz eine Strategie zur Konzeption eines "Biosensors" für AGEs verfolgt. Hierfür wurde sowohl der membranständige Rezeptor (flRAGE) als auch löslicher RAGE (sRAGE) in Säugerzellen exprimiert und in zahlreichen Bindungs- und Funktionsanalysen getestet. Hierbei konnte keine spezifische Interaktion der AGE-modifizierten Moleküle mit RAGE nachgewiesen werden. Auch die in der Literatur beschriebene Expression von RAGE auf Endothelzellen konnte mit Hilfe neu generierter monoklonaler Antikörper, sowie in quantitativen "real time" RT-PCR-Analysen nicht nachvollzogen werden. Diese Ergebnisse warfen Zweifel an der grundlegenden Bedeutung von RAGE als proinflammatorischer Rezeptor in AGE-bedingten Krankheiten auf und stellten damit auch dessen Eignung für einen AGE-Biosensor in Frage. Gleichzeitig warf diese Skepsis die Frage nach einer möglichen physiologischen Funktion dieses Rezeptors auf. Eine vergleichende Analyse der RAGE-Expression in verschiedenen gesunden Geweben mittels "real time" RT-PCR ergab eine nahezu selektive Expression in Lungengewebe. Wichtige Anhaltspunkte für die Funktion von RAGE in der Lunge ergaben sich aus der selektiven Lokalisation des Rezeptors auf Alveolarepithelzellen Typ I (AT I) sowohl in Gefrierschnitten der Lunge als auch nach in vitro-Kultur von Lungenzellen. RAGE konnte als neuer, hoch spezifischer Marker für die lang gestreckten AT 1 Zellen, die einen Teil der Blut-Luft-Schranke bilden, definiert werden. In folgenden Funktionsanalysen konnte RAGE als spezifischer Interaktionspartner für Kollagen IV, einer Hauptkomponente der Alveolar-Basalmembran, identifiziert werden. Membranständiger RAGE verstärkte nicht nur die Adhärenz von Zellen an Kollagen IV-beschichtete Oberflächen, er induzierte auch Zell-"Spreading". Dies gab Anlass für die Vermutung, dass die beobachtete präferentielle Interaktion von RAGE mit Kollagen IV maßgeblich zu der funktionellen Morphologie der AT I Zellen in vivo beitragen könnte, die die Voraussetzung für einen effektiven bidirektionalen Gasaustausch darstellt. Durch die Ergebnisse dieser Arbeit wurde ein neuer, bisher unbeachteter Aspekt der Biologie des RAGE aufgedeckt, der vermutlich entscheidend zur phänotypischen Ausprägung und Funktion des normalen humanen Lungengewebes beiträgt.

The presented thesis summarizes the results of research on craniofacial morphology in patients with facial cleft defects in relation to therapeutic approaches (Caganova et al., 2014; Dadáková et al., 2016; Hoffmannova et al., 2016; Hoffmannova et al., 2018; Moslerová et al., 2018). The effect of therapy in individuals with pathological growth disorders cannot be evaluated without detailed auxological studies of control subjects whose facial morphology, longitudinal changes, or manifestations of sexual dimorphism were evaluated upon similar methodology (Koudelová et al. 2015). Therefore, the thesis was conceived as a volume of six publications complemented with a general synthetic introduction into the area of study. Together, the thesis includes probands in a broad age spectrum from birth to 15 years with a total of 294 facial 3D scans, 36 tele-X-ray face images, 3D scans of 112 gypsum palate castings. The methods of geometric morphometry and multidimensional statistics prevail in the assessment. The main clinical part of the thesis deals with the influence of two types of surgery on the facial growth and development of patients with cleft palate, namely secondary spongioplasty (SS) and neonatal cheiloplasty (NCH). Neonatal cheiloplasty (NCH) is the surgery whose effects were studied from several...

Intravital microscopy (IVM) is a well-established imaging technique for real-time monitoring of microscale lung tissue dynamics. Although accepted as a gold standard in respiratory research, its characteristic image features are scarcely understood, especially when trying to determine the actual position of alveolar walls. To allow correct interpretation of these images with respect to the true geometry of the lung parenchyma, we analyzed IVM data of alveoli in a mouse model in comparison with simultaneously acquired optical coherence tomography images. Several IVM characteristics, such as double ring structures or disappearing alveoli in regions of liquid filling, could be identified and related to the position of alveoli relative to each other. Utilizing a ray tracing approach based on an idealized geometry of the mouse lung parenchyma, two major reflection processes could be attributed to the IVM image formation: partial reflection and total internal reflection between adjacent alveoli. Considering the origin of the reflexes, a model was developed to determine the true position of alveolar walls within IVM images. These results allow thorough understanding of IVM data and may serve as a basis for the correction of alveolar sizes for more accurate quantitative analysis within future studies of lung tissue dynamics.

Récemment reconnus comme cancérogènes certains pour l'homme par l’IARC, la pollution atmosphérique et les particules fines (PM₂.₅) peuvent être inhalées et pourraient être retenues au niveau pulmonaire ou passer dans la circulation systémique. Ceci peut causer ou renforcer de nombreuses pathologies auxquelles les personnes âgées sont souvent plus sensibles. Cette thèse s’inscrit dans une démarche d’identification des processus impliqués dans la modulation du potentiel cancérogène des PM₂.₅, en lien avec l’âge ou le statut tabagique. Les particules ont été collectées à Dunkerque, agglomération présentant des influences maritimes mais également caractérisée par des activités industrielles et un trafic automobile importants. Pour évaluer l'influence de l'âge, des lymphocytes sanguins prélevés chez 90 patients issus de trois classes d'âge (25-30, 50-55 et 75-80 ans) ont été exposés ex vivo à des PM₂.₅ d’origine urbaine. Les lymphocytes isolés ont été exposés aux PM₂.₅ pendant 72 heures, avant de mesurer l'activité télomérase et la modulation d'expression de gènes tels que P16INK4A et MGMT. Les PM₂.₅ entraînent des variations de l'activité télomérase et de la longueur des télomères dans toutes les tranches d'âge indifféremment. L’expression du gène P16INK4A est significativement augmentée avec l'âge après exposition aux PM₂.₅. L'âge augmenterait l'expression du gène MGMT après exposition aux particules, en diminuant le niveau de méthylation de son promoteur uniquement dans le groupe des patients les plus âgés. Concernant le rôle du statut tabagique, 26 lavages broncho-alvéolaires ont été réalisés chez des patients fumeurs et non-fumeurs. Les macrophages issus de ces prélèvements ont été mis en culture avec des cellules épithéliales bronchiques BEAS-2B, avant exposition aux PM₂.₅ (3 et 15 µg/cm², 72 h). L’activité télomérase et la longueur des télomères varient après exposition aux PM2.5 et le statut tabagique modifie ces paramètres dans les cellules BEAS-2B et les macrophages alvéolaires. La méthylation des promoteurs et l’expression des gènes P16INK4A et MGMT ne sont pas modifiées dans les cellules BEAS-2B, alors que dans les macrophages alvéolaires les particules induisent l’expression de ces gènes par une diminution de la méthylation de leurs promoteurs. Le statut tabagique fumeur semble au contraire accroître la méthylation et limite l’expression de ces deux gènes. En conclusion, il apparaît que l’échantillon de PM₂.₅ étudié peut induire ex vivo plusieurs lésions décrites dans les étapes d’initiation et de promotion de la cancérogenèse broncho-pulmonaire. L’âge et le tabagisme sont susceptibles de moduler les effets toxiques des particules. Alors que les symptômes du cancer du poumon apparaissent seulement à une étape avancée de la maladie, nos résultats pourraient aider à la découverte de nouveaux marqueurs de diagnostic précoce permettant ainsi d’améliorer la survie. / Recently recognized as carcinogenic to human by IARC, air pollution and fine particulate matter (PM₂.₅) can be inhaled and could be retained into the lung or reach the systemic circulation. This can cause or worsen many diseases for which the elderly are often more sensitive. The PhD objective corresponds to the identification of the mechanisms of action involved in the modulation of carcinogenic potential of PM₂.₅, in connection with age or smoking status. PM₂.₅ were collected in Dunkerque, a French seaside city characterized by important industrial activities and heavy motor vehicle traffic. In order to estimate the influence of age, blood lymphocytes sampled from 90 patients from age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to PM₂.₅ during 72 hours, before evaluation of telomerase activity and gene expression modulation of P16INK4A and MGMT. PM₂.₅ modulated telomerase activity and telomeres length in all age groups without any influence of age. P16INK4A gene expression increased significantly with age after exposure to PM₂.₅. Age could enhance MGMT gene expression after exposure to particles by decreasing the level of promoter methylation in the oldest group. Regarding the role of smoking status, 26 broncho-alveolar lavage were performed in smoker and non-smoker people. Macrophages were cultured with bronchial epithelial BEAS-2B cells before PM₂.₅ exposure (3 or 15µg/cm²; 72h). The telomerase activity and telomere length vary after exposure and the tobacco modify these parameters in BEAS-2B cells and alveolar macrophages. Methylation of P16INK4A and MGMT genes promoters and their expression are not modified in BEAS-2B cells. In alveolar macrophages, particles lead to a decrease of methylation of P16INK4A gene promoter. The smoking status seems also to increase methylation and to down-regulate expression of these two genes. In conclusion, it seems that the studied PM₂.₅ sample can induce ex vivo modifications described in the initiation and promotion of lung carcinogenesis. The age and smoking status may modulate the toxic effects of particles. Since lung cancer symptoms appear only at an advanced stage, our results could help in proposing new biomarkers of carcinogenesis allowing an early diagnosis to improve survival.

Le « porcine respiratory disease complex » (PRDC) est un trouble multifactoriel dû à une infection simultanée ou séquentielle de divers micro-organismes pouvant intensifier ou prolonger les signes cliniques des porcs. On retrouve dans ce complexe Mycoplasma hyopneumoniae, un des agents initiateurs du PRDC et agent primaire de la pneumonie enzootique (EP) chez les porcs. Streptococcus suis est l’un des agents pathogènes secondaires du PRDC, c’est également un agent pathogène important induisant principalement des méningites, des septicémies et la mort subite des porcelets post-sevrés. Mycoplasma hyorhinis est également l’un des agents pathogènes secondaires du PRDC, et va induire des inflammations sérofibrineuses chez les porcelets. Comme ces trois pathogènes sont retrouvés au sein du PRDC et au niveau des voies respiratoires supérieures des porcs, il pourrait exister un effet positif des mycoplasmes sur la pathogénèse de S. suis. C’est pourquoi différentes expériences in vitro ont été réalisées avec les cellules épithéliales porcines (NPTr), les macrophages alvéolaires porcins (PAMs) et les cellules dendritiques porcines (BM-DCs) qui ont été pré-infectés par les mycoplasmes puis infectés avec S. suis. Il a été observé que la cytotoxicité et l’inflammation des cellules porcines ont été significativement augmentées lorsqu’elles ont été pré-infectées par les mycoplasmes puis infectées par S. suis. Cependant, la pré-infection des cellules n’a pas joué de rôle sur l’adhésion et l’invasion de S. suis, sur la phagocytose et la survie intracellulaire de la bactérie. Cette étude semble montrer que la pré-infection des mycoplasmes pourraient induire un contexte inflammatoire favorisant la pathogenèse de S. suis. / Porcine respiratory disease complex (PRDC) is a multifactorial disorder due to simultaneous or sequential infection with various microorganisms that can intensify or prolong clinical signs in pigs. Included in this complex is Mycoplasma hyopneumoniae, one of the initiating agents of PRDC and the primary agent of enzootic pneumonia (EP) in pigs. Streptococcus suis is one of the secondary pathogens of PRDC and is also an important pathogen, mainly causing meningitis, septicemia, and sudden death in post-weaned piglets. Mycoplasma hyorhinis is also one of the secondary pathogens of PRDC and will also induce serofibrinous inflammation in piglets. As all three pathogens are found in the PRDC and in the upper respiratory tract of pigs there may be a positive effect of mycoplasma on the pathogenesis of S. suis. Therefore, different in vitro experiments were performed with newborn pig tracheal cells (NPTr), primary porcine alveolar macrophages (PAMs) and porcine bone-marrow-derived dendritic cells (BM-DCs) that were pre-infected with mycoplasma and then infected with S. suis. It was observed that cytotoxicity and inflammation of pig cells were significantly increased when they were pre-infected with mycoplasma and then infected with S. suis. However, pre-infection of the cells did not play a role in the adhesion and invasion of S. suis and in the phagocytosis and intracellular survival of the bacteria. This study suggests that pre-infection with mycoplasma may induce an inflammatory context favoring the pathogenesis of S. suis.

Aim: The aim of this literature study was to systematically review the scientific evidence on the most effective donor sites and/or bone substitute material for secondary alveolar cleft grafting in alveolar cleft patients. Material and method: In order to acquire a systematic and transparent reporting this literature review was conducted according to the PRISMA statement. The literature search was performed in the following four databases; PubMed, CENTRAL, Web of Science and Scopus.The quality of the included studies was assessed using the revised Cochrane Risk of Bias 2 tool (RoB 2 tool). Result: The search identified 4754 studies. Five RCT studies was included in this systematic review and assessed different donor site or bone substitute materials. Two studies showed low risk of bias and three moderate risk of bias. Only one study showed a statistically significant difference when comparing iliac bone to substitute material however all studies presented substitute materials with satisfactory results. Conclusion: According to the data from this systematic review no clear conclusion can be drawn regarding what the most effective bone donor site and/or tissue engineered bone substitute material to use in secondary bone grafts. Based on the available evidence iliac bone could still be regarded as a benchmark, but more research and RCT’s of high quality are required, especially for artificial bone substitute materials. / Syfte: Syftet med denna litteraturstudie var att systematiskt granska den vetenskapliga evidensen gällande det mest effektiva bentagningsstället och/eller bensubstitutmaterialet vid sekundär bentransplantation hos patienter med käkspalt. Material och metod: För att uppnå en systematisk och transparent rapportering av denna litteraturstudie följdes PRISMA statement. Litteratursökningen gjordes i följande fyra databaser; PubMed, CENTRAL, Web of Science och Scopus. Kvaliteten av inkluderade studier granskades med hjälp av Cochrane Risk of Bias 2 tool (Rob 2 tool). Resultat: Sökningen identifierade 4754 studier. Fem RCT studier inkluderades i denna systematiska översikt vilka värderade olika bentagningsställen eller bensubstitut. Två studier bedömdes ha låg risk för bias och tre artiklar måttlig risk för bias. Endast en studie visade på en statistiskt signifikant skillnad vid jämförelse av höftben med bensubstitut däremot presenterade samtliga studier substitutmaterial med tillfredsställande resultat. Konklusion: Denna systematiska översikt visade att ingen klar slutsats kan dras gällande vilken det mest effektiva bentagningsstället eller bensubstitutsmaterialet är för sekundär bentransplantation hos patienter med käkspalt. Baserat på tillgänglig evidens kan transplantat från höftbenet fortfarande anses vara bäst lämpat men mer forskning samt RCT studier av hög kvalité erfordras, särskilt för artificiella bensubstitutmaterial.

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Background: Mechanical ventilation (MV) inflicts stress on the lungs, initiating or increasing lung inflammation, so-called ventilator-induced lung injury (VILI). Besides overdistention, cyclic opening-and-closing of alveoli (atelectrauma) is recognized as a potential mechanism of VILI. The dynamic stretch may be reduced by positive endexpiratory pressure (PEEP), which in turn increases the static stretch. We investigated whether static stretch modulates the inflammatory response of rat type 2 alveolar epithelial cells (AECs) at different levels of dynamic stretch and hypothesized that static stretch increases pro-inflammatory response of AECs at given dynamic stretch. - Methods: AECs, stimulated and not stimulated with lipopolysaccharide (LPS), were subjected to combinations of static (10, 20, and 30%) and dynamic stretch (15, 20, and 30%), for 1 and 4 h. Non-stretched AECs served as control. The gene expression and secreted protein levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) were studied by real-time polymerase chain reaction (RTqPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The effects of static and dynamic stretch were assessed by two-factorial ANOVA with planned effects post-hoc comparison according to Šidák. Statistical significance was considered for p < 0.05. - Results: In LPS-stimulated, but not in non-stimulated rat type 2 AECs, compared to nonstretched cells: 1) dynamic stretch increased the expression of amphiregulin (AREG) (p < 0.05), MCP-1 (p < 0.001), and MIP-2 (<0.05), respectively, as well as the protein secretion of IL-6 (p < 0.001) and MCP-1 (p < 0.05); 2) static stretch increased the gene expression of MCP-1 (p < 0.001) and MIP-2, but not AREG, and resulted in higher secretion of IL-6 (p < 0.001), but not MCP-1, while MIP-2 was not detectable in the medium. - Conclusion: In rat type 2 AECs stimulated with LPS, static stretch increased the proinflammatory response to dynamic stretch, suggesting a potential pro-inflammatory effect of PEEP during mechanical ventilation at the cellular level.