Increased airway smooth muscle (ASM) mass contributes to airway hyperresponsiveness (AHR) in asthma and is orchestrated by growth factors, cytokines and chemokines. Airway contractile responses are influenced by neuromediators, such as acetylcholine, and glutamate released by parasympathetic and sympathetic airway nerves. Hyperactivity of these neural elements, termed neurogenic inflammation, is linked with hypercontractility and AHR. Glutamate is a non-essential amino acid derivative, and its physiological role is traditionally considered with respect to its being the primary excitatory neurotransmitter in brain, and regulation of neuronal development and memory. In allergic inflammation, immune cells including dendritic cells, neutrophils and eosinophils, constitutively synthesize and release glutamate, which signals through activation of glutamate receptors, most important among which are ionotrophic N-methyl D-aspartate receptors (NMDA-R). We hypothesized that glutamatergic signaling mediated through NMDA-Rs plays an important role in inducing functional Ca2+ responses in human (H) ASM cells that can underpin airway hypercontractility. We investigated the expression and function of NMDA-Rs in HASM cells, and assessed the effects of pro-inflammatory cytokines on NMDA-R expression and functional responses. Moreover, we measured airway responses to NMDA in mice, murine thin cut lung slice preparations, and floating collagen gels seeded with HASMs. Our data reveal that airway myocytes express multi-subunit NMDA-R complexes that function as receptor-operated calcium channels (ROCCs), mobilizing intracellular Ca2+ in ASM in vitro and airway contraction ex vivo. Individual airway myocytes treated with NMDA-R agonist exhibit disparate temporal patterns of intercellular Ca2+ flux that can be partitioned into four discrete function sub-groups. Further we show that tumor necrosis factor (TNF) exposure modulates NMDA-R subunit expression, and these changes are associated with a shift in the distribution of myocytes in individual Ca2+-mobilization sub-groups in vitro. Further, post-TNF exposure, NMDA-R agonists’ treatment induced Ca2+-dependent airway dilation in murine lung slice preparations, an effect that was prevented by co-treatment with inhibitors of nitric oxide synthase (NOS) or cyclooxygenase (COX). Taken together, we conclude that NMDA-R regulate HASM-mediated airway contraction and their role can be affected upon exposure to asthma-associated inflammatory mediators. Thus, NMDA-Rs are of relevance to mechanisms that determine airway narrowing and AHR associated with chronic respiratory diseases. / October 2015

<p>Positron Emission Tomography, PET, can be used for dynamic studies in humans. In such studies a selected part of the body, often the whole brain, is imaged repeatedly after administration of a radiolabelled tracer. Such studies are performed to provide sequences of images reflecting the tracer’s kinetic behaviour, which may be related to physiological, biochemical and functional properties of tissues. This information can be obtained by analyzing the distribution and kinetic behaviour of the administered tracers in different regions, tissues and organs. Each image in the sequence thus contains part of the kinetic information about the administered tracer. </p><p>Several factors make analysis of PET images difficult, such as a high noise magnitude and correlation between image elements in conjunction with a high level of non-specific binding to the target and a sometimes small difference in target expression between pathological and healthy regions. It is therefore important to understand how these factors affect the derived quantitative measurements when using different methods such as kinetic modelling and multivariate image analysis.</p><p>In this thesis, a new method to explore the properties of the noise in dynamic PET images was introduced and implemented. The method is based on an analysis of the autocorrelation function of the images. This was followed by proposing and implementing three novel approaches for application of Principal Component Analysis, PCA, on dynamic human PET studies. The common underlying idea of these approaches was that the images need to be normalized before application of PCA to ensure that the PCA is signal driven, not noise driven. Different ways to estimate and correct for the noise variance were investigated. Normalizations were carried out Slice-Wise (SW), for the whole volume at once, and in both image domain and sinogram domain respectively. We also investigated the value of masking out and removing the area outside the brain for the analysis. </p><p>The results were very encouraging. We could demonstrate that for phantoms as well as for real image data, the applied normalizations allow PCA to reveal the signal much more clearly than what can be seen in the original image data sets. Using our normalizations, PCA can thus be used as a multivariate analysis technique that without any modelling assumptions can separate important kinetic information into different component images. Furthermore, these images contained optimized signal to noise ratio (SNR), low levels of noise and thus showed improved quality and contrast. This should allow more accurate visualization and better precision in the discrimination between pathological and healthy regions. Hopefully this can in turn lead to improved clinical diagnosis. </p>

Positron Emission Tomography, PET, can be used for dynamic studies in humans. In such studies a selected part of the body, often the whole brain, is imaged repeatedly after administration of a radiolabelled tracer. Such studies are performed to provide sequences of images reflecting the tracer’s kinetic behaviour, which may be related to physiological, biochemical and functional properties of tissues. This information can be obtained by analyzing the distribution and kinetic behaviour of the administered tracers in different regions, tissues and organs. Each image in the sequence thus contains part of the kinetic information about the administered tracer. Several factors make analysis of PET images difficult, such as a high noise magnitude and correlation between image elements in conjunction with a high level of non-specific binding to the target and a sometimes small difference in target expression between pathological and healthy regions. It is therefore important to understand how these factors affect the derived quantitative measurements when using different methods such as kinetic modelling and multivariate image analysis. In this thesis, a new method to explore the properties of the noise in dynamic PET images was introduced and implemented. The method is based on an analysis of the autocorrelation function of the images. This was followed by proposing and implementing three novel approaches for application of Principal Component Analysis, PCA, on dynamic human PET studies. The common underlying idea of these approaches was that the images need to be normalized before application of PCA to ensure that the PCA is signal driven, not noise driven. Different ways to estimate and correct for the noise variance were investigated. Normalizations were carried out Slice-Wise (SW), for the whole volume at once, and in both image domain and sinogram domain respectively. We also investigated the value of masking out and removing the area outside the brain for the analysis. The results were very encouraging. We could demonstrate that for phantoms as well as for real image data, the applied normalizations allow PCA to reveal the signal much more clearly than what can be seen in the original image data sets. Using our normalizations, PCA can thus be used as a multivariate analysis technique that without any modelling assumptions can separate important kinetic information into different component images. Furthermore, these images contained optimized signal to noise ratio (SNR), low levels of noise and thus showed improved quality and contrast. This should allow more accurate visualization and better precision in the discrimination between pathological and healthy regions. Hopefully this can in turn lead to improved clinical diagnosis.

Lokale Pyramidenzellen sind die Hauptneurone des Hippocampus und können durch ihre Position und die Morphologie ihrer Dendriten als CA1 und CA3 Pyramidenzellen identifiziert werden. Die Dendriten der exzitatorischen Pyramidenzellen sind mit postsynaptischen Vorwölbungen, den so genannten Spines, bedeckt, welche in einem spezifischen Verteilungsmuster angeordnet sind. Neurotoxine wie das C3 Toxin von Clostridium botulinum sind funktionelle Substanzen, die die neuronale Morphologie verändern und die neuronale Funktion beeinflussen können. In dieser Studie wurden die morphologischen Veränderungen von intrazellulär mit Biocytin gefüllten CA1 Pyramidenzellen qualitativ und quantitativ analysiert. Die hippocampalen Schnittkulturen, in denen sich bekanntermaßen Pyramidenzellen ähnlich entwickeln wie in vivo, wurden dazu herangezogen, die Effekte der C3bot Toxin-Applikation auf die Verzweigung der Dendriten sowie Anzahl und Dichte der dendritischen Spines zu untersuchen. Drei Gruppen von Zellen wurden verglichen: Erstens Neurone, die in serumhaltigem Medium inkubiert worden waren, zweitens Nervenzellen, die in einem Medium ohne Serum inkubiert worden waren und drittens Zellen, die unter Serumentzug dem C3bot Toxin ausgesetzt worden waren. Die Inkubation dauerte 14 Tage, während die Dauer der Toxinexposition zwischen vier und sechs Stunden betrug. Mit Hilfe eines Computers wurden zweidimensionale Nachbildungen der biocytin-markierten CA1 Pyramidenzellen erstellt, und die Gesamtlänge der Dendriten, die Anzahl der dendritischen Verzweigungspunkte und die Gesamtzahl und Dichte der dendritischen Spines gemessen und statistisch ausgewertet. Signifikante Unterschiede wurden zwischen der mit C3 Toxin behandelten Gruppe und der serumhaltig inkubierten Kontrollgruppe beobachtet. Diese signifikanten morphologischen Veränderungen traten selektiv an den Apikaldendriten der toxinbehandelten CA1 Pyramidenzellen auf. Aus der Behandlung resultierte eine Reduktion der Anzahl apikaler Verzweigungspunkte, der Anzahl der apikalen Spines, der Gesamtzahl (basal und apikal addiert) der Spines sowie der Gesamtspinedichte. Im Gegensatz dazu ergaben sich keine signifikanten Unterschiede zwischen der toxinbehandelten Gruppe und der ohne Serum inkubierten Kontrollgruppe, obwohl der Serumentzug im Vergleich zur serumhaltig inkubierten Kontrollgruppe die Entwicklung der Zellen beeinflusste. Auf Grundlage der beobachteten Veränderungen können wir schließen, dass die Behandlung mit C3 bot einen starken Einfluss selektiv auf die Morphologie der Apikaldendriten ausübt. Der Mechanismus, der dieser selektiven Empfindlichkeit der Apikaldendriten gegenüber dem C3 bot Toxin zugrunde liegt, wird Gegenstand weiterer Untersuchungen sein.

Maschinelle Beatmung kann neben den gewünschten Effekten eine vorbestehende Lungenerkrankung weiter aggravieren und sogar das Lungenparenchym zuvor lungengesunder Patienten schädigen. Mit Hilfe der quantitativen Computertomographie (qCT) können pathologische Belüftungszustände und gegebenenfalls durch maschinelle Beatmung verursachte Schäden analysiert werden. Solche auf der qCT basierende Analysen der Lungenbelüftung werden jedoch potentiell durch CT-Akquisitions- und Bildrekonstruktionsparameter beeinflusst. Um die Ergebnisse vor allem von Analysen des überbelüfteten Lungenvolumens richtig bewerten zu können, müssen solche Einflüsse untersucht werden. Bei 10 Versuchstieren (Schweine) wurden bei einem konstanten Atemwegsdruck von 25 cm H2O zuerst bei gesunder Lunge und dann erneut nach experimenteller Lungenschädigung CT-Bildserien mit zwei unterschiedlichen Strahlendosen angefertigt. Von diesen Rohdaten wurden Bildserien mit unterschiedlichen Rekonstruktionsparametern angefertigt und in jeder dieser Bildserien das überbelüftete Lungenvolumen bestimmt. Sowohl die Schichtdicke, der Filter als auch die Stromstärke hatten einen signifikanten Einfluss auf das eigentlich konstante überbelüftete Lungenvolumen, der jedoch nur teilweise klinisch relevant war. Bei der Interpretation von Messungen des überbelüfteten Lungenvolumens sollten dennoch die Einflüsse der genannten Parameter beachtet und für Vergleichsuntersuchungen gleiche Parametereinstellungen verwendet werden. Eine Dosisreduktion scheint dabei für Messungen des überbelüfteten Lungenvolumens praktikabel.

Κύριος στόχος της εργασίας ήταν η συγκριτική μελέτη της παλίνδρομης αναστολής μεταξύ του ραχιαίου και του κοιλιακού πόλου του ιππόκαμπου αρουραίου. Χρησιμοποιήθηκε η μεθοδολογία της in vitro διατήρησης τομών ιππόκαμπου και εξωκυττάριων καταγραφών προκλητών δυναμικών πεδίου. Τα αποτελέσματά μας έδειξαν ότι η GABAA εξαρτώμενη παλίνδρομη αναστολή είναι ασθενέστερη, έχει μικρότερη διάρκεια και φθίνει πιο γρήγορα στον κοιλιακό σε σχέση με το ραχιαίο ιππόκαμπο. Χρησιμοποιώντας διάφορα φάρμακα που δρουν ενισχυτικά στον GABAA υποδοχέα δείξαμε ότι υπάρχει λειτουργική διαφοροποίηση του GABAA εξαρτώμενου ανασταλτικού μηχανισμού μεταξύ των δύο πόλων του ιππόκαμπου, ενισχύοντας την υπόθεση της λειτουργικής διαφοροποίησης στο επίπεδο του υποδοχέα μεταξύ των δύο πόλων. Στην in vivo μελέτη, χρησιμοποιώντας το μοντέλο επαγωγής επιληπτικών κρίσεων με χορήγηση πεντυλενοτετραζόλης, δείξαμε ότι η ενίσχυση της GABAA εξαρτώμενης αναστολής απο τα κατασταλτικά φάρμακα συσχετίζεται με το μέγεθος της αντιεπιληπτικής τους δράσης. Επιπλέον, η βιταμίνη D δεν παρουσίασε αντιεπιληπτική δράση στους C57BL/6J μύες, ούτε ενίσχυσε την αναστολή, κάτι που δείχνει ότι δεν έχει επίδραση στον GABAA υποδοχέα ή, τουλάχιστον, στους υπότυπούς του στον ιππόκαμπο. / The major aim of this work was the comparative study of recurrent inhibition between the dorsal and ventral pole of the rat hippocampus. We used the methodology of in vitro maintenance of hippocampal slices and recording of evoked field potentials. We showed that the GABAA mediated recurrent inhibition is weaker, lasts less and decays faster in ventral than in dorsal hippocampus. Using various drugs that act as positive allosteric modulators of the GABAA receptor, we showed that there is a functional differentiation of the GABAA inhibitory mechanism between the two hippocampal poles, strengthening the hypothesis of the functional differentiation at the level of the receptor between the two poles. In the in vivo study, using the pentylenetetrazole model for inducing epileptic seizures, we showed that the enhancement of the GABAA mediated recurrent inhibition correlates with the strength of antiepileptic action of the sedative drugs used. In addition vitamin D did not show antiepileptic action in C57BL/6J mice. Moreover it didn’t enhance recurrent inhibition, showing that it doesn’t have any action on the GABAA receptor or, at least, on its subtypes in hippocampus.

No description available.

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Perfect slice sampling is a method to turn Markov Chain Monte Carlo (MCMC) samplers into exact generators for independent random variates. The originally proposed method is rather slow and thus several improvements have been suggested. However, two of them are erroneous. In this article we give a short introduction to perfect slice sampling, point out incorrect methods, and give a new improved version of the original algorithm. (author's abstract) / Series: Preprint Series / Department of Applied Statistics and Data Processing

Affective disorders represent one of the greatest global burdens of disease. Work in patients with affective disorders demonstrates that serotonin (5-HT) signaling within the prefrontal cortex, particularly at the level of the 5-HT receptors, plays an integral role in both the pathology and treatment of these diseases. Surprisingly, the characterization of the prefrontal 5-HT receptors under both healthy and pathological conditions remains incomplete. The technique of whole cell electrophysiological recording provides an unparalleled tool for investigating the functional effects of these 5-HT receptors on neurons in acute prefrontal cortical slices. The objectives of my thesis were to delve deeper into the 5-HT receptor subtypes that modulate the prefrontal cortex in the healthy control rodents and to examine how this modulation was disrupted in a rodent model of affective disorders. In work from healthy control rodents, I examined two prefrontal 5-HT receptor-mediated currents. I show for the first time the presence of the 5-HT1A receptor during the early postnatal period, a critical developmental window during which this receptor programs adult anxiety behaviors. In adulthood, I characterized an inhibitory current mediated by the 5-ht5A receptor; findings that will permit the classification of this receptor within the 5-HT receptor family. Collectively, this investigation of functional early 5-HT1A receptors and adult 5-ht5A receptors offers a novel conceptual framework for understanding 5-HT receptor modulation of the healthy prefrontal cortex. To model vulnerability to affective disorder in the rodent, I used the early stress of maternal separation. In early stress rodents, I observed a marked increase in 5-HT1A receptor currents during the early postnatal period, the critical time window for the programming of anxiety. By comparison, in adulthood I found that rodents exposed to early stress displayed increased 5-HT2A receptor currents. These findings provide novel insight into the developmental and long-lasting pathology underlying early stress, indicating that the early prefrontal 5-HT1A receptor and adult prefrontal 5-HT2A receptors as a potential therapeutic target in treatment of affective disorders At a fundamental level, the findings provided herein offer critical insight into the cellular mechanisms underlying affective disorders, one of the most debilitating and costly diseases worldwide.