Techn. Universiẗat, Diss., 2005--München.

Universiẗat, Diss., 2004--Leipzig.

In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles and is responsible for transmitting and coordinating atrial and ventricular contractions. The AVN electrically connects the atria and ventricles of the heart and is part of the electrical conduction system. Conduction within this system is highly regulated by the autonomic nervous system. The complex neurochemical anatomy of this region has been studied extensively in mice and other small animals but not in humans. The goal of this study was to provide detailed neurochemical characterization of parasympathetic (cholinergic) and sympathetic (noradrenergic) innervation of the human AVN, which is the lead component of the conducting system. Using immunohistochemistry, we have investigated the innervation of the AVN region in samples collected from human hearts that were rejected for transplantation. Tissues were fixed in 4% paraformaldehyde, cryoprotected, and sectioned frozen at 30um thickness. Sections through the AVN were cut in the horizontal plane and collected in representative sets on charged slides. Each set of slides was stained for a different phenotypic marker using the VECTOR Elite ABC kits and ImmmPACT VIP Chromogen. To aid in locating the AVN, we used anti-Connexin-43(Cx-43). Unlike surrounding myocardium, nodal tissue lacks significant Cx-43. Cholinergic nerves were stained with anti-vesicular acetylcholine transporter (VAChT).Noradrenergic nerves were stained with anti-tyrosine hydroxylase (TH). We evaluated tissue from two patients and obtained similar results. The AVN received prominent input from cholinergic and noradrenergic nerves, with cholinergic being dominant. The AVN displayed a greater density of parasympathetic and sympathetic innervation compared to surrounding regions. Sympathetic innervation is what causes an increase in conduction speed in the AVN, while parasympathetic innervation is what causes a decrease in conduction speed. Based on previous pharmacological evidence and observation of innervation in other species, the AVN is a highly controlled area for cardiac regulation. Our research implies that parasympathetic innervation is more highly regulated in the AVN and that parasympathetic innervation could play a larger role in cardiac conduction than sympathetic innervation.

Dans le cadre de ce projet de maîtrise, le but principal était de poursuivre le développement d'un modèle de peau psoriasique reconstruit par génie tissulaire et de caractériser le rôle de l'innervation sensorielle dans le phénotype psoriasique de ces derniers. Le développement de cet équivalent vise avant tout à mieux comprendre la pathogénèse du psoriasis et potentiellement ouvrir la porte à de nouvelles voies thérapeutiques permettant de contrôler la maladie ou ses symptômes. Avec nos protocoles, nous avons pu démontrer que l'ajout d'innervation à un équivalent de peau reconstruit par génie tissulaire exacerbait le phénotype psoriasique en tout ou en partie. Jusqu'à présent, la caractérisation des cytokines étudiées dans nos protocole nous laisse croire que le facteur de croissance neuronal (NGF) est le principal médiateur de la prolifération kératinocytaire caractérisant les peaux psoriasiques.

Includes bibliography / 135 leaves, 7 leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986

La peau est un organe sensoriel qui permet notamment à l’organisme de s’adapter à son environnement. Ainsi, de nombreuses fibres nerveuses sont présentes au sein de cette peau permettant de détecter différents stimuli tels que la pression, la douleur ou encore une variation de température. A la suite d’une brûlure profonde, ces terminaisons nerveuses sont détruites et la repousse de ces fibres lors du processus de cicatrisation est inadéquate conduisant à des handicaps souvent sérieux chez les patients. Dans un premier travail, la réalisation d’une brûlure chez des animaux présentant ou non une neuropathie périphérique induite par la résinifératoxine a permis d’étudier le rôle de l’innervation lors de la cicatrisation. Chez les animaux traités, une cicatrisation plus lente et un défaut de réinnervation par rapport aux animaux contrôles étaient observés. Pour approfondir le rôle de l’innervation dans la cicatrisation, des études in vitro ont ensuite été réalisées afin d’évaluer les interactions possibles entre les cellules neuronales et les fibroblastes dermiques. Même en l’absence de contacts directs, il a été montré que les cellules neuronales sont capables d’induire la différenciation des fibroblastes en myofibroblastes. Au vue de l’implication de l’innervation sur la différenciation des fibroblastes et sur la cicatrisation, il semble important de pouvoir proposer aux patients brûlés, pour leur traitement, un nouveau concept de substitut cutané favorisant une repousse axonale fonctionnelle au sein du tissu cicatriciel. / The skin is a sensitive organ which notably allows the body to adapt to its environment. Many nerve fibers are present in the skin and are implicated in the detection of various stimuli such as pressure, pain or temperature variation. After a deep burn injury, these nerve fibers are destroyed and their regrowth during the wound healing process is imperfect which induces serious disabilities for patients. In a first study, a burn model was developed on animals presenting or not a peripheral neuropathy induced with resiniferatoxin and had allowed to study the role of sensory innervation on wound healing. On animals treated with resiniferatoxin, wound healing was delayed and nerve regeneration was imperfect compared to control animals. To further investigate the role of innervation in wound healing, in vitro studies were then performed to evaluate possible interactions between neuronal cells and dermal fibroblasts. Even in the absence of direct contacts, it has been shown that neuronal cells are able to induce the differentiation of fibroblasts into myofibroblasts. Considering the involvement of innervation on the differentiation of fibroblasts and on wound healing, it seems important to provide burned patients with a new concept of skin substitute promoting functional axonal regrowth in the scar tissue.

L’identification de signaux impliqués dans la formation des cellules endocrines pancréatiques permettra d’apporter de nouvelles connaissances sur les mécanismes régissant la différenciation des cellules endocrines et de nouvelles applications dans le domaine de la génération de cellules sécrétrices d’insuline afin de traiter des patients atteints de diabète. C’est dans ce contexte que nous avons étudié l’expression et la fonction du récepteur membranaire Glial cell line derived neurotrophic factor Family Receptor α 3 (Gfrα3) et de son ligand Artemin au cours du développement du pancréas et dans le pancréas adulte chez la souris. Des techniques de PCR quantitatives, d’hybridations in situ et d’immunofluorescences nous ont permis de caractériser l’expression de Gfrα3, d’Artn mais aussi d’autres ligands et récepteurs de cette famille. Nous avons aussi utilisé un modèle de souris perte de fonction et générer un modèle de souris transgénique sur exprimant Artn dans le pancréas afin d’étudier la fonction de la voie de signalisation Artn/Gfrα3 dans le pancréas. Nous avons ainsi découvert que le récepteur Gfrα3 est exprimé au cours du développement du pancréas par les progéniteurs endocrines, les cellules insulines-positives et glucagon-positives ainsi que les cellules embryonnaires neuronales. Au stade adulte, Gfrα3 n’est pas exprimé par les cellules à insuline et est exprimé par quelques cellules à glucagon. A ce stade, son expression est aussi observée au niveau des cellules gliales, ainsi que des neurones du système nerveux sympathique et parasympathique. Les différentes expériences de perte et de gain de fonction réalisées afin de comprendre le rôle pancréatique de Gfrα3, ont révélé que ce récepteur n’est pas essentiel à la différenciation et au maintien des cellules endocrines, ni à la formation et au maintien de l’innervation endocrine. En conclusion, nous avons découvert et caractérisé un nouveau marqueur de surface exprimé dans les cellules endocrines pancréatiques en développement. Cette caractéristique pourrait s’avérer utile pour purifier par cytométrie en flux et étudier des sous populations cellulaires générées au cours des protocoles de différentiation in vitro visant à générer de nouvelles cellules sécrétrices d’insuline pour une thérapie cellulaire du diabète. / The generation of therapeutic ß-cell from human embryonic stem cells relies on the identification of growth factors that faithfully mimic pancreatic ß-cell development in vitro. In this context, the aim of the study was to determine the expression and function of a novel endocrine progenitor surface marker, the Glial cell line derived neurotrophic factor receptor α3 (Gfrα3) and its ligand Artemin in islet cell development and function.RT-PCR, In situ hybridization and immunochemistry were used to characterize the expression of Gfra3 and Artn mRNAs and proteins as well as of other members of the GDNF receptor and ligand family. We used Gfra3-deficient mice to study Gfrα3 function and generated a transgenic mice over expressing Artn in the embryonic pancreas to study Artn function. We found that Gfrα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and ß-cells, while Artn is found in the pancreatic mesenchyme. Adult ß-cell lack Gfrα3, but rare ß cell express the receptor. Gfrα3 is also found in parasympathetic and sympathetic intra islets neurons as well as in glial cell in the embryonic and adult pancreas. The loss of Gfrα3 or overexpression of Artn has no impact on Ngn3-and islet cell formation and maintenance in the embryo. Islet organisation and innervation as well as glucose homeostasis is normal in Gfrα3-deficient mice. Our data show that Gfrα3 is dispensable for islet cell differentiation and innervation suggesting functional redundancy. Gfrα3 could be instrumental as a surface marker for antibody–mediated sorting and characterization of relevant cell population during islet cell differentiation.

La cornée est le tissu le plus densément innervé du corps humain. Cette innervation joue un rôle dans la régulation de la sécrétion du film lacrymal et exerce un rôle trophique direct sur l'épithélium cornéen. Les axones cornéens expriment différents types de récepteurs et répondent à des fonctions de mécano-nocicepteurs, de récepteurs au froid ou de récepteurs polymodaux. Nous avons pu identifier de nouvelles lignées de souris transgéniques et les utiliser pour caractériser ces différentes populations axonales. L'innervation cornéenne débute à E12.5 chez la souris et est régulée par les molécules de Slits et de Sémaphorines et leurs récepteurs Robo et Plexines/Neuropilines respectivement. Nous avons pu étudier le rôle de ces deux familles dans le développement de l'innervation cornéenne. Les mutants Slits et Robos présentent une réduction du nombre et de la taille des terminaisons axonales épithéliales cornéennes. A l'âge adulte, les mutants Robos présentent une dégénérescence précoce des terminaisons épithéliales. Les mutants plexine-A4 et Neuropiline-1 présentent à l'inverse une augmentation du nombre de divisions des troncs stromaux cornéens. A l'âge adulte les mutants Plexine-A4 retrouvent une organisation classique de l'innervation alors que les mutants Neuropiline-1 conservent la désorganisation de l'innervation cornéenne. Nous avons également étudié la régénération de l'innervation après lésions de grattage de l'épithélium cornéen. Nos résultats préliminaires semblent en faveur d'une augmentation de la régénération de l'innervation cornéenne chez les mutants Neuropiline-1. / The cornea is the most densely innervated tissue in the entire body. Corneal innervation plays a role in regulating the secretion of lacrimal film and exerts a direct trophic role on the corneal epithelium. Corneal axons express different types of sensory receptors ranging between mechano-, thermo-, and polymodal nociceptors. We identified transgenic mouse lines to characterize these different axonal populations. Corneal innervation begins at E12.5 in mice and is regulated by a range of axon guidance cues such as Slits and Semaphorins, which respond to their receptors Robo and Plexin/Neuropillin respectively. We studied the role of these two families in the development of corneal innervation. The Slits and Robos mutants show a reduction in the number and size of the corneal epithelial nerves endings. In adult, Robos mutants exhibit early degeneration of the epithelial nerves endings. Plexin-A4 and Neuropilin-1 mutants, on the other hand, show an increase in the number of divisions of the corneal stromal nerve trunks. In adult, Plexin-A4 mutants regain a classical organization of innervation whereas Neuropilin-1 mutants retain the disorganization of corneal innervation. Following a lesion, corneal innervation is able to regenerate, however the axons never regain their initial morphology or complexity. Due to the increased corneal innervation observed in the Neuropilin-1 mutants, we wondered whether the regeneration of innervation after scrapping lesions of the corneal epithelium could be enhanced in Neuropilin-1 loss of function. Our preliminary results support an increase in corneal innervation regeneration in Neuropilin-1 mutants.

Bei der vorliegenden Arbeit handelt es sich um eine experimentelle Untersuchung zum Einfluss von zwei Ko-Transmittern des autonomen Nervensystems, Neuropeptid Y und dem Pituitary Adenylate Cyclase-activating Polypeptide (PACAP), auf den intrazellulären Kalziumspiegel und die Insulinsensitivität von 3T3-L1 Adipozyten. Mittels Polymerasekettenreaktion und Western Blot Analyse konnte die Expression des NPY-1 (Y1) Rezeptors als auch die der PACAP Rezeptoren PAC1 und VPAC2 nachgewiesen werden. Die Aktivierung des Y1 oder des PAC1 Rezeptors durch ihre Agonisten führte zur Erhöhung des intrazellulären Kalziumspiegels. Im Weiteren führte NPY nach Ko-Applikation mit Insulin zu einer abgeschwächten Insulinsensitivität der Adipozyten, da sowohl die insulin-stimulierte Translokation von Glukosetransporter 4 zur Zelloberfläche als auch die Glukoseaufnahme durch NPY abgeschwächt wurde. Dieser Effekt konnte als Y1 spezifisch beschrieben werden. Diese Ergebnisse gewähren somit neue Einblicke über den peptidergen Einfluss auf den Adipozytenstoffwechsel und erlauben Rückschlüsse über die Rolle des autonomen Nervensystems in der Entwicklung von Adipositas und Diabetes mellitus Typ 2.

While prolonged nearwork is considered to be an environmental risk factor associated with myopia development, an underlying genetic susceptibility to nearwork-induced accommodative adaptation may be one possible mechanism for human myopia development. As the control of accommodation by the autonomic system may be one such genetically predetermined system, this research sought to investigate whether an anomaly of the autonomic control of accommodation may be responsible for myopia development and progression. The emphasis of this work was determining the effect of altering the sympathetic input to the ciliary muscle on accommodation responses such as tonic accommodation and nearwork-induced accommodative adaptation in myopes and non-myopes. The first study of the thesis was based on observations of Gilmartin and Winfield (1995) which suggested that a deficit in the sympathetic inputs to the ciliary muscle may be associated with a propensity for myopia development. The effect of ß-antagonism with timolol application on accommodation characteristics was studied in different refractive error groups. Our results support the previous findings that a deficit of sympathetic facility during nearwork was not a feature of late-onset myopia. However it was found that classifying myopes according to stability of their myopia and their ethnic background was important and this allowed differentiation between accommodation responses and characteristics of the ciliary muscle autonomic inputs, with the greatest difference observed between Caucasian stable myopes and Asian progressing myopes. Progressing myopes, particularly those with an Asian background, demonstrated enhanced susceptibility to nearwork-induced accommodative adaptation and this was suggested to result from a possible parasympathetic dominance and a relative sympathetic deficit to the ciliary muscle. In contrast, stable myopes, particularly those with an Asian background, demonstrated minimal accommodation changes following nearwork (counter-adaptation in some cases), and increased accommodative adaptation with ß-antagonism, suggesting sympathetic dominance as the possible autonomic accommodation control profile. As ethnic background was found to be an important factor, a similar study was also conducted in a group of Hong Kong Chinese children to investigate if enhanced susceptibility to nearwork-induced changes in accommodation may explain in part the high prevalence of myopia in Hong Kong. Despite some minor differences in methodology between the two studies, the Hong Kong stable myopic children demonstrated counter-adaptive changes and greater accommodative adaptation with timolol, findings that were consistent with those of the adult Asian stable myopes. Both Asian progressing myopic children and adults also showed greater accommodative adaptation than the stable myopes and similar response profiles following ß-adrenergic antagonism. Thus a combination of genetically predetermined accommodation profiles that confer high susceptibility and extreme environmental pressures is a likely explanation for the increase in myopia over the past decades in Asian countries. The hypothesis that a sympathetic deficit is linked to myopia was also investigated by comparing the effect of â-stimulation with salbutamol, a ß-agonist, on accommodation with that of ß-antagonism using timolol. It was hypothesized that salbutamol would have the opposite effect of timolol, and that it would have a greater effect on subjects who demonstrated greater accommodative adaptation effects, i.e. the progressing myopes, compared to those who showed minimal changes in accommodation following nearwork. Consistent with the hypothesis, the effect of sympathetic stimulation with salbutamol application was only evident in the progressing myopes whom we hypothesized may have a parasympathetic dominance and a relative sympathetic deficit type of autonomic imbalance while it did not further enhance the rapid accommodative regression profile demonstrated by the stable myopes. Characteristics of the convergence system and the interaction between accommodation and convergence were also investigated in the Hong Kong children. No significant differences in response AC/A ratios between the emmetropic, stable and progressing myopic children were found and it was concluded that elevated AC/A ratios were not associated with higher myopic progression rate in this sample of Hong Kong children. However, ß-adrenergic antagonism with timolol application produced a greater effect on accommodative convergence (AC) in stable myopic children who presumably have a more adequate, robust sympathetic input to the ciliary muscle, but had little effect on AC of progressing myopic children. This finding again points to the possibility that the autonomic control of the accommodation and convergence systems may be different between stable and progressing myopia. The primary contribution of this study to the understanding of myopia development is that differences in the autonomic control of the ciliary muscle may be responsible for producing anomalous accommodation responses. This could have significant impact on retinal image quality and thus results in myopia development. This knowledge may be incorporated into computer models of accommodation and myopia development and provides scope for further investigation of the therapeutic benefits of autonomic agents for myopia control.