Boron arsenide is a compound main group semiconductor with a theoretical band gap in the range of 1.1 to 1.6 eV. Despite this ideal band gap, experimental studies of boron arsenide are very limited. In the present work, single source precursors with covalent bonds between boron and arsenic and labile ligands have been designed and synthesized. These precursors underwent thermal or chemical treatment to produce boron arsenide materials. Boron arsenide has also been prepared as a thin layer deposited on a boron substrate and a p-type photoelectrode was prepared from this material. The structure of the product was identified on the basis of X-ray diffraction and scanning electron microscopy, and the surface composition was determined by means of X-ray photoelectron spectroscopy. The electrode was found to be photoactive under both visible and UV-visible light irradiation and displayed a photocurrent of approximately 0.1 mA/cm² under UV-visible light irradiation at an applied potential of -0.25 V vs. Ag/AgCl. The valence band was estimated to be -5.1 eV. The indirect band gap, as determined from incident photo-to-electron conversion efficiency plots, is 1.46 eV. An ester-fuctionalized salophen aluminum complex that features a polymerizable bithiophene as the ester R group has been designed and synthesized. Metallopolymers of this type offer the additional advantages of processability and uniformity of the resulting films. The new salophen complex exhibited emission in the blue region at 491 nm with a quantum yield of 8.19%, which is significantly larger than that of the isolated ligand. Electropolymerization of this complex on a platinum button electrode resulted in the formation of an electrically conductive polymer that is also ionically conductive at low scan rates. In the polymeric form, the emission wavelength was found to be red-shifted to 505 nm. / text

Despite intense interest in the proteolysis of Amyloid Precursor Protein (APP) in Alzheimer’s disease (AD), how the normal processing and function of this type I receptor-like glycoprotein is regulated remains ill-defined. APP is reported to function in neurodevelopment, including migration of neuronal precursor cells into the cortical plate. In recent years, several candidate ligands for APP, including F-spondin, Reelin, \(\beta1\) Integrin, Contactins, and Lingo-1 have been reported. However, a cognate ligand for APP that regulates its function or processing has yet to be widely confirmed in multiple laboratories. First, in an unbiased approach to reveal novel ligands, Pancortin was identified by a mass spectrometry-based screen for factors that bind to the APP ectodomain in rodent brain. Each of the Pancortin isoforms was confirmed to interact with APP. However, only specific Pancortin isoforms reduced \(\beta\)-secretase but not \(\alpha\)-secretase cleavage of endogenous APP. Using in utero electroporation to overexpress or knockdown Pancortin isoforms in rodent cortex, a previously unidentified role for Pancortin in cortical cell migration with evidence for a functional interaction with APP was discovered. Next, I developed new assays in an effort to confirm a role for one or more of the published candidate ligands in regulating APP ectodomain shedding in a biologically relevant context. A comprehensive quantification of APPs\(\alpha\) and APPs\(\beta\), the immediate products of secretase processing, in both non-neuronal cell lines and primary neuronal cultures expressing endogenous APP yielded no evidence that any of these published candidate ligands stimulate ectodomain shedding. Rather, Reelin, Lingo-1 and Pancortin emerged as the most consistent ligands for significantly inhibiting ectodomain shedding. These studies clarify mechanisms regulating the function and processing of APP, which is needed to understand consequences of chronically altering APP proteolysis to treat AD and to develop new potential drug targets.

No description available.

No description available.

Die Inzidenz von neurodegenerativen Erkrankungen und Schlaganfällen steigt in Folge der Überalterung der westlichen Gesellschaft immer weiter an. Die Behand-lung von Schlaganfall-, Alzheimer und Parkinsonpatienten ist bisher aber meist unbefriedigend bzw. weitgehend erfolglos. Ein neues Modell in der Schlaganfallforschung wurde daher am Schaf entwickelt. In diesem wird auch der in den letzten zwei Jahrzehnten verstärkt verfolgte zelltherapeutische Ansatz untersucht (BOLTZE et al. 2011, DREYER et al. 2012). Neurale Vorläuferzellen gelten dabei, auf Grund ihrer wichtigen Rolle bei den endogenen Reparaturmechanismen nach einem Schlaganfall, als besonders vielversprechend. Die Gewinnung dieser Zellen für eine autologe Transplantation ist jedoch aufwendig und nur eingeschränkt möglich. Im Vergleich zu Nervengewebe stellt die Haut eine sowohl beim Tier als auch beim Menschen leicht zugängliche und in ausreichendem Maß verfügbare Quelle verschiedener Stamm- und Vorläuferzellen dar. Bei verschiedenen Spezies wurde die Isolation spezieller, dermaler Vorläuferzellen beschrieben, die als skin-derived precursor cells (SKPs) bezeichnet werden. SKPs wiesen dabei ein ähnliches Differenzierungspotential auf wie neurale Vorläuferzellen (TOMA et al. 2001, FERNANDES et al. 2006). Ein Einsatz der SKPs in der Schlaganfalltherapie wäre somit denkbar, muss aber zunächst im Schafmodell erforscht werden. SKPs wurden jedoch noch nicht bei der Spezies Schaf isoliert. Ziel der vorliegenden Arbeit war es daher, ein Isolationsprotokoll für SKPs aus der ovinen Dermis zu etablieren und diese morphologisch und immunzytologisch zu charakterisieren. Im Rahmen dieser Arbeit wurden verschiedene in der Literatur beschriebene Isolati-onsverfahren an ovinen Hautproben getestet und modifiziert. Es wurden verschiedene Körperregionen auf ihre Eignung zur Probenentnahme und zur anschließenden Isolierung untersucht. Des Weiteren wurde der Effekt einer Rasur eine Woche vor Exzision des Hautareals auf die Sphäroidbildung überprüft. Der Einsatz von Enzymen in Kombinationslösungen oder singulär wurde variiert und eine unterschiedlich intensive mechanische Aufbereitung der Proben durchgeführt. Der Erfolg der zwei vielversprechendsten Isolationsprotokolle wurde statistisch validiert. Außerdem wurde der Effekt einer initialen Fibronektinbeschichtung analysiert. Die von den isolierten Zellen gebildeten sphärenartigen Zellaggregate wurden unter morphologischen Gesichtspunkten sechs und neun Wochen nach Isolation ausgewertet. Dabei wurden die Anzahl der Sphäroide/cm², die Größe und die Form berücksichtigt. Des Weiteren erfolgte eine immunzytologische Analyse der Sphäroide mit Fokus auf das in der Literatur beschriebene Expressionsmuster von SKPs und neuralen Vorläuferzellen. Für die Isolation von ovinen SKPs erwies sich die Regio nasofrontalis als das geeignetste Hautareal. Dabei war die Isolation eine Woche nach Rasur des beprobten Areals zuverlässiger als ohne diese. Bei vergleichender Betrachtung der Methoden erwies sich ein enzymatisch orientiertes Isolationsverfahren modifiziert nach FERNANDES und MILLER (2009) als zielführend. Neben einer hohen Anzahl an isolierten Zellen erfolgte in jedem Versuchsdurchgang eine Zusammenlagerung der Zellen in frei flotierenden Aggregaten. Diese waren im Median 70,97 µm groß. Auf Grund ihrer Geometrie ist es korrekter sie als Sphäroide und nicht, wie bei anderen Spezies üblich, als Sphären zu bezeichnen. Eine anfängliche Beschichtung der Zellkulturplatten mit Fibronektin hatte keinen fördernden Effekt auf die Bildung und die Größe der Sphäroide. Lediglich eine anfänglich höhere Proliferationsrate war bemerkbar. Immunzytologisch konnte gezeigt werden, dass in den Sphäroiden eine heterogene Zellpopulation vorlag. Die Sphäroide wurden überwiegend von Zellen gebildet, in denen neben mesenchymalen Markern auch klassische Vorläuferantigene wie Nestin und Sox2 nachgewiesen wurden. Das immunzytologische Expressionsmuster ist damit vergleichbar mit dem von SKPs anderer Spezies. Außerdem wurden in unterschiedlicher Ausprägung Antigene detektiert, die typischerweise in neuralen Vorläuferzellen der ventrikulären und subventrikulären Zone vorkommen. Dies konnte auch in den Positivkontrollen für das ovine Gehirn bestätigt werden. Die Anzahl proliferierender Zellen in den Sphäroiden war relativ gering und die Anzahl an kokultivierter Keratinozyten minimal. Die Zusammenfassung der heterogenen Vorläuferzellpopulation unter dem Begriff skin-derived precursor cells ist auf Grund ihres dermalen Ursprungs und ihrer morphologischen und immunzytologischen Eigenschaften gerechtfertigt. Somit ist es in dieser Arbeit gelungen, zum ersten Mal SKPs aus der ovinen Dermis zu isolieren und über neun Wochen zu kultivieren. Es wurde ein Isolationsprotokoll entwickelt, das eine Sphäroidbildung reproduzierbar ermöglicht und an die Gegebenheiten beim Schaf angepasst ist. Bevor eine autologe Transplantation von diesen SKPs etwa im Schlaganfallmodell am Schaf vorgenommen werden kann, ist eine intensivere Untersuchung der isolierten Zellen etwa mittels PCR durchzuführen und eine fluoreszenzbasierte Zellsortierung der heterogenen Vorläuferzellen zu entwickeln. / In consequence of the demographic changes in modern western society, the inci-dence of neurodegenerative diseases and stroke is increasing. Unfortunately, there is still no successful or at least satisfactory treatment available for patients who suffer from stroke Alzheimer’s or Parkinson’s disease. Therefore, a new animal model in stroke research has been established in sheep (BOLTZE et al. 2011, DREYER et al. 2012). First cell therapy studies have already been performed in this model. Especially neural precursor cells seem to be promising as they play an important role in endogenous repair processes in the brain after stroke. However, the extraction of these cells prior to an autologous transplantation is elaborate and of limited success. Compared to neural tissue, skin is an easily accessible and sufficiently available source of a variety of stem and precursor cells in animals as well as in humans. Thus, the isolation of a specific type of dermal precursor cells, called skin-derived precursor cells (SKPs), seems to be easier compared to neural precursor cells and in vitro SKPs are capable of neural differentiation as well (TOMA et al. 2001, FERNANDES et al. 2006). According to these findings, a therapeutic application of SKPs after stroke seems to be promising. Prior to that, however, intensive studies in the ovine stroke model are necessary. Thus, SKPs have to be isolated from the dermis of sheep for an autologous transplantation. Therefore, the aim of this dissertation has been the establishment of an optimal isolation protocol for SKPs from the ovine dermis as well as the morphological and by immunocytochemical characterisation of those cells. Within this study, several previously described isolation protocols were modified for ovine skin. Skin samples were taken from several body regions to assess the local suitability for excision and isolation. Additionally, the effect of shaving the areas one week before sampling on spheroid forming was tested. A variety of enzymes was used alone and in combination. Furthermore, the effectiveness of an isolation protocol using enhanced mechanical treatment was analysed. The two most promising protocols were evaluated statistically and compared to each other. In these experiments, the influence of an initial fibronectin coating was determined as well. The isolated cells formed spheroids, which were assessed after six and nine weeks of cultivation considering the amount of spheroids per cm², their size and form. Moreover, immunocytochemical tests were conducted, focusing on expression patterns described for SKPs and neural precursor cells. According to these experiments, it is advisable to take skin samples from the naso-frontal region one week after shaving. Comparing all tested protocols, a predominantly enzymatic isolation protocol modified according to FERNANDES and MILLER (2009) was most successful. A high cell yield was achieved and free-floating spheroids formed spontaneously in all test runs. The median diameter of these spheroids was 70.97 µm. Due to their three-dimensional shape, it is more correct to use the term “spheroid” instead of the commonly used term “sphere”. Growing the isolated cells initially on fibronectin coated culture plates does not support both formation and size of the spheroids. Only a higher cell proliferation at the beginning of cultivation can be noticed. Immunocytochemical assays demonstrated that the formed spheroids consisted of a heterologous cell population. Besides mesenchymal antigens the cells in the spheroids expressed characteristic antigens of precursor cells, like Nestin and Sox2. Thus, the immunocytochemical expression pattern is comparable to SKPs isolated from other species. Furthermore, common markers of neural precursor cells of the ventricular and subventricular zone, whose existence in the ovine brain was also proven in this study, were detected in the spheroid forming cells. There were only a few proliferating cells and a minimal amount of keratinocytes in the spheroids. Due to the dermal origin and the given morphological and immunocytochemical characteristics, the heterogeneous cell population can be addressed by the term “skin-derived precursor cells”. In conclusion, in this study ovine SKPs were isolated for the first time and cultured successfully over nine weeks. An isolation protocol was established, which guarantees reproducible formation of spheroids in cell isolates from ovine dermis. Further intensive examinations of the isolated cells, for example using PCR, have to be conducted before SKPs can be applied in autologous transplantation in the ovine stroke model. Additionally, the usage of fluorescence-activated cell sorting of the heterogeneous precursor cells should be considered.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

<p>Hypoxia is a profound stressor of the central nervous system implicated in numerous neurodegenerative diseases. While it is increasingly evident that the early effects of hypoxia cause impairment at the level of the axon, the precise mechanisms through which hypoxia compromises axonal structure and function remain unclear. However, links between hypoxia-induced axonopathic disease and the amyloid cascade, as well as the upregulation of amyloid precursor protein (APP) and amyloid beta (A&beta;) by hypoxic stress, give rise to the hypothesis that proteolytic cleavage of APP into A&beta; may be specifically responsible for axonopathy under conditions of hypoxia. </p><p>The goal of this dissertation was thus to understand dependence of hypoxia-induced axonal morphological and functional impairment on APP cleavage and the production of A&beta;. I have developed a model of hypoxia-induced axonopathy in retinal explants. Using this model, I have experimentally addressed the core hypothesis that APP cleavage, and in particular the formation of A&beta;, is necessary and sufficient to mediate morphological and functional axonopathy caused by hypoxia. I have found that there is a dissociation between the mechanisms responsible for hypoxia-induced morphological and functional impairment of the axon in the explanted retina, with the former being dependent on APP-to-A&beta; processing and the latter likely being dependent on cleavage of a non-APP substrate by the enzyme BACE1. These findings shed light on mechanisms of hypoxia-induced axonopathy.</p> / Dissertation

Spinal cord injury (SCI) is uniquely devastating. Cellular transplantation strategies for SCI are showing promise. Little, however, is known about how transplanted neural precursor cells (NPCs) enhance functional recovery or the mechanisms by which they interact with the host spinal cord. Better understanding of these critical issues may lead to improved strategies to enhance recovery after SCI. Given this background, I hypothesized that NPCs mediate functional recovery by a number of mechanisms including trophin production, neuroprotection, modulation of the host inflammatory response or glial scarring, and/or remyelination. I thus endeavored to characterize trophin production by NPCs in vitro and in vivo in rats with clip compression SCI of the thoracic spinal cord, to determine if preservation of host cells and tissue contribute to functional recovery and to determine how NPC transplantation influences the host inflammatory response and glial scarring. Here I present unique and novel insights into NPC-host interactions following SCI. We show that NPCs are poised to provide trophic support to the injured spinal cord. We also show that the combination of NPCs, pharmacotherapy and trophin infusion is associated with sparing of grey and white matter, enhanced numbers of oligodendrocytes but not axons as well as an increased inflammatory response. To assess the potential impact of myelination as a mechanism underlying NPC-mediated functional recovery after SCI, experiments were undertaken using NPCs derived from shiverer mutant mice unable to produce central myelin. These experiments showed that while NPCs from wild-type mice generate myelin and mediate functional recovery after SCI; transplanted shiverer NPCs impede neurobehavioural recovery. In summary, my work provides unique insights into the functional effects of NPC transplantation after SCI. Of importance, this thesis provides novel evidence that remyelination is a key mechanism of action by which NPCs mediate recovery after SCI. Hence, this work has important implications for patients with SCI.

Spinal cord injury (SCI) is uniquely devastating. Cellular transplantation strategies for SCI are showing promise. Little, however, is known about how transplanted neural precursor cells (NPCs) enhance functional recovery or the mechanisms by which they interact with the host spinal cord. Better understanding of these critical issues may lead to improved strategies to enhance recovery after SCI. Given this background, I hypothesized that NPCs mediate functional recovery by a number of mechanisms including trophin production, neuroprotection, modulation of the host inflammatory response or glial scarring, and/or remyelination. I thus endeavored to characterize trophin production by NPCs in vitro and in vivo in rats with clip compression SCI of the thoracic spinal cord, to determine if preservation of host cells and tissue contribute to functional recovery and to determine how NPC transplantation influences the host inflammatory response and glial scarring. Here I present unique and novel insights into NPC-host interactions following SCI. We show that NPCs are poised to provide trophic support to the injured spinal cord. We also show that the combination of NPCs, pharmacotherapy and trophin infusion is associated with sparing of grey and white matter, enhanced numbers of oligodendrocytes but not axons as well as an increased inflammatory response. To assess the potential impact of myelination as a mechanism underlying NPC-mediated functional recovery after SCI, experiments were undertaken using NPCs derived from shiverer mutant mice unable to produce central myelin. These experiments showed that while NPCs from wild-type mice generate myelin and mediate functional recovery after SCI; transplanted shiverer NPCs impede neurobehavioural recovery. In summary, my work provides unique insights into the functional effects of NPC transplantation after SCI. Of importance, this thesis provides novel evidence that remyelination is a key mechanism of action by which NPCs mediate recovery after SCI. Hence, this work has important implications for patients with SCI.

Alzheimer�s disease (AD) is a neurodegenerative disorder, charaeterised by progressive loss of memory. It is important to understand what factors initiate the onset of AD so that effective therapeutic treatments can be developed to target the precise mechanisms that initiate this disease. Currently, synaptic dysfunction is widely believed to be the first significant alteration preceding the onset of AD, and is thought to be initiated by an intracellular accumulation of amyloid-β (Aβ), or a free radical-induced increase of oxidative stress. As Aβ levels rise during the onset of AD, a concomitant reduction of secreted amyloid precursor protein-α (sAPPα) is observed, as the two proteins exist in equilibrium. Intriguingly, the neuroprotective and neurotrophic properties of sAPPα indicate that it is intimately involved in the physiological pathways of the major hypotheses for the cause of AD, and may also be involved in the mechanisms that underlie learning and memory. Therefore, it is possible that during the onset of AD, the decrease of sAPPα may contribute to synaptic dysfunction by disrupting the mechanisms of synaptic plasticity. Long-term potentiation (LTP) is the leading experimental model for investigating the neural substrate of memory formation, and describes the molecular mechanisms that underlie an increase in the strength of synaptic transmission. The role sAPPα may play in the induction and maintenance of LTP has not previously been addressed in vivo. Therefore, the aim of this thesis was to investigate whether sAPPα affects the induction of LTP in the hippocampus of the anaesthetised rat. The present findings are the first to suggest that sAPPα may modulate the induction of LTP in vivo. Decreasing the function of endogenous sAPPα (with sAPPα-binding antibodies and a pharmacological inhibition of α-secretase) significantly reduced the magnitude of LTP induced in the dentate gyrus. Therefore, the reduction of sAPPα during AD is likely to have a detrimental impact on the mechanisms of synaptic plasticity, and by extension, learning and memory. The present investigation has also found that the application of recombinant, purified sAPPα to the rat hippocampus has an �inverted U-shaped� dose-response effect on the magnitude of LTP. Low concentrations of sAPPα significantly enhanced LTP, supporting previous findings that exogenous sAPPα can facilitate in vitro LTP and enhance memory performance in animals. On the other hand, comparatively high concentrations of sAPPα significantly decreased the magnitude of LTP. This observation is also consistent with previous findings, in which high concentrations of sAPPα have been shown to be less synaptogenic and memory enhancing than lower doses. These results are the first to suggest that sAPPα modulates in vivo synaptic plasticity, and have important implications for the development of strategies to treat AD.