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Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.

alpha2-adrenerge Rezeptoren, von denen drei verschiedene Subtypen (alpha2A, alpha2B, alpha2C) kloniert wurden, gehören zur Familie der G-Protein-gekoppelten Rezeptoren und vermitteln vielfältige physiologische Funktionen der Transmitter Adrenalin und Noradrenalin. Im Rahmen dieser Arbeit sollte untersucht werden, inwieweit Rezeptorsubtypen, die subzelluläre Lokalisation von Rezeptoren oder der Differenzierungsstatus einer Zelle für die funktionelle Diversität der alpha2-Rezeptor-Effekte in vivo verantwortlich sind. Im ersten Teil des Projektes wurde ein transgenes Mausmodell untersucht, bei dem selektiv alpha2A-Rezeptoren unter Kontrolle des Dopamin-beta-Hydroxylase Promotors in adrenergen Neuronen exprimiert wurden. In diesem Modell sollte getestet werden, ob ein einzelner Rezeptorsubtyp in den verschiedenen Neuronen des sympathischen Nervensystems in vivo identische Funktionen hat. Transgene alpha2A-Rezeptoren hemmten in vivo zwar die Freisetzung von Noradrenalin aus sympathischen Nervenfasern nicht aber die Exozytose von Adrenalin aus dem Nebennierenmark. Deshalb stellte sich die Frage, ob die Rezeptorfunktion von der Morphologie, dem Differenzierungsstatus der Zellen oder von der subzellulären Lokalisation der Rezeptoren abhängt. Hierfür wurden alpha2A-Rezeptoren durch Varianten des grün fluoreszierenden Proteins markiert und mittels FRET-Fluoreszenzmikroskopie untersucht. In PC12 Phäochromozytomzellen, die durch NGF zum Auswachsen neuronaler Fortsätze stimuliert wurden, waren die Agonist-bedingten Konformationsänderungen von alpha2A-Rezeptoren jedoch weder vom Differenzierungsstatus der Zellen noch von deren subzellulärer Lokalisation abhängig. Lediglich in transient transfizierten Zellen waren im Vergleich zu stabil transfizierten Zellen höhere Agonist-Konzentrationen zur Rezeptoraktivierung erforderlich. Diese Befunde zeigen, dass zusätzlich zur Diversität der Rezeptorsubtypen auf Proteinebene der zelluläre Kontext, in dem ein Rezeptor exprimiert wird, eine ganz wesentliche Rolle für dessen Funktion spielt. / alpha2-adrenergic receptors of which three different subtypes were cloned (alpha2A, alpha2B, alpha2C), are part of the family of G-protein coupled receptors and mediate many physiological functions of the transmitters adrenaline and noradrenaline. This study was initiated to determine whether receptor subtypes, their subcellular localization or the status of differentiation of a cell are responsible for the functional diversity of effects of alpha2-adrenergic receptors in vivo. In the first part of this project a transgenic mouse model was characterized, in which alpha2-adrenergic receptors were expressed under control of the dopamine-beta-hydroxylase-promotor in adrenergic neurons selectively. This model was used to test whether a single receptor subtype has identical functions in different neurons of the sympathetic nervous system in vivo. Transgenic alpha2A-adrenergic receptors inhibited the release of noradrenaline from sympathetic nerves in vivo, but not the exocytosis of adrenaline from the adrenal medulla. Therefore the question arose whether the functions of receptors are dependent on cell morphology, the status of differentiation of cells or the subcellular localization of receptors. To address this question, alpha2A-receptors were tagged with variants of the green fluorescent protein and investigated by means of FRET fluorescence microscopy. In PC12 rat pheochromocytoma cells which were stimulated by nerve growth factor to develop neurites, the conformational changes of alpha2A-adrenergic receptors upon agonist activation, however, did not dependent on the status of cellular differentiation or on the subcellular localization of receptors. Only in transiently transfected cells, higher agonist concentrations were necessary for the activation of receptors as determined by FRET microscopy. These findings demonstrate that the cellular context in which receptor subtypes are expressed play an essential role for their function.

Pathogenesis, diagnosis and treatment, the essential domains in medical practice, seem helpless to address Alzheimer's disease (AD). With a huge mortality rate, it is looming and threatening the socioeconomic barrier. Despite many different studies, the pathogenesis of AD remains inconclusive. However, growing numbers of studies suggest oxidative stress to contribute to the initiation and progression of AD. We propose an iron hypothesis: iron mediated oxidative damage by reactive oxygen species (ROS), which induces protective roles of amyloid beta and hyper-phosphorylated tau (HP-tau) to sequester iron and limit the disease. We propose to study such mechanism using transgenic mice models for AD, inducing oxidative stress to elevate intracellular iron, and analyze its co-localization with proteins using Magnetic Resonance Imaging (MRI), 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Western blot. We report three primary findings: 1) a significant loss in T2 signal over bilateral hippocampi of transgenic mice compared to the wild types (WT) by three months, corresponding to early disease and the ability of proteins to sequestration iron. Ability of rescue treatments to impede disease progression reflected as preserved T2 signal intensities over these areas throughout our study period of nine months. 2) Concentration of zinc and its dual role in the presence or absence of oxidative stress reflected as loss of 1H NMR T2 measurement showed that higher concentrations of zinc were neuro protective when there was an active oxidative stress inducing condition, but neurotoxic and promote oxidative damage in normal condition. And 3) Different strains of mice, according to their transgene, expressed various proteins associated with AD. However, these expressions were in accordance with our iron-hypothesis.

The lung is an organ that contains a vast system of airways carefully constructed to achieve maximal surface area in a confined space, requiring guidance from a multitude of developmental factors. The Shh pathway is one such signaling mechanism that is critical to proper lung formation, guiding branching morphogenesis and cellular proliferation through its downstream Gli transcription factors. Additionally, Foxf1 has been shown to be a key developmental factor required for proper lung formation during embryogenesis. Although theorized that the Gli transcription factors are responsible for regulating foxf1 levels, their exact relationship has yet to be revealed. Using five different models for Shh signaling (gli2 null, gli2 over-expressor [hVER-Gli2], gli3 null, Gli3 constitutive repressor [Gli3Δ699] and cyclopamine treated lung explants), I compared and contrasted the role of Gli2 and Gli3 in terms of their effect on cell cycle regulation, and on the expression levels of foxf1 and its potential downstream target genes tbx4, tbx5 and fgf10. I found that ectopic over-expression of gli2 resulted in increased Shh pathway activation, and increased expression of G1/S phase cyclins, which was associated with increased cellular proliferation and lung growth. However, no change in the levels of G1/S phase cyclins due to altered Gli3 signaling was observed. Foxf1 levels positively correlate with the levels of gli2, and appear to be independent of Gli3 activity. The amount of tbx4, tbx5, and fgf10 transcripts were observed to follow the levels of gli2 in the different gli2 mouse models, however, there was no significant change in gli3 null or Gli3Δ699 mice. Finally, by analyzing gene expression at different time points during gestation, I found that while gli2 levels affect foxf1 throughout gestation, the relationship to tbx4, tbx5 and fgf10, occurs only during the latter stages of lung development. I conclude, that Gli2 and not Gli3 appears to be the primary transducer of Shh signaling influencing cyclin regulation, leading to changes in embryonic lung growth. Furthermore, that Gli2 and not Gli3 appears to regulate foxf1 expression levels, and that this may extend downstream to influence tbx4, tbx5 and fgf10 expression.

The lung is an organ that contains a vast system of airways carefully constructed to achieve maximal surface area in a confined space, requiring guidance from a multitude of developmental factors. The Shh pathway is one such signaling mechanism that is critical to proper lung formation, guiding branching morphogenesis and cellular proliferation through its downstream Gli transcription factors. Additionally, Foxf1 has been shown to be a key developmental factor required for proper lung formation during embryogenesis. Although theorized that the Gli transcription factors are responsible for regulating foxf1 levels, their exact relationship has yet to be revealed. Using five different models for Shh signaling (gli2 null, gli2 over-expressor [hVER-Gli2], gli3 null, Gli3 constitutive repressor [Gli3Δ699] and cyclopamine treated lung explants), I compared and contrasted the role of Gli2 and Gli3 in terms of their effect on cell cycle regulation, and on the expression levels of foxf1 and its potential downstream target genes tbx4, tbx5 and fgf10. I found that ectopic over-expression of gli2 resulted in increased Shh pathway activation, and increased expression of G1/S phase cyclins, which was associated with increased cellular proliferation and lung growth. However, no change in the levels of G1/S phase cyclins due to altered Gli3 signaling was observed. Foxf1 levels positively correlate with the levels of gli2, and appear to be independent of Gli3 activity. The amount of tbx4, tbx5, and fgf10 transcripts were observed to follow the levels of gli2 in the different gli2 mouse models, however, there was no significant change in gli3 null or Gli3Δ699 mice. Finally, by analyzing gene expression at different time points during gestation, I found that while gli2 levels affect foxf1 throughout gestation, the relationship to tbx4, tbx5 and fgf10, occurs only during the latter stages of lung development. I conclude, that Gli2 and not Gli3 appears to be the primary transducer of Shh signaling influencing cyclin regulation, leading to changes in embryonic lung growth. Furthermore, that Gli2 and not Gli3 appears to regulate foxf1 expression levels, and that this may extend downstream to influence tbx4, tbx5 and fgf10 expression.

abstract: The effects of specific histone deacetylase inhibitors (HDACi) on transgene expression in combination with a novel polymer as a delivery vehicle are investigated in this research. Polymer vectors, although safer than viruses, are notorious for low levels of gene expression. In this investigation, the use of an emerging chemotherapeutic anti-cancer drug molecule, HDACi, was used to enhance the polymer-mediated gene expression. HDACi are capable of inhibiting deacetylation activities of histones and other non-histone proteins in the cytoplasm and nucleus, as well as increase transcriptional activities necessary for gene expression. In a prior study, a parallel synthesis and screening of polymers yielded a lead cationic polymer with high DNA-binding properties, and even more attractive, high transgene expressions. Previous studies showed the use of this polymer in conjunction with cytoplasmic HDACi significantly enhanced gene expression in PC3-PSMA prostate cancer cells. This led to the basis for the investigation presented in this thesis, but to use nuclear HDACi to potentially achieve similar results. The HDACi, HDACi_A, was a previously discovered lead drug that had potential to significantly enhance luciferase expression in PC3-PSMA cells. The results of this study found that the 20:1 polymer:plasmid DNA weight ratio was effective with 1 uM and 2 uM HDACI_A concentrations, showing up to a 9-fold enhancement. This enhancement suggested that HDACi_A was effectively aiding transfection. While not an astounding enhancement, it is still interesting enough to investigate further. Cell viabilities need to be determined to supplement the results. / Dissertation/Thesis / M.S. Bioengineering 2012

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