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The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

The thermoregulatory responses of subjects wearing two different forms of rugby league jersey, one with plastic sponsorship recognition and numbering (trial Gl) and one without (trial G2), and a lightweight alternative (trial G3), were compared with a trial without any form of upper body garment (trial GO). Ten male volunteers, mean age 20.9 (±2.3) years, height 179.8 (±4.7) cm, weight 80.2 (±8.9) kg, and body surface area 1.99 (±0.13) m2, participated in this study. Subjects had a mean maximal oxygen uptake capacity of 56.0 (±6.3) ml.kg.min-1 and a sum of 8 skinfolds of 80.6 (±23.8) mm. Subjects were exercised at approximately 50% of maximal oxygen uptake in a warm humid environment for 50 minutes. Mean ambient temperature was 27.6°C (±0.32) with a relative humidity of 64.7% (±1.44). Measurements of core and skin (7 sites) temperature, heart rate, oxygen uptake, plasma volume, peak lactate concentration, and pre- and post-trial body weight, hematocrit and garment weight were recorded. The statistical results showed that all subjects experienced significant (p ≤.0001) decreases in body weight representing a percentage decrease ranging from 1.2-1.3%. No significant difference was found between trials with respect to body weight change. No significant effect of garment type was found on pre- and post-trial hematocrit, plasma volume changes or peak blood lactic acid concentration. However, mean peak lactate was highest for trial Gl (5.6 mmol.L-1 ±2.2) and lowest for trial G3 (4.6 mmol.L-1 ±1.27). Post-trial core temperature was significantly (p≤ .0001) higher than the resting value; no significant difference was found between trials. The mean absolute increase for all experimental trials was 0.9°C. A significant (p≤.005) difference between mean total (7 sites) skin temperature was found with a post-hoc test revealing that trials Gl and G2 were significantly higher than trial GO; no significant difference was found when comparing trial G3 with trial GO or when comparing the garments between each other. Mean skin temperature under the garment (4 sites) was found to be significantly (p≤.05) higher for all trials involving a garment when compared with mean skin temperature outside (3 sites) the garment; no significant difference was found between trials. Mean oxygen uptake was significantly different between trials (p≤.005), with trial Gl and G3 found to be significantly lower than trial GO; no difference was found when comparing the garments with each other. Post-trial garment weights were significantly (p≤.001) heavier than pre-trial and were significantly (p≤.0001) different when compared with each other. There was no significant effect on heart rate, haematocrit, plasma volume changes, peak blood lactic acid concentration, or core temperature due to garment type. However, differences in skin temperature suggest that the garment used in trial G3 may have a benefit. Further research should consider the impact of increased exercise intensity and/or environmental temperature and humidity on the measured parameters while wearing the garments described in this study.

München, Techn. University, Diss., 2006.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Long-term stimuli of many systems leads to decreased cellular responsiveness, or desensitization. We characterized the desensitization of angiotensin II (Ang 11)-mediated inositol phospholipid (IP) hydrolysis in cultured human aortic smooth muscle cells (HASMC). Although it has been suggested that the desensitization induced by long-term Mg II exposure may result partially from down-regulation of Ang II receptor, this is not sufficient to explain fully desensitization in many systems. Post-receptor desensitization of IP hydrolysis may also result from phosphorylation or changes in protein levels of the effector enzyme, PLC-β. We identified the major PLC-β isoenzymes expressed by HASMC as PLC-β1 and PLC-β3. Ang II pretreatment reduced IP accumulation induced by Ang II (1μM) in a time-dependent manner. Phorbol ester-12-myristrate-13-acetate (PMA), a protein kinase C (PKC) activator, also reduced Ang II-stimulated IP accumulation. These results suggest that PKC activation may negatively regulate Ang II-stimulated IP signaling in HASMC, similar to rat cells. In addition, PKC also reduced IP accumulation stimulated by A1F₄⁻, directly activating the G protein. It suggests that the majority of PKC-induced desensitization of Ang II-stimulated IP signaling occurs downstream of the Ang II receptor in HASMC. However, both PLC-β1 and PLC-β3, expected candidates for PKC phosphorylation, were phosphorylated independently of PKC activation or inhibition, indicating that PKC might not be involved in direct phosphorylation of PLC-β1 and PLC-β3. Furthermore, PLC-β1, but not PLC-β3, was highly phosphorylated under basal conditions, suggesting that PLC-β1 and PLC-β3 may play different roles in IP signaling in HASMC. / Graduation date: 2003

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 87-102.

Thesis (M. P. A.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.

Der vorliegende Beitrag untersucht das Potential und die Bedingungen alternativer Regulierungsformen vor dem Hintergrund von Better Regulation in Deutschland. Nahezu alle EU- und OECD-Staaten betreiben heute umfassende politische Reformprogramme zur Modernisierung der Regulierungsarchitekturen, die unter dem Label Better Regulation subsumiert werden. Die Zielsetzung dieser Programme besteht zum einen in der wirtschaftsfreundlichen Reduktion von Regulierungskosten und zum anderen in der Effektivitätssteigerung von Regulierung durch Vermeidung unintendierter Nebeneffekte. Better Regulation ist ein Toolkit verschiedener metaregulativer Policy-Instrumente, deren programmatische Zusammensetzung vom nationalen politischen Kontext abhängt. Im Rahmen der parallel verlaufenden Reformagenden werden überdies alternative Regulierungsformen von verschiedenen Advokaten besserer Regulierung thematisiert, und als Alternative zur rein staatlichen, hierarchischen Command-and-Control-Regulierung (CaC-Regulierung) promotet, jedoch weder näher auf deren Bedingungen noch deren Konsequenzen einzugehen. Den optimistischen Vorstellungen eines evidenzbasierten Regulatory Managements folgend, erfolgt die Prüfung und Analyse alternativer Regelungsformen im Zuge der Gesetzesfolgenabschätzung (GFA; Regulatory Impact Assessment RIA). Bisher fristen alternative Regulierungsformen allerdings ein Dasein im Schatten von Standardkosten-Modell, RIA und Co. und werden von den Policy-Makern nicht systematisch in Betracht gezogen oder eingesetzt. CaC-Regulierung ist und bleibt das dominante Steuerungsinstrument des Staates. Es existieren jedoch zahlreiche diskursive Anknüpfungspunkte einschließlich zahlreicher Kritiken an der hierarchisch-regulativen Steuerung, welche die Auseinandersetzung mit alternativen Regulierungsformen begründen. Ziel dieses Papieres ist es daher, die in der Kritik stehende CaC-Regulierung Modellen alternativer Regulierungsformen entgegenzustellen, wobei alternative Regulierung mit den Konzepten Selbstregulierung, Koregulierung und regulierter Selbstregulierung eng definiert wird, und nur jene Konstellationen betrachtet werden, die kooperativ zwischen Regierung und Wirtschaft operieren. Ebenso wird der Versuch unternommen, die mannigfaltigen Konzepte und Erscheinungsformen alternativer Regulierung zu definieren und zu kategorisieren. Im Anschluss an den theoretischen Part erfolgt im nächsten Schritt die Identifikation der Einsatz- und Erfolgsbedingungen alternativer Regulierung. Dies geschieht anhand zweier empirischer Fallbeispiele aus den Politikfeldern Berufsbildungspolitik („der Ausbildungspakt der deutschen Wirtschaft“) sowie Umweltpolitik („die Mehrwegquotenverpflichtung“), wobei gezielt ein erfolgreiches und gescheitertes Beispiel alternativer Regulierung verglichen werden und Erfolgsfaktoren abgeleitet werden. Das Ergebnis ist, dass alternative Regulierung gewisse Potentiale und Anreize für staatliche wie privatwirtschaftliche Akteure bietet, aber der Einsatz dieser Steuerungsformen höchst voraussetzungsvoll ist und nur in wenigen Politikfeldern möglich und zu empfehlen ist. So besteht das Potential alternativer Regulierungsformen vor allem in der ganzheitlichen Senkung von Regulierungskosten (Befolgungs- und Vollzugskosten) und in der Erhöhung der Steuerbarkeit der Adressaten. Allerdings sind die korporativen Akteure auf beiden Seiten zum einen nicht immer hinreichend auf diese Form indirekter Steuerung vorbereitet. Zum anderen wird alternative Regulierung durch die Fragmentierung und Schwäche von Wirtschaftsverbänden sowie durch Interessengegensätze unter den Regelungsadressaten gehindert, die in Trittbrettfahrertum und dem letztlichen Scheitern selbstregulativer Verpflichtungen münden. Als entscheidende, aber anspruchsvolle Erfolgskomponente erweisen sich die politische Kommunikation und die Durchsetzbarkeit staatlicher Sanktionen sowie die Erfolgskontrolle, um die Regelungseinhaltung durch die Wirtschaft zu gewährleisten. Generell ist zu konstatieren, dass alternative Regulierungsformen dort zustande kommen, wo sich konzentrierter Widerstand auf Seiten der Adressaten gegen geplante, autoritative Steuerung abzeichnet, eine gewisse Pfadabhängigkeit bzw. eine Historie sektoraler Selbstregulierung vorliegt und eine Win-Win-Situation bei der Kooperation für Politik und Wirtschaft ersichtlich ist. In der Konsequenz zeigen die Ergebnisse, dass alternative Regulierung nicht nach einem synoptisch-rationalen Modell politischer Prozesse entsteht, sondern ein ursprünglich nicht intendiertes Produkt Garbage Can-artiger Verhandlungen mit einem hohen Konfliktniveau darstellt. Folglich ist das Resultat für die Vorstellung einer „Rational Regulatory Choice“ induzierenden Better Regulation-Agenda desillusionierend. / This paper analyses the potentials and the preconditions of alternative forms of regulation against the background of Better Regulation in Germany. Today, almost every OECD- and EU-country runs extensive regulatory reform programs to modernize its regulatory governance structures, which are summarized under the label Better Regulation. The goal of this type of agenda can be regarded, firstly, as the business-friendly reduction of overall regulatory costs and secondly as the increase of regulatory efficiency by removing unintended side-effects. Better Regulation is a toolkit consisting of several meta-regulatory instruments, whose programmatic composition and use varies with the national political context. Moreover, within these parallel proceeding reform agendas alternative forms of regulation are constantly promoted by advocates of Better Regulation and portrayed as alternative to purely governmental command-and-control approaches (CaC), though neither responding to their preconditions nor addressing their consequences. According to the optimistic notion of an evidence-based regulatory management, the examination and analysis of regulatory alternatives is supposed to be carried out within regulatory impact assessments (RIA). However, hitherto alternative forms of regulations exist unnoted in the shadow of the standard cost model, RIA and co. and they are not systematically considered and applied by policy-makers. CaC-regulation is and remains the dominant and favoured policy-instrument of the state. Yet, there are several discursive connections including broad criticism on hierarchical-regulative steering, which give reason to the deeper analysis of alternative forms of regulation. Therefore the aim of this paper is to contrast alternative regulation with the criticized CaC-technique, whereas alternative regulation is defined narrowly as the concepts of self-regulation, co-regulation and regulated self-regulation which operate as cooperative substitutes of CaC between government and business. Moreover, this paper attempts to define and to categorize the diverse concepts and natures of alternative regulation. Subsequent to the theoretical part the preconditions and critical success factors for alternative regulation are identified in the next section. For this purpose two empirical case studies from two different policies, under which one is successful and the other has failed, are examined and compared to deduce success factors. The case studies are from vocational training policy (“The Training Pact of the German Business”) and waste policy (“refill quota for beverage containers”). The result is that alternative forms of regulation offer several potential und incentives for state and business actors, but that the use of these policy instruments is highly demanding and in addition only possible and recommendable in few policies. Undoubtedly its potential lies in the holistic reduction of regulatory costs (compliance and enforcement costs) and in the enhancement of the governability of the regulated business. However, the corporate actors on both sides are not always adequately prepared for this indirect mode of steering. Furthermore, alternative regulation is hindered by the fragmentation and weakness of business associations and opposing interests among the regulated sectors and companies, which may lead to free-riding and could result in the final failure of alternative regulation. The political communication and assertiveness of public sanctions as well as the measurement of success, which ensure self-regulatory compliance, turned out to be decisive, but very demanding constituents of success. Generally speaking, alternative forms of regulation emerge in those cases and policy areas, where concentrated resistance against planned CaC-measures becomes apparent, where a certain path dependency or history of sectoral self-regulation exists, and where a win-win-situation becomes obvious for politicians and business. To sum up, this paper argues that alternative regulations don’t emerge by a synoptic-ration model of policy process, but that they present an unintended product of garbage can like policy processes, further characterized by a high level of conflict. Consequentially, this paper’s findings disillusion the (exaggeratedly) optimistic notion of a rational regulatory choice inducing Better Regulation agenda.