Philosophiae Doctor - PhD (Industrial Psychology) / Organizations exist for the primary aim of meeting particular objectives: innovation and advancement, customer satisfaction, profit making and delivery of quality goods and services. These goals are mostly channelled with the intent of demonstrating high performance crucial for the continued existence of the organization especially in these rapidly changing global economies. This target, however, cannot be achieved without the aid of employees in the organization. A plethora of previous studies have proven that efficiency, productivity, high performance and stability on the job can be better achieved when the employees are dedicated, committed to their work roles and experience work engagement. The experience of work engagement on the part of the employees is not a random event; it depends on a myriad of factors that include authentic leadership. Leaders have a cumulative change effect on their followers; hence, leaders in an organization can be termed as core drivers of employee engagement. Hence, it becomes imperative to seek to understand what authentic leadership as a construct has to offer to the workplace.

Lun Samantha Wei Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 142-174). / Abstracts in English and Chinese. / Contents --- p.ii / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.xi / Chapter Section 1. --- Introduction / Chapter 1.1 --- General review of the epididymis --- p.1 / Chapter 1.1.1 --- Structure of the epididymis --- p.1 / Chapter 1.1.2 --- Function of the epididymis --- p.3 / Chapter 1.1.3 --- Regulation of the epididymal function --- p.5 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.8 / Chapter 1.2.1 --- VEGF peptides --- p.8 / Chapter 1.2.2 --- Biological activities of VEGF --- p.10 / Chapter 1.2.3 --- Hormonal regulation of VEGF --- p.11 / Chapter 1.3 --- VEGF receptors --- p.12 / Chapter 1.3.1 --- Flt-1 or VEGFR1 --- p.12 / Chapter 1.3.2 --- Flk-1 or VEGFR2 --- p.13 / Chapter 1.4 --- Caveolae --- p.15 / Chapter 1.4.1 --- Overview of caveolae --- p.15 / Chapter 1.4.2 --- Caveolins/caveolin-1 --- p.16 / Chapter 1.4.3 --- Caveolae and VEGF --- p.18 / Chapter 1.4.4 --- Caveolae and the epididymis --- p.20 / Chapter 1.5 --- VEGF/ VEGF receptors in the epididymis --- p.20 / Chapter 1.6 --- Aims of study --- p.22 / Chapter Section 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.2 --- Animal surgery --- p.35 / Chapter 2.2.1 --- Animals --- p.35 / Chapter 2.2.2 --- Castration and hemi-castration --- p.35 / Chapter 2.2.3 --- Efferent duct ligation (EDL) --- p.36 / Chapter 2.2.4 --- Tissue collection --- p.37 / Chapter 2.3 --- Epididymal cell culture --- p.38 / Chapter 2.4 --- Sample preparation --- p.40 / Chapter 2.4.1 --- Collection of epididymal plasma and sperm --- p.40 / Chapter 2.4.2 --- Purification of caveolae fraction --- p.41 / Chapter 2.5 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.43 / Chapter 2.5.1 --- Preparation of RNA from epididymal tissues --- p.43 / Chapter 2.5.2 --- Quantitation of total RNA --- p.44 / Chapter 2.5.3 --- Reverse transcription (RT) and polymerase chain reaction (PCR) --- p.44 / Chapter 2.5.4 --- Purification and authenticity confirmation of PCR products --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.53 / Chapter 2.6.1 --- Preparation of protein --- p.53 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Western immunoblotting --- p.55 / Chapter 2.7 --- Immunohistochemistry --- p.56 / Chapter 2.7.1 --- Preparation of tissue sections --- p.56 / Chapter 2.7.2 --- Immunohistochemical staining of tissue sections --- p.57 / Chapter 2.7.3 --- Immunostaining of cultured cells --- p.59 / Chapter 2.8 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.59 / Chapter 2.9 --- Statistical analyses --- p.60 / Chapter Section 3. --- Results / Chapter 3.1 --- Expression and localization of VEGF in the rat epididymis --- p.62 / Chapter 3.1.1 --- RT-PCR of VEGF in the rat epididymis --- p.62 / Chapter 3.1.2 --- Western immunoblot of VEGF in the rat epididymis --- p.63 / Chapter 3.1.3 --- Developmental changes in VEGF expression in the rat epididymis --- p.66 / Chapter 3.1.4 --- Immunolocalization of VEGF in the rat epididymis --- p.66 / Chapter 3.1.5 --- Summary of the localization and expression of VEGF in the rat epididymis --- p.72 / Chapter 3.2 --- Expression and localization of VEGF receptors in the rat epididymis --- p.74 / Chapter 3.2.1 --- RT-PCR of Flt-1 and sFlt-1 in the rat epididymis --- p.74 / Chapter 3.2.2 --- Western immunoblot of Flt-1 in the rat epididymis --- p.74 / Chapter 3.2.3 --- Immunolocalization of Flt-1 in the rat epididymis --- p.75 / Chapter 3.2.4 --- RT-PCR of Flk-1 in the rat epididymis --- p.75 / Chapter 3.2.5 --- Western immunoblot of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.6 --- Immunolocalization of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.7 --- Summary on the localization and expression of VEGF receptors in the rat epididymis --- p.83 / Chapter 3.3 --- Detection of VEGF immunoreactivity in epididymal plasma and sperm lysate collected from cauda epididymidis --- p.83 / Chapter 3.4 --- Effect of castration on VEGF and VEGF receptor expression in the rat epididymis --- p.84 / Chapter 3.4.1 --- Effect of castration with or without testosterone replacement on VEGF expression in the rat epididymis --- p.84 / Chapter 3.4.2 --- Effect of castration with or without testosterone replacement on Flt-1 expression in the rat epididymis --- p.94 / Chapter 3.4.3 --- Effect of castration with or without testosterone replacement on Flk-1 expression in the rat epididymis --- p.98 / Chapter 3.5 --- Effect of efferent duct ligation and hemicastration on VEGF peptide levels in the rat epididymis --- p.102 / Chapter 3.5.1 --- Effect of efferent duct ligation on VEGF expression in the rat epididymis --- p.102 / Chapter 3.5.2 --- Effect of hemi-castration on VEGF expression in the rat epididymis --- p.106 / Chapter 3.6 --- "Summary on the effects of castration, efferent duct ligation, and hemicastration on the epididymal weight, and VEGF/VEGF receptor expression in the rat epididymis" --- p.107 / Chapter 3.6.1 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on the epididymal weight" --- p.107 / Chapter 3.6.2 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on VEGF and VEGF receptor expression in the rat epididymis" --- p.112 / Chapter 3.7 --- Localization and expression of caveolin-1 and 226}0ؤ2 in the rat epididymis --- p.113 / Chapter 3.7.1 --- RT-PCR of caveolin-1 and caveolin-2 in the rat epididymis --- p.113 / Chapter 3.7.2 --- Western immunoblot of caveolin-1 and caveolin-2 in the rat epididymis --- p.114 / Chapter 3.7.3 --- Immunolocalization of caveolin-1 in the rat epididymis --- p.117 / Chapter 3.7.4 --- Summary on the localization and expression of caveolin-1 and -2 in the rat epididymis --- p.119 / Chapter 3.8 --- Co-localization of VEGF receptors with caveolae in the rat epididymis --- p.119 / Chapter Section 4. --- Discussion --- p.124 / Chapter 4.1 --- VEGF expression and localization --- p.124 / Chapter 4.2 --- VEGF receptors expression and localization --- p.129 / Chapter 4.3 --- Possible VEGF action in the rat epididymis --- p.133 / Chapter 4.4 --- Regulation of VEGF and its receptor expression by androgen and/or other testicular factors --- p.136 / References

by Chan Po Kwok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 132-140). / Abstract --- p.i / Acknowledgement --- p.iii / Publication --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter Section a --- Blood Coagulation --- p.1 / Chapter Section b --- The role of factor XII --- p.4 / Chapter Section c --- Genomic organisation of the human factor XII gene --- p.8 / Chapter Section d --- Protein structure of the human factor XII --- p.12 / Chapter Section e --- Mutations in factor XII --- p.16 / Chapter Section f --- Methods to detect mutations --- p.25 / Chapter Section g --- About the patient with factor XII deficiency --- p.29 / Chapter Section h --- Strategies used in this study --- p.30 / Chapter Chapter 2 --- Methods and Materials / Chapter Section a --- Genomic DNA extraction --- p.33 / Chapter Section b --- Polymerase chain reaction amplification --- p.34 / Chapter Section c --- Cycle sequencing --- p.35 / Chapter Section d --- Restriction enzyme digestion and cloning of PCR products --- p.36 / Chapter Section e --- Reverse transcription and polymerase chain reaction of factor XII ectopic transcript --- p.36 / Chapter Section f --- Subcloning of 95-bp novel fragment --- p.38 / Chapter Section g --- Gel mobility shift assay --- p.39 / Chapter Chapter 3 --- Results / Chapter Section a --- Analysis of the catalytic region of the human factor XII --- p.41 / Chapter Section b --- Ectopic transcript of factor XII in peripheral blood lymphocytes --- p.65 / Chapter Section c --- Analysis of 3'end untranslated region of factor XII gene --- p.70 / Chapter Section d --- Analysis of 5' flanking region of factor XII gene --- p.75 / Chapter Section e --- Analysis of intron B --- p.94 / Chapter Section f --- Analysis of 5'-b PCR product --- p.98 / Chapter Chapter 4 --- Discussions / Chapter Section a --- Mutations in the coding sequence of factor XII gene --- p.107 / Chapter Section b --- Ectopic transcript of factor XII in lymphocytes --- p.113 / Chapter Section c --- The 3'untranslated region of factor XII gene --- p.116 / Chapter Section d --- The 5'flanking region of factor XII gene --- p.117 / Chapter Section e --- Other possible explanations of undetectable factor XII mRNA --- p.120 / Chapter Section f --- The 95-bp novel sequence in ´5ة flanking region --- p.123 / Chapter Section g --- Technical difficulties --- p.126 / Chapter Section h --- Future study --- p.130 / Chapter Chapter 5 --- References --- p.132 / List of Tables --- p.137 / List of Figures --- p.138

Yung, Kam Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 107-129). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / PUBLICATION --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- "The biology, diagnostics and management of lung cancer" --- p.2 / Chapter 1.1 --- "Basic biology, classification and diagnostics" --- p.2 / Chapter 1.1.1 --- Epidemiology and etiology of lung cancer --- p.2 / Chapter 1.1.2 --- Clinical Presentation and Diagnostics of Lung Cancer --- p.3 / Chapter 1.2 --- Treatment of lung cancer --- p.9 / Chapter 1.2.2 --- Radiotherapy --- p.10 / Chapter 1.2.3 --- Chemotherapy --- p.11 / Chapter CHAPTER 2: --- Epidermal Growth Factor Receptor Mutations in Lung Cancer --- p.13 / Chapter 2.1 --- The Epidermal Growth Factor Receptor --- p.13 / Chapter 2.2 --- Overexpression of EGFR in NSCLC --- p.14 / Chapter 2.3 --- The development of EGFR inhibitors --- p.15 / Chapter 2.3.1 --- Monoclonal Antibodies --- p.16 / Chapter 2.3.2 --- Small-molecule inhibitors --- p.17 / Chapter 2.3.2.1 --- Gefitinib --- p.17 / Chapter 2.3.2.2 --- Erlotinib --- p.19 / Chapter 2.3.2.3 --- Other small-molecule inhibitors --- p.20 / Chapter 2.4 --- Mutations of EGFR in NSCLC --- p.21 / Chapter 2.4.1 --- Activating Mutations conferring sensitivity to tyrosine kinase inhibitors --- p.21 / Chapter 2.4.2 --- Secondary mutations associated with resistance to tyrosine kinase inhibitors --- p.23 / Chapter 2.5 --- EGFR gene amplification --- p.24 / Chapter 2.6 --- Detection of EGFR mutations --- p.25 / Chapter 2.7 --- Aim of the thesis --- p.31 / Chapter SECTION II: --- DETECTION OF EGFR MUTATIONS IN TUMOR AND PLASMA SAMPLES BY MASS SPECTROMETRY AND DIGITAL PCR --- p.33 / Chapter CHAPTER 3: --- Detection of EGFR mutations by mass spectrometric methods --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Principles of Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.34 / Chapter 3.1.2 --- The MassARRAY Homogenous MassEXTEND (hME) assay --- p.35 / Chapter 3.1.3 --- The Single-Allele Base Extension Reaction (SABER) and the Allele-Specific Base Extension Reaction (ASBER) --- p.36 / Chapter 3.2 --- Materials and Methods --- p.36 / Chapter 3.2.1 --- The protocol for the detection of EGFR exon 21 point mutation by Mass Spectrometric Methods --- p.37 / Chapter 3.3 --- Results --- p.42 / Chapter 3.4 --- Discussion --- p.49 / Chapter CHAPTER 4: --- Evaluation of the detection limit and sensitivity of the digital PCR assays --- p.51 / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.1.1 --- The theoretical basis of digital PCR quantification and the relationship with the Poisson distribution --- p.51 / Chapter 4.1.2 --- Assessment of Assay Detection Limit --- p.54 / Chapter 4.1.3 --- Comparing Digital PCR with sequencing after conformation sensitive gel electrophoresis (CSGE) --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Design of digital PCR assay for the detection of EGFR exon21 L858R point mutation --- p.59 / Chapter 4.2.2 --- Design of digital PCR assay for the detection of EGFR exon19 deletion --- p.60 / Chapter 4.2.3 --- The protocols of digital PCR assays for EGFR mutation detection --- p.64 / Chapter 4.2.4 --- Single molecule detection test --- p.65 / Chapter 4.2.5 --- Artificial mixtures of mutant and wild-type DNA --- p.66 / Chapter 4.2.6 --- Sequencing after CSGE --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Results of the single molecule detection test and artificial mixture analysis --- p.67 / Chapter 4.3.2 --- Results of CSGE and sequencing compared with digital PCR --- p.73 / Chapter 4.4 --- Discussion --- p.75 / Chapter CHAPTER 5: --- Detection of EGFR mutations in prospectively collected tumor samples of NSCLC patients --- p.77 / Chapter 5.1 --- Introduction --- p.77 / Chapter 5.2 --- Materials and Methods --- p.78 / Chapter 5.2.1 --- Sample preparation and DNA extraction of tumor tissues --- p.78 / Chapter 5.3 --- Results --- p.79 / Chapter 5.4 --- Discussion --- p.82 / Chapter CHAPTER 6: --- Detection of EGFR mutations in prospectively collected plasma samples of NSCLC patients --- p.85 / Chapter 6.1 --- Introduction --- p.85 / Chapter 6.2 --- Materials and Methods --- p.87 / Chapter 6.2.1 --- Sample preparation and DNA extraction of plasma samples --- p.87 / Chapter 6.3 --- Results --- p.88 / Chapter 6.3.1 --- Digital PCR analysis of EGFR mutations in plasma samples of NSCLC patient --- p.88 / Chapter 6.3.2 --- Variations in plasma EGFR mutation concentration after TKI treatment --- p.93 / Chapter 6.4 --- Discussion --- p.96 / Chapter SECTION III: --- CONCLUDING REMARKS --- p.100 / Chapter CHAPTER 7: --- Conclusion and future perspectives --- p.101 / Chapter 7.1 --- Mass spectrometric analysis --- p.101 / Chapter 7.2 --- Microfluidics Digital PCR --- p.102 / Chapter 7.3 --- Future perspectives --- p.105 / References --- p.107

En la actualidad existen cargas inductivas dentro de un sistema eléctrico, generando el consumo de potencia reactiva, este consumo genera pérdidas económicas para la empresa Electro Pangoa S.A., que en cumplimiento de la norma de opciones tarifarías, se aplicara cuando el consumo de energía reactiva son mayores al 30% de energía activa total mensual, aplicando para la facturación el sobrexcedo de la energía reactiva inductiva, el problema general de la presente tesis es, ¿Cuál es la influencia de un banco de condensadores en la reducción del consumo de potencia reactiva en el sistema eléctrico EPASA, San Martin de Pangoa, 2018?. Siendo el objetivo general, determinar la influencia del banco un condensadores, para reducir por lo menos un 30% el consumo de potencia reactiva en el sistema eléctrico EPASA, San Martin de Pangoa, 2018., con la hipótesis qué, el banco de condensadores influye positivamente en la reducción del consumo de potencia reactiva en el sistema eléctrico EPASA, San Martin de Pangoa, 2018. El propósito de la investigación es que en base a los datos obtenidos se analizará la influencia de un banco de condensadores para mejorar la problemática encontrada en la unidad de análisis. Este trabajo de tesis, reporta un estudio básico, con un nivel de investigación descriptivo analítico, método de investigación cuantitativo; y se trabajará teniendo en cuenta las lecturas mensuales que se registra en el medidor del punto de entrega de la empresa EPASA, que generaliza a las 13 subestaciones del sistema eléctrico y la información recopilada se analizara a través de diagramas de cargas diarios de energía reactiva y así poder calcular el sobre exceso de este tipo de energía. Las principales conclusiones son: La influencia de un banco de condensadores es positivo respecto a la reducción de consumo de potencia reactiva, se obtuvo en la condición actual, en horas punta un consumo de 257.90 kVAR y en fuera de punta 230.53 kVAR, como valores máximos, logrando reducir a 167.90 kVAR en hora punta y fuera de punta a 120.52 kVAR.

This study analyzed sex differences in methylphenidate (MPH) sensitization and corresponding changes in glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotprhic factor protein (BDNF) in adolescent male and female rats. After habituation to a locomotor arena, animals were sensitized to MPH (5mg/kg) or saline from postnatal day (P) 33–49, tested every second day. On P50, one group of animals were injected with saline and behavior assessed for conditioned hyperactivity. Brain tissue was harvested on P51 and analyzed for GDNF protein. A second group of animals was also sensitized to MPH from P33 to 49, and expression of behavioral sensitization was analyzed on a challenge given at P60, and BDNF protein analyzed at P61. Females demonstrated more robust sensitization to MPH than males, but only females given MPH during sensitization demonstrated conditioned hyperactivity. Interestingly, MPH resulted in a significant increase in striatal and accumbal GDNF with no sex differences revealed. Results of the challenge revealed that females sensitized and challenged with MPH demonstrated increased activity compared to all other groups. Regarding BDNF, only males given MPH demonstrated an increase in dorsal striatum, whereas MPH increased accumbal BDNF with no sex differences revealed. A hierarchical regression analysis revealed that behavioral sensitization and the conditioned hyperactivity test were reliable predictors of striatal and accumbal GDNF, whereas sensitization and activity on the challenge were reliable predictors of accumbal BDNF, but had no relationship to striatal BDNF. These data have implications for the role of MPH in addiction and dopamine system plasticity.

In this study I investigate the role of Schwann cell and axon-derived trophic signals as modifiers of axonal integrity and sprouting in motoneuron disease and diabetic neuropathy (DNP). The first part of this thesis focuses on the role of the Schwann-cell-derived ciliary neurotrophic factor (CNTF) for compensatory sprouting in a mouse model for mild spinal muscular atrophy (SMA). In the second part, the role of the insulin-like growth factor 1 (IGF-1) and its binding protein 5 (IGFBP-5) is examined in the peripheral nerves of patients with DNP and in two corresponding mouse models. Proximal SMA is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5. The different forms of SMA can be divided into four groups, depending on the levels of SMN protein produced from a second SMN gene (SMN2) and the severity of the disease. Patients with milder forms of the disease, type III and type IV SMA, normally reach adulthood and regularly show enlargement of motor units, signifying the reinnervation of denervated muscle fibers. However, the underlying mechanisms are not understood. Smn+/- mice, a model of type III/IV SMA, are phenotypically normal, but they reveal progressive loss of motor neurons and denervation of motor endplates starting at 4 weeks of age. The progressive loss of spinal motor neurons reaches 50% at 12 months but muscle strength is not reduced. The first evidence for axonal sprouting as a compensatory mechanism in these animals was the more than 2-fold increase in amplitude of single motor unit action potentials (SMUAP) in the gastrocnemius muscle. Confocal analysis confirmed pronounced sprouting of innervating motor axons. As CNTF is highly expressed in Schwann cells and known to be involved in sprouting, its role for this compensatory sprouting response and the maintenance of muscle strength in Smn+/- mice was investigated. Deletion of CNTF in this mouse model results in reduced sprouting and decline of muscle strength in Smn+/- Cntf-/- mice. These findings indicate that CNTF is necessary for a sprouting response and thus enhances the size of motor units in skeletal muscles of Smn+/- mice. DNP afflicting motor and sensory nerve fibers is a major complication in diabetes mellitus. The underlying cellular mechanisms of motor axon degeneration are poorly understood. IGFBP-5, an inhibitory binding protein for IGF-1, is highly upregulated in peripheral nerves in patients with DNP. The study investigates the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP-5 in motor axons. These mice develop motor axonopathy similar to that seen in DNP. Motor axon degeneration is also observed in mice in which the IGF-1 receptor (IGF-1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF-1 on IGF-1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP-5 in diabetic nerves reduces the availability of IGF-1 for IGF-1R on motor axons leading to progressive neurodegeneration, and thus offers novel treatment strategies. / In dieser Arbeit habe ich die Rolle der neurotrophen Faktoren Ciliary neurotrophic factor (CNTF) und Insulin-like-growth factor 1 (IGF-1), die in Schwannzellen gebildet werden, als Modulatoren der axonalen Integrität bei einer degenerativen Motoneuronenerkrankung und bei diabetischer Neuropathie (DNP) untersucht. Im ersten Teil dieser Arbeit wird gezeigt, dass CNTF für ein kompensatorisches Sprouting von motorischen Axonen in einem Mausmodell für spinale Muskelatrophie (SMA) verantwortlich ist. Im zweiten Teil wird die Rolle von IGF-1 und dessen Bindeprotein, IGFBP-5, in Axonen motorischer Nerven bei Patienten mit DNP und zwei korrespondieren Mausmodellen gezeigt. Die proximale SMA wird durch einen homozygoten Verlust oder Mutation des SMN1 Gens auf dem Chromosom 5 verursacht. Bei der spinalen Muskelatrophie unterscheidet man verschiedene Schweregrade, abhängig von der Menge an SMN Protein, das vom zweiten SMN Gen (SMN2) produziert werden kann. Patienten mit einer milderen Form von SMA (Typ III und IV) erreichen das Erwachsenenalter und zeigen oft vergrößerte motorische Einheiten, im Gegensatz zu Patienten mit den schweren kindlichen Formen der Erkrankung. Smn+/- Mäuse, ein Modell für die leichten SMA Formen Typ II und IV, zeigen denervierte Endplatten bereits 4 Wochen nach der Geburt und einen fortschreitenden Verlust von Motoneuronen, der nach 12 Monaten mehr als 50% beträgt, ohne dass sich die Muskelkraft der Tiere verringert. Die Amplitude der Summenpotenziale von einzelnen motorischen Einheiten (Single motor unit action potential, SMUAP) im Wadenmuskel ist mehr als 2-fach erhöht. Konfokale Aufnahmen bestätigen ausgeprägtes Sprouting der noch innervierenden Axone. Smn+/- Mäuse ohne CNTF, das normalerweise stark in Schwann-Zellen exprimiert ist, zeigen reduziertes Sprouting und verringerte Muskelkraft. Diese Ergebnisse sprechen dafür, dass CNTF für das Sprouting und die vergrößerten motorischen Einheiten in Smn+/- Mäusen verantwortlich ist. Dieser kompensatorische Mechanismus könnte neue Behandlungs-möglichkeiten für Motoneuronerkrankungen eröffnen. Die Diabetische Neuropathie (DNP), eine der Hauptkomplikationen bei Diabetes Mellitus, betrifft sowohl motorische als auch sensorische Nervenfasern. Die zugrunde liegenden zellulären Mechanismen, die zur Degeneration motorischer Axone in Spätstadien der Erkrankung führen, sind größtenteils noch ungeklärt. IGFBP-5, ein IGF-1 hemmendes Bindeprotein, ist in peripheren Nervbiopsien von DNP Patienten stark überexprimiert. Diese potenzielle pathogene Relevanz wurde bei IGFBP-5 überexprimierenden transgenen Mäusen untersucht. Diese Mäuse entwickeln ähnlich wie die DNP Patienten eine motorische Axonopathie. Diese Axondegeneration zeigen auch Mäuse, bei denen der IGF-1 Rezeptor (IGF-1R) neuronenspezifisch ausgeschaltet wurde. Das bedeutet, dass reduzierte Wirkung von IGF-1 am IGF-1R auf Axonen von Motoneuronen für die beobachtete Axonopathie verantwortlich ist. Zusammenfassend zeigen diese Daten, dass erhöhtes IGFBP-5 in diabetischen Nerven die Verfügbarkeit von IGF-1 für den IGF-1R reduziert und zu progressiver Neurodegeneration führt. Diese Erkenntnis könnte neue Behandlungsstrategien für Patienten mit DNP eröffnen.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1994

xxiii, 222 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Insulin-like growth factor-I circulates at high concentrations in blood, mainly complexed with IGF-binding proteins. The main objective of the thesis is to determine the general role played by plasma IGF-binding proteins in the regulation of IGF transfer from blood to tissues. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1994