No description available.

No description available.

<p>α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor (serpin) in plasma. The α₁-PI M358R mutant exhibits greatly increased rates of thrombin inhibition compared to wild type α₁-PI, which predominantly inhibits neutrophil elastase. M358R (P1) lies at the reactive centre (P1-P1’) bond of the reactive centre loop (RCL) of α₁-PI, cleaved by cognate proteases as they become trapped in the serpin-type inhibitory complex. The relationship between RCL structure and serpin inhibitor function is incompletely understood and has not been subjected to saturation mutagenesis. α₁-PI M358R is a less potent inhibitor of thrombin than natural thrombin-inhibitory serpins, suggesting room for engineered improvement into an antithrombotic protein drug.</p> <p>Phage display is a powerful tool for screening mutant protein libraries, but only one serpin (PAI-1) has previously been mutated and expressed in this manner. In this study the T7Select10-3b (Novagen) phage display system was used to express α₁-PI variants and PAI-1, fused to the first 348 residues of the T7 10B coat protein. Following confirmation that α₁-PI M358R retained inhibitory activity when fused to T7Select10-3b phage, this system was used to express a library of α₁-PI mutant proteins with all possible codon combinations at positions P2 (P357) and P1 (M358) (441 mutants). The library was biopanned using a novel technique in order to amplify only the α₁-PI P2P1 mutants capable of forming stable complexes with thrombin. The P357/M358R mutant was the only P2P1 mutant enriched, indicating that the α₁-PI M358R protein has the optimal P2P1 sequence for thrombin inhibition.</p> <p>A second T7Select10-3b library of α₁-PI mutant proteins was generated to identify the optimal sequence at positions P7 through to P3 (amino acids 352-356) for thrombin inhibition. The P2 and P1 positions were maintained at P357/M358R, while all possible codon combinations at positions P7 through to P3 were represented (>4.08 million mutants). The library was biopanned using the protocol developed for the P2P1 library, before sequences were inserted into an <em>E. coli</em> expression vector and α₁-PI M358R P7-P3 mutants were screened for thrombin inhibitory activity. 80 individual colonies were screened, yielding 22 unique P7-P3 mutants with thrombin inhibitory activity greater than the M358R RCL sequence. The consensus observed in sequences with improved activity matched thrombin’s known substrate specificity and also general RCL trends: P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic.</p> <p>Kinetic characterization of selected mutants with improved thrombin inhibitory activity yielded two mutants, P7-P3 sequence DITMA and AAFVS, with a second order rate constant of 1.0 x 10⁶ M^-1s^-1. This represents a >2-fold increase in the rate of thrombin inhibition versus α₁-PI M358R. Both the DITMA and AAFVS mutants were found to have a lower stoichiometry of inhibition compared to α₁-PI M358R, indicating that an improved thrombin inhibitory mechanism was also enriched during biopanning.</p> <p>These findings suggest that based on the scaffold of the α₁-PI protein, improved thrombin inhibitory activity can be engineered and selected via phage display. Additionally, this work represents a proof-of-principle for the application of this system to screen libraries of up to 10 million mutants in order to better engineer serpins towards a desired activity.</p> / Master of Health Sciences (MSc)

INTRODUÇÃO: A HPN é uma patologia na qual a atividade do sistema complemento, sem oposição, leva a complicações sistêmicas. Ela é caracterizada por anemia hemolítica adquirida com hemoglobinúria intermitente, falência medular e fenômenos tromboembólicos (TE). A trombose venosa é a sua principal causa de mortalidade, entretanto o seu mecanismo fisiopatológico é apenas parcialmente elucidado. O grande número de pacientes com trombocitopenia dificulta o manejo da profilaxia antitrombótica secundária e primária. Optou-se por evidenciar o desequilíbrio hemostático associado ao clone HPN através de um teste de avaliação global da coagulação. MÉTODOS: Para a detecção do potencial hemostático de cada indivíduo foi utilizado um ensaio fluorogênico de geração de trombina, em amostra de plasma pobre em plaquetas na presença e na ausência de trombomodulina (TM). A eficiência na redução do potencial de trombina endógeno (ETP) e da concentração máxima de trombina (pico) pela TM foi utilizada para a identificação do estado de hipercoagulabilidade. O tempo para o início da geração de trombina (tempo de latência) e para a concentração máxima de trombina foram utilizados para a detecção do fenótipo hemorrágico. Os indivíduos foram categorizados em três grupos: HPN, se clone HPN >= 10%; anemia aplástica idiopática adquirida ou associada a clone < 10%, e normais. Os pacientes e controles foram submetidos a avaliação laboratorial que incluiu pesquisa de trombofilia (TB) e do clone HPN, hemograma, testes habituais de avaliação da hemostasia, e no grupo de pacientes análise bioquímica. Os participantes foram avaliados para a identificação da presença de fatores de risco para TE através de questionário. A análise dos resultados foi realizada em duas fases: a primeira incluiu apenas indivíduos com pesquisa de TB negativa e sem fatores de risco para TE; a segunda, realizada apenas no grupo de pacientes, também incluiu indivíduos em uso de contraceptivo hormonal, diagnóstico de infecção assintomática, evento de TE associado a fator de risco temporário, em período superior a 1 ano da inclusão no estudo, e com pesquisa de TB positiva. Esta última fase da análise teve como objetivo incluir um maior número de pacientes com o diagnóstico destas patologias, de baixa prevalência populacional. RESULTADOS: A presença do clone >= 10% foi associada à ineficiência da ação da TM em reduzir o ETP e o pico. O primeiro, de maior relevância científica e clínica, apresentou correlação positiva e negativa, respectivamente, com a atividade do fator von Willebrand (FvW:RCo) e com níveis plasmáticos de proteína C (PC). O grupo HPN apresentou menor tempo para atingir o pico. Na segunda fase, o tempo de latência apresentou correlação negativa com o número de plaquetas no grupo HPN, e houve correlação positiva do clone com a ineficiência da ação da TM na redução do ETP. CONCLUSÕES: o teste de geração de trombina é eficaz na detecção do fenótipo protrombótico associado ao clone HPN. As correlações encontradas com o FvW:RCo e a PC sugerem que a ativação endotelial e o sistema da PC, respectivamente, podem estar comprometidos. A inflamação secundária à ativação do sistema complemento pode levar à redução de expressão endotelial da TM e do receptor da PC. Entretanto os nossos achados, associados à descrição recente da redução de expressão e de atividade da TM, secundária à depleção de óxido nítrico (em estudos com estatinas), podem justificar a característica agressiva da trombofilia na HPN, e o desenvolvimento de TE mesmo nos pacientes em anticoagulação oral. Os parâmetros que avaliam o \'tempo\' no trombograma (tempo de latência e tempo para o pico) podem auxiliar na identificação do risco hemorrágico eventualmente associado à HPN / INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. The pathology is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic event (TE). Venous thrombosis is the main cause of death, however, its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We chose to demonstrate the hemostatic unbalance associated with PNH clone through a global evaluation of the coagulation test. METHODS: To detect the hemostatic potential, we used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without throbomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential (ETP) and of the upper limit of thrombin concentration (peak) was done to identify the hypercoagulable state. Times to initiate thrombin generation (latency time-LT) and for reaching the peak were used to identify hemorrhagic phenotypes. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%), and controls. Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood test, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TE through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TE; the second step, done only in patients, included individuals using hormonal contraceptive, diagnosis of any asymptomatic infection, TE associated to temporary risk factors, and which have occurred in a period longer than one year since inclusion in the study, and positive TB. The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. RESULTS: The presence of the clone >= 10% was associated with TM inefficiency in reducing the ETP (ETP+TM) and peak. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP+TM and the activity of the von Willebrand factor (FvW:RCo), whereas a negative correlation was observed with the levels of protein C (PC). The PNH group presented the shortest time to reach the peak. In the second step of the analysis, the LT showed negative correlation with platelet counts in the PNH group, whereas a positive correlation between the clone and ETP+TM was observed. CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The correlation found with both FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Secondary inflammation to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor. However, our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statin) may explain the aggressive nature of thrombophilia in PNH and the development of TE in these patients, even in those taking oral anticoagulants. The parameters that measure the time of thrombin generation may help identify the hemorrhagic risk that might be associated with PNH

INTRODUÇÃO: A HPN é uma patologia na qual a atividade do sistema complemento, sem oposição, leva a complicações sistêmicas. Ela é caracterizada por anemia hemolítica adquirida com hemoglobinúria intermitente, falência medular e fenômenos tromboembólicos (TE). A trombose venosa é a sua principal causa de mortalidade, entretanto o seu mecanismo fisiopatológico é apenas parcialmente elucidado. O grande número de pacientes com trombocitopenia dificulta o manejo da profilaxia antitrombótica secundária e primária. Optou-se por evidenciar o desequilíbrio hemostático associado ao clone HPN através de um teste de avaliação global da coagulação. MÉTODOS: Para a detecção do potencial hemostático de cada indivíduo foi utilizado um ensaio fluorogênico de geração de trombina, em amostra de plasma pobre em plaquetas na presença e na ausência de trombomodulina (TM). A eficiência na redução do potencial de trombina endógeno (ETP) e da concentração máxima de trombina (pico) pela TM foi utilizada para a identificação do estado de hipercoagulabilidade. O tempo para o início da geração de trombina (tempo de latência) e para a concentração máxima de trombina foram utilizados para a detecção do fenótipo hemorrágico. Os indivíduos foram categorizados em três grupos: HPN, se clone HPN >= 10%; anemia aplástica idiopática adquirida ou associada a clone < 10%, e normais. Os pacientes e controles foram submetidos a avaliação laboratorial que incluiu pesquisa de trombofilia (TB) e do clone HPN, hemograma, testes habituais de avaliação da hemostasia, e no grupo de pacientes análise bioquímica. Os participantes foram avaliados para a identificação da presença de fatores de risco para TE através de questionário. A análise dos resultados foi realizada em duas fases: a primeira incluiu apenas indivíduos com pesquisa de TB negativa e sem fatores de risco para TE; a segunda, realizada apenas no grupo de pacientes, também incluiu indivíduos em uso de contraceptivo hormonal, diagnóstico de infecção assintomática, evento de TE associado a fator de risco temporário, em período superior a 1 ano da inclusão no estudo, e com pesquisa de TB positiva. Esta última fase da análise teve como objetivo incluir um maior número de pacientes com o diagnóstico destas patologias, de baixa prevalência populacional. RESULTADOS: A presença do clone >= 10% foi associada à ineficiência da ação da TM em reduzir o ETP e o pico. O primeiro, de maior relevância científica e clínica, apresentou correlação positiva e negativa, respectivamente, com a atividade do fator von Willebrand (FvW:RCo) e com níveis plasmáticos de proteína C (PC). O grupo HPN apresentou menor tempo para atingir o pico. Na segunda fase, o tempo de latência apresentou correlação negativa com o número de plaquetas no grupo HPN, e houve correlação positiva do clone com a ineficiência da ação da TM na redução do ETP. CONCLUSÕES: o teste de geração de trombina é eficaz na detecção do fenótipo protrombótico associado ao clone HPN. As correlações encontradas com o FvW:RCo e a PC sugerem que a ativação endotelial e o sistema da PC, respectivamente, podem estar comprometidos. A inflamação secundária à ativação do sistema complemento pode levar à redução de expressão endotelial da TM e do receptor da PC. Entretanto os nossos achados, associados à descrição recente da redução de expressão e de atividade da TM, secundária à depleção de óxido nítrico (em estudos com estatinas), podem justificar a característica agressiva da trombofilia na HPN, e o desenvolvimento de TE mesmo nos pacientes em anticoagulação oral. Os parâmetros que avaliam o \'tempo\' no trombograma (tempo de latência e tempo para o pico) podem auxiliar na identificação do risco hemorrágico eventualmente associado à HPN / INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. The pathology is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic event (TE). Venous thrombosis is the main cause of death, however, its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We chose to demonstrate the hemostatic unbalance associated with PNH clone through a global evaluation of the coagulation test. METHODS: To detect the hemostatic potential, we used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without throbomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential (ETP) and of the upper limit of thrombin concentration (peak) was done to identify the hypercoagulable state. Times to initiate thrombin generation (latency time-LT) and for reaching the peak were used to identify hemorrhagic phenotypes. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%), and controls. Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood test, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TE through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TE; the second step, done only in patients, included individuals using hormonal contraceptive, diagnosis of any asymptomatic infection, TE associated to temporary risk factors, and which have occurred in a period longer than one year since inclusion in the study, and positive TB. The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. RESULTS: The presence of the clone >= 10% was associated with TM inefficiency in reducing the ETP (ETP+TM) and peak. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP+TM and the activity of the von Willebrand factor (FvW:RCo), whereas a negative correlation was observed with the levels of protein C (PC). The PNH group presented the shortest time to reach the peak. In the second step of the analysis, the LT showed negative correlation with platelet counts in the PNH group, whereas a positive correlation between the clone and ETP+TM was observed. CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The correlation found with both FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Secondary inflammation to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor. However, our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statin) may explain the aggressive nature of thrombophilia in PNH and the development of TE in these patients, even in those taking oral anticoagulants. The parameters that measure the time of thrombin generation may help identify the hemorrhagic risk that might be associated with PNH

Nanostrukturen haben in den letzten Jahrzehnten durch konsequente Förderung wie der im Jahr 2000 gestarteten National Nanotechnology Initiative der USA oder des deutschen Pendants Aktionsplan Nanotechnologie erhebliches Aufsehen, nicht nur in der Wissenschaft, sondern auch in der technischen und wirtschaftlichen Umsetzung erfahren. In Kombination mit biologischen Systemen, deren Funktionalität sich auf der Größenordnung von Nanometern abspielt, finden nanotechnologische Entwicklungen auf dem Gebiet der Medizin ein großes technisches Anwendungsgebiet. Diese Arbeit widmet sich der Untersuchung und technischen Entwicklung von Siliziumnanodrähten als Sensoren für zukünftige medizinische Anwendungen. Im Gegensatz zu Sensoren die auf dotierten Nanodrähten basieren, wurden hier undotierte Nanodrähte untersucht, die mit geringerem Produktionsaufwand auskommen und mittels Schottky-Barrieren als Feldeffekttransistoren nutzbar sind. Deren Eigenschaften wurden im Hinblick auf pH und Biosensorik theoretisch und experimentell untersucht, sowie technisch in ein lab-on-chip sowie ein kompaktes Multiplexer-Messgerät integriert. In einem zweiten, separaten Teil wurden die Eigenschaften undotierter Nanodrähte für die optische Spektroskopie theoretisch modelliert. Die Inhalte beider Teile werden im folgenden kurz zusammengefasst. Um die elektrischen Sensoreigenschaften der Siliziumnanodrähte zu untersuchen, wurden zunächst Computermodelle der Drähte erstellt, mit deren Hilfe der Elektronentransport in flüssiger Umgebung quantenmechanisch modelliert wurde. Die dafür erstellten Modellvorstellungen waren für die sich daran anschließenden experimentellen Untersuchungen des Rauschverhaltens, der pH-Sensitivität sowie der Biosensoreigenschaften sehr vorteilhaft. Mit Hilfe einer neu entwickelten Messmethode konnte der optimale Arbeitspunkt der Sensoren ermittelt werden, sowie die hohe Sensorqualität mittels einer empirischen mathematischen Beschreibung des zu erwartenden Sensorsignals eingeordnet werden. Weiterhin wurden für die Medizintechnik relevante Messungen von Thrombin durchgeführt. Damit ist für den hier beschriebenen Sensortyp ein proof-of-concept für neuartige medizinische Messelemente gelungen. Um die kleinen Abmessungen der Sensoren darüber hinaus technisch nutzbar zu machen, wurden sie in ein lab-on-chip System integriert, in welchem sie als Sensoren für den pH-Wert sowie die ionische Konzentration in Nanoliter-Tropfen verwendet wurden. Desweiteren wurde in Kooperation mit dem Institut für Aufbau- und Verbindungstechnik ein portables Messgerät entwickelt, welches die parallele Messung mehrerer Nanodrahtsensoren ermöglicht. Im zweiten Teil der Arbeit wird eine theoretische Untersuchung zur Eignung von Silizium-Nanodrähten als Messsonden (Probes) für die optische Spektroskopie vorgestellt. Dazu wurde eine Methode entwickelt mittels derer es möglich ist, Raman und Infrarotspektren von Nanostrukturen mittels Molekulardynamik zu berechnen. Die Methode wurde auf undotierte Silizium-Nanodrähte augewendet und zeigt, dass die Oberflächenbeschaffenheit der Drähte die optischen Spektren entscheidend beeinflusst. Damit konnte die Relevanz von Halbeiter-Nanostrukturen auch für Anwendungen in der optischen Spektroskopie gezeigt werden. / Nanostructures have attracted great attention not only in scientific research, but also in engineering applications during the last decades. Especially in combination with biological systems, whose complex function is controlled from nanoscale building blocks, nanotechnological developments find a huge field of applications in the medical sector. This work is dedicated to the functional understanding and technical implementation of silicon nanowires for future medical sensor applications. In contrast to doped silicon nanowire based sensors, this work is focussed on pure, undoped silicon nanowires, which have lower demands on production techniques and use Schottky-barriers as electric field detectors. The pH and biosensing capabilities of such undoped silicon nanowire field effect transistors were investigated theoretically and experimentally and further integrated in a lab-on-a-chip device as well as a small-scale multiplexer measurement device. In a second separate part, the optical sensing properties of undoped silicon nanowires were theoretically modeled. The main contents of both parts are shortly described in the following paragraphs. A multiscale model of silicon nanowire FETs to describe the charge transport in liquid surrounding in a quantum mechanical framework was developed to investigate the sensing properties of the nanowire sensors in general. The model set the basis for the understanding of the subsequent experimental investigations of noise characterization, pH sensitivity and biosensing properties. With the help of a novel gate sweeping measurement method the optimal working point of the sensors was determined and the high sensor quality could be quantified in terms of an empirical mathematical model. The sensor was then used for measurements of medically relevant concentrations of the Thrombin protein, providing a proof-of-concept for medical applications for our newly developed sensor. In order to exploit the small size of our sensors for technical applications we integrated the devices in lab-on-a-chip system with a microfluidic droplet generation module. There they were used to measure the pH and ionic concentration of droplets. Finally a portable multiplex measurement device for silicon nanowire sensors as well as other ion sensitive FETs was developed in cooperation with the IAVT at TU Dresden (Institut für Aufbau- und Verbindungstechnik). The second part of this thesis investigates the usability of silicon nanowires for optical sensor applications from a theoretical point of view. Therefore a method for the extraction of Raman and Infrared spectra from molecular dynamics simulations was developed. The method was applied to undoped silicon nanowires and shows that the surface properties of the nanowires has a significant effect on optical spectra. These results demonstrate the relevance of semiconductor nanostructures for applications in optical spectroscopy.

Nanostrukturen haben in den letzten Jahrzehnten durch konsequente Förderung wie der im Jahr 2000 gestarteten National Nanotechnology Initiative der USA oder des deutschen Pendants Aktionsplan Nanotechnologie erhebliches Aufsehen, nicht nur in der Wissenschaft, sondern auch in der technischen und wirtschaftlichen Umsetzung erfahren. In Kombination mit biologischen Systemen, deren Funktionalität sich auf der Größenordnung von Nanometern abspielt, finden nanotechnologische Entwicklungen auf dem Gebiet der Medizin ein großes technisches Anwendungsgebiet. Diese Arbeit widmet sich der Untersuchung und technischen Entwicklung von Siliziumnanodrähten als Sensoren für zukünftige medizinische Anwendungen. Im Gegensatz zu Sensoren die auf dotierten Nanodrähten basieren, wurden hier undotierte Nanodrähte untersucht, die mit geringerem Produktionsaufwand auskommen und mittels Schottky-Barrieren als Feldeffekttransistoren nutzbar sind. Deren Eigenschaften wurden im Hinblick auf pH und Biosensorik theoretisch und experimentell untersucht, sowie technisch in ein lab-on-chip sowie ein kompaktes Multiplexer-Messgerät integriert. In einem zweiten, separaten Teil wurden die Eigenschaften undotierter Nanodrähte für die optische Spektroskopie theoretisch modelliert. Die Inhalte beider Teile werden im folgenden kurz zusammengefasst. Um die elektrischen Sensoreigenschaften der Siliziumnanodrähte zu untersuchen, wurden zunächst Computermodelle der Drähte erstellt, mit deren Hilfe der Elektronentransport in flüssiger Umgebung quantenmechanisch modelliert wurde. Die dafür erstellten Modellvorstellungen waren für die sich daran anschließenden experimentellen Untersuchungen des Rauschverhaltens, der pH-Sensitivität sowie der Biosensoreigenschaften sehr vorteilhaft. Mit Hilfe einer neu entwickelten Messmethode konnte der optimale Arbeitspunkt der Sensoren ermittelt werden, sowie die hohe Sensorqualität mittels einer empirischen mathematischen Beschreibung des zu erwartenden Sensorsignals eingeordnet werden. Weiterhin wurden für die Medizintechnik relevante Messungen von Thrombin durchgeführt. Damit ist für den hier beschriebenen Sensortyp ein proof-of-concept für neuartige medizinische Messelemente gelungen. Um die kleinen Abmessungen der Sensoren darüber hinaus technisch nutzbar zu machen, wurden sie in ein lab-on-chip System integriert, in welchem sie als Sensoren für den pH-Wert sowie die ionische Konzentration in Nanoliter-Tropfen verwendet wurden. Desweiteren wurde in Kooperation mit dem Institut für Aufbau- und Verbindungstechnik ein portables Messgerät entwickelt, welches die parallele Messung mehrerer Nanodrahtsensoren ermöglicht. Im zweiten Teil der Arbeit wird eine theoretische Untersuchung zur Eignung von Silizium-Nanodrähten als Messsonden (Probes) für die optische Spektroskopie vorgestellt. Dazu wurde eine Methode entwickelt mittels derer es möglich ist, Raman und Infrarotspektren von Nanostrukturen mittels Molekulardynamik zu berechnen. Die Methode wurde auf undotierte Silizium-Nanodrähte augewendet und zeigt, dass die Oberflächenbeschaffenheit der Drähte die optischen Spektren entscheidend beeinflusst. Damit konnte die Relevanz von Halbeiter-Nanostrukturen auch für Anwendungen in der optischen Spektroskopie gezeigt werden.:I Introduction: Sensing with Nanostructures 1 Introduction 2 Field effect transistors as electronic sensor elements 3 Packaging: Connecting Nano and Macro 4 Nanostructures as transducers in optical spectroscopy II Electronic sensing with Schottky Barrier silicon nanowires 5 Schottky-Barrier silicon nanowire field effect transistors 6 ISFET measurement principles 7 pH and Biosensing with silicon nanowires 8 Thrombin sensing 9 Silicon nanowire FETs in a Lab-on-a-Chip device 10 Multiplexer sensing platform 11 Experimental methods III Simulating optical spectra of silicon nanowires 12 Theoretical fundamentals 13 Computational Methods 14 Results 15 Bibliography 16 Anhang / Nanostructures have attracted great attention not only in scientific research, but also in engineering applications during the last decades. Especially in combination with biological systems, whose complex function is controlled from nanoscale building blocks, nanotechnological developments find a huge field of applications in the medical sector. This work is dedicated to the functional understanding and technical implementation of silicon nanowires for future medical sensor applications. In contrast to doped silicon nanowire based sensors, this work is focussed on pure, undoped silicon nanowires, which have lower demands on production techniques and use Schottky-barriers as electric field detectors. The pH and biosensing capabilities of such undoped silicon nanowire field effect transistors were investigated theoretically and experimentally and further integrated in a lab-on-a-chip device as well as a small-scale multiplexer measurement device. In a second separate part, the optical sensing properties of undoped silicon nanowires were theoretically modeled. The main contents of both parts are shortly described in the following paragraphs. A multiscale model of silicon nanowire FETs to describe the charge transport in liquid surrounding in a quantum mechanical framework was developed to investigate the sensing properties of the nanowire sensors in general. The model set the basis for the understanding of the subsequent experimental investigations of noise characterization, pH sensitivity and biosensing properties. With the help of a novel gate sweeping measurement method the optimal working point of the sensors was determined and the high sensor quality could be quantified in terms of an empirical mathematical model. The sensor was then used for measurements of medically relevant concentrations of the Thrombin protein, providing a proof-of-concept for medical applications for our newly developed sensor. In order to exploit the small size of our sensors for technical applications we integrated the devices in lab-on-a-chip system with a microfluidic droplet generation module. There they were used to measure the pH and ionic concentration of droplets. Finally a portable multiplex measurement device for silicon nanowire sensors as well as other ion sensitive FETs was developed in cooperation with the IAVT at TU Dresden (Institut für Aufbau- und Verbindungstechnik). The second part of this thesis investigates the usability of silicon nanowires for optical sensor applications from a theoretical point of view. Therefore a method for the extraction of Raman and Infrared spectra from molecular dynamics simulations was developed. The method was applied to undoped silicon nanowires and shows that the surface properties of the nanowires has a significant effect on optical spectra. These results demonstrate the relevance of semiconductor nanostructures for applications in optical spectroscopy.:I Introduction: Sensing with Nanostructures 1 Introduction 2 Field effect transistors as electronic sensor elements 3 Packaging: Connecting Nano and Macro 4 Nanostructures as transducers in optical spectroscopy II Electronic sensing with Schottky Barrier silicon nanowires 5 Schottky-Barrier silicon nanowire field effect transistors 6 ISFET measurement principles 7 pH and Biosensing with silicon nanowires 8 Thrombin sensing 9 Silicon nanowire FETs in a Lab-on-a-Chip device 10 Multiplexer sensing platform 11 Experimental methods III Simulating optical spectra of silicon nanowires 12 Theoretical fundamentals 13 Computational Methods 14 Results 15 Bibliography 16 Anhang

Zusätzlich zu seiner Funktion innerhalb des Gerinnungssystems vermittelt Thrombin inflammatorische Reaktionen. Mastzellen (MZ) sind durch die Freisetzung von proinflammatorischen Mediatoren wie Maus-Mastzell Proteasen (MCPTs mouse mast cell proteases auch als mMCPs bekannt), Zytokinen und Chemokinen. charakterisiert. Da Thrombinrezeptoren, auch als Proteinase-aktivierbare Rezeptoren (PAR) bekannt, von MZ exprimiert werden, wurde untersucht ob eine MZ-Aktivierung über die Thrombin/PAR Interaktion bei einer dermalen Entzündung eine Rolle spielt. Die intrakutane Injektion von Thrombin in die Ohren von C57BL/6 Kit+/+ Mäusen löste eine sofortige kutane Entzündung, einhergehend mit einer starken Ohrschwellung, aus. Im Vergleich dazu war diese Schwellung in MZ-defizienten C57BL/6 KitW-sh/W-sh Mäusen deutlich stärker, was darauf hindeutet, dass MZ anti-inflammatorisch wirken. Die lokale Rekonstitution von C57BL/6 KitW-sh/W-sh Mäusen mit knochenmarksgenerierten MZ normalisierte diesen Effekt. Die quantitative histomorphometrische Untersuchung der MZ bestätigte zusätzlich eine starke Degranulation der MZ nach Thrombininjektion nach. PCR-Analysen der MZ wiesen die Expression aller bekannten Thrombin-Rezeptoren. Die Stimulation mit verschiedenen Konzentrationen von Thrombin oder PAR-agonistischen Peptiden führte zu einer dosis-abhängigen Degranulation der MZ, was nahe legt, dass die Degranulation der MZ für die Limitierung der thrombin-induzierten Entzündung nötig ist. Gestützt wird die Hypothese durch die Tatsache, dass Zellkulturüberstand von degranulierten MZ zu einer Inaktivierung der Thrombinaktivität führt. Des Weiteren führte die Injektion von Thrombin in die Ohren von MCTP4-defizienten Mäusen zu einer deutlich erhöhten Ohrschwellung im Vergleich zur korrespondierenden Wildtyp-Maus. Zusammengenommen zeigen die Ergebnisse, dass die sofortige thrombin-induzierte Entzündungsreaktion durch kutane MZ kontrolliert wird. Dieser Mechanismus wird teilweise durch MCPT4 vermittelt. / In addition to its function in the coagulation system, thrombin mediates inflammatory reactions. Mast cells (MCs) are characterized by releasing inflammatory mediators like mouse mast cell proteases (MCPTs, also designated mMCPs), cytokines, and chemokines, upon activation. Since thrombin-receptors, also known as Proteinase-activated receptors (PAR), are expressed by MCs, it was questioned whether MC activation via the thrombin/PAR axis plays a role in skin inflammation. Intracutaneous injection of thrombin in ears of C57BL/6 Kit+/+ mice induced immediate inflammatory skin reactions associated with a distinct ear swelling. This swelling was more pronounced in MC-deficient C57BL/6 KitW-sh/W-sh mice, indicating that MCs are anti-inflammatory, local reconstitution of C57BL/6 KitW-sh/W-sh mice with C57BL/6 Kit+/+ bone marrow-derived MCs normalized this effect. Additionally thrombin injection resulted in a strong degranulation of MCs assessed by quantitative histomorphometry. PCR analysis of MCs displayed expression of all known thrombin receptors. Stimulation with thrombin or PAR agonistic peptides resulted in a dose-dependent degranulation of MC, suggesting MC degranulation could be necessary for the limitation of thrombin-induced inflammatory responses. Supporting this hypothesis, supernatant from degranulated MCs inactivated thrombin activity. Furthermore, injection of thrombin in ears of C57BL/6 MCPT4-deficient mice, resulted in markedly increased ear swelling compared to the corresponding wild type mice. Together our results suggest that thrombin-induced immediate inflammatory skin reactions are controlled by cutaneous MCs, a mechanism partly mediated via MCPT4.

Einige Patienten mit Raynaud-Syndrom, Urtikaria, koronarer Herzkrankheit, Angina pectoris, oder Pulmonaler Hypertonie haben funktionelle Autoantikörper gegen die Thrombin-Rezeptoren PAR1/2 und/oder gegen den Endothelin-Rezeptor ET(A). In dieser Arbeit wurde die Wirkung solcher Patienten-IgG-Präparate auf Funktionen von Ventrikel-Kardiomyozyten neonataler Ratten; humanen, glatten Muskelzellen aus Coronararterien (hCASMC), frisch isolierten, humanen Thrombozyten sowie von Monozyten untersucht. Zum Vergleich wurden die PAR-Agonisten Thrombin und das stimulierende Peptid SFLLRN sowie Endothelin-1 verwendet. Während aufgereinigte ET(A)-Autoantikörper ERK1/2 in Kardiomyozyten nicht aktivierten, bewirkten IgG-Präparate mit PAR-Autoantikörpern hier eine ähnliche Aktivierung wie das Peptid SFLLRN. Überraschenderweise bewirkte Kontroll-IgG eine starke Aktivierung von ERK1/2. Die Coinkubation der Kardiomyozyten mit Antikörper-Präparaten und IL-1beta erhöhte die Phosphorylierung von ERK1/2 in allen Fällen. In hCASMCs bewirkten IgG-Präparate mit PAR-Autoantikörpern und Kontroll-IgGs eine Aktivierung von ERK1/2, ET(A)-Autoantikörper nicht. Die Ergebnisse der Thrombozytenaktivierung durch Patienten-IgG waren unterschiedlich. Versuche mit vorstimulierten Thrombozyten zeigten, dass ein stimulierender Einfluss der Autoantikörper auf präaktivierte Thrombozyten nicht ausgeschlossen werden kann. Ohne Vorstimulation jedoch schien Patienten-IgG eher einen hemmenden Einfluss auf die Thrombozytenfunktion zu haben. Eine Vorinkubation von Monozyten mit Patienten-IgG hatte keinen Einfluss auf die PMA-induzierte Produktion von Superoxidanion im Vergleich zu Kontroll-IgG. Nur bei zwei von fünf Patienten-IgGs konnte eine stimulierende Wirkung anhand der monozytären ERK1/2-Phosphorylierung gefunden werden. Die Schlussfolgerung aus dieser Arbeit liegt darin, dass PAR1/2- und ET(A)-Autoantikörper keine allgemeine Wirkung auf die Funktion von glatten Gefäßmuskelzellen, Thrombozyten und Monozyten zeigten. Die Unterscheidung von Autoantikörper-positiven und -negativen IgG-Präparaten war nur über die Bestimmung der Pulsationsrate von Kardiomyozyten möglich. / Some patients with Raynaud’s syndrome, urticaria, coronary artery disease, Angina or pulmonary hypertension have functional autoantibodies against thrombin receptors PAR1/2 and/or against endothelin receptor ET(A). In this work the effects of such patients’ IgG preparations on functions of ventricular cardiomyocytes of neonatal rats, human smooth muscle cells from coronary arteries (hCASMC); freshly isolated, human platelets as well as monocytes were investigated. For comparison, the PAR agonists thrombin and the stimulating peptide SFLLRN as well as endothelin-1 were used. While purified autoantibodies against ET(A) did not activate ERK1/2 in cardiomyocytes, IgG preparations with autoantibodies against PAR1/2 resulted in a similar activation as the peptide SFLLRN. Surprisingly, control IgG also caused a strong activation of ERK1/2. Coincubation of cardiomyocytes with antibody preparations and IL-1beta increased the phosphorylation of ERK1/2 in all cases. In hCASMCs, IgG preparations with PAR-autoantibodies and control IgGs caused activation of ERK1/2, whereas ET(A)-autoantibodies did not. The results of platelet activation with patients’ IgG were varying. Tests with prestimulated platelets showed, that a stimulating effect of the autoantibodies on preactivated platelets can not be excluded. However, without prestimulation patients’ IgG rather seemed to have an inhibiting effect on platelet function. Preincubation of monocytes with patients’ IgG had no influence on PMA-induced production of superoxide anion compared with control IgG. Only two of five patients’ IgGs showed a stimulating effect on monocytic ERK1/2 phosphorylation. In conclusion, PAR1/2- and ET(A)-autoantibodies showed no common effects on the function of vascular smooth muscle cells, platelets and monocytes. The differentiation of autoantibody-positive and -negative preparations of IgG only was possible by determining the beating rate of cardiomyocytes.

Die vorliegende Arbeit untersucht in einer Population von 132 gesunden Probanden die Hämostase mittels Calibrated Automated Thrombogram (CAT) und Rotationsthromboelastometrie (ROTEM). CAT wurde im plätchenarmen Plasma mit einer tissue factor (TF) von 1 und 5 pM durchgeführt. Lag time, Thrombin peak, Time to thrombin peak und das endogene Thrombin Potential (ETP) wurden ermittelt. ROTEM wurde ohne Aktivator durchgeführt (NATEM) und die Daten für Gerinnungszeit (clotting time, CT), Gerinnselbildungszeit, Alpha Winkel und maximale Gerinnselfestigkeit (MCF) mit den Daten der Thrombingenerierung korreliert. Es zeigte sich eine positive aber nicht lineare Korrelation bezüglich Alter versus lag time und time to peak, sowie eine annähernd lineare Korrelation bezüglich Alter versus thrombin peak und ETP. Für ROTEM konnte eine positive Korrelation bezüglich Alter versus MCF und Alpha Winkel, aber eine negative Korrelation bezüglich Alter versus CT dargestellt werden. In der Gegenüberstellung beider Assays korrelierten Thrombin peak und ETP (aktiviert mit einer TF Konzentration von 5 pM) signifikant mit dem Alpha Winkel und der MCF. Alle signifikanten Korrelationen zeigten lediglich eine moderate Regressionssteigung. / Published data on thrombin generation variables and their correlation with thromboelastometry in the healthy population are scarce. This study aimed at assessing thrombin generation in adults and its correlation to classical rotational thromboelastometry (ROTEM). Methods: Thrombin generation was measured in platelet-poor plasma from healthy volunteers using the calibrated automated thrombogram (CAT) with 1 and 5 pmol/l tissue factor final concentration. Lag time, thrombin peak, time to thrombin peak and endogenous thrombin potential (ETP) were analyzed. ROTEM was performed without activator (NATEM) and data for clotting time, alpha angle, clot formation time and maximum clot firmness were correlated with those of thrombin generation. Results: Altogether 132 persons (72 men, 60 women; median age: 48.0 years) were included. There was a positive non-linear correlation for age versus lag time (p < 0.001) and time to peak (p = 0.001), and almost linear correlation for age versus thrombin peak (p = 0.024) and ETP (p = 0.001), although with a moderate regression slope. Regarding ROTEM, there was a positive correlation between age and maximum clot firmness and alpha angle (p = 0.001), but a negative correlation between age and clotting time (p = 0.039). Comparing both assays, thrombin peak and ETP measured with a final tissue factor concentration of 5 pmol/l correlated significantly with alpha angle and maximum clot firmness. Conclusion: The age-related changes in CAT and ROTEM variables among adults are not linear. There is a significant correlation, although with a moderate slope, between data from CAT measured with 5 pmol/l tissue factor and ROTEM.