Pandrol Rail Fastenings Limited are a designer and manufacturer of railway rail-fastening systems. As an organisation they have the capability to reduce the noise impact of bridges using resilient track components. They also have a commercial interest in providing such technology. Knowledge of the processes behind bridge noise is important to Pandrol in two ways; to aid the engineers within the organisation in the design of fastening systems and to demonstrate a state-of-the-art understanding of the problem of railway bridge noise to customers, as this will aid in the sale of Pandrol products. The fitting of new rail components to an existing track form, or failure to meet noise regulations with a new track form, can be costly. It is important to be able to predict accurately the effectiveness of noise reduction techniques. Currently, Pandrol’s knowledge of the problem consists almost entirely of experience gained and data gathered while working on existing bridge projects. To expand their knowledge base, Pandrol perform noise and vibration measurements on railway bridges and viaducts and then use the measured data to predict the performance of their systems on other bridges. This completely empirical approach to predicting bridge noise is both costly and situation specific results cannot be provided before the installation of the fastening system. ii Another approach to predicting bridge noise is through the application of analytical models. Limited analytical modelling in the context of bridge noise is currently conducted within the organisation. For these reasons, Pandrol are sponsoring research into bridge noise in the form of this EngD project. Here an existing rapid calculation approach is identified that relies less on the exact geometry of the bridge and more on its general characteristics. In this approach an analytical model of the track is coupled to a statistical energy analysis (SEA) model of the bridge. This approach forms a suitable basis from which to develop a better model here by concentrating on its weaknesses. A mid-frequency calculation for the power input to the bridge via a resilient track system has been developed by modelling the track-bridge system as two finite Timoshenko beams continuously connected by a resilient layer. This has resulted in a power input calculation which includes the important effects of coupling between the rail and bridge and the resonance effects of the finite length of a bridge. In addition, a detailed study of the frequency characteristics of deep I-section beams has been performed using Finite Element, Boundary Element and Dynamic stiffness models. It is shown that, at high frequencies, the behaviour of the beam is characterised by in-plane motion of the beam web and bending motion in the flange. This knowledge has resulted in an improved calculation for the mobility of a bridge at high frequencies. The above improvements are included in an improved model for use by Pandrol in their general activities. Data from real bridges is compared to predictions from the improved model in order to validate different aspects of the model. The model is then used to study the effect on noise of varying many bridge design parameters. It is shown that the parameter that has most influence on the noise performance of a bridge is the dynamic stiffness of the resilient rail fastening system. Additionally it is demonstrated that for a given bridge and noise receiver location, an optimum fastener stiffness exists where the noise radiated by the bridge and track is at a minimum.

The noise level associated with a train travelling on a bridge is normally greater than that for a train travelling on plain track. It is sometimes the bridge noise that causes the highest levels of disturbance to people in the vicinity or triggers action under regulations such as the Environmental Noise Directive. Consequently, there is a need to study means of predicting noise levels from proposed bridges, noise control measures for existing structures and principles of low-noise bridge design. This thesis describes a programme of work in which an existing calculation model for bridge noise and vibration has been tested and alternative calculation methods have been developed where required. The existing model is based on analytical models for wheel-rail interaction and the calculation of the power input to the bridge. The response of the various component parts of the bridge for this power input is found using a simplified SEA scheme. In this work, the existing model has been tested against measurements made on railway bridges and the results of an advanced method of structural analysis, the Waveguide Finite Element (WFE) method. This method is well-suited to modelling some important types of railway bridge. Specifically, it allows a numerical modelling approach to be used up to higher frequency than conventional Finite Element methods. It has been found to offer some significant advantages over the existing bridge noise model, particularly for concrete-steel composite bridges and concrete box-section viaducts. The track support structure has an important influence on bridge noise and vibration, through its role in the transmission of vibration from the rail to the bridge. Laboratory measurements have been made in this work to characterise the vibration transmission properties of two important types of track support structure on bridges; ballasted track and two-stage resilient baseplate track. Improved methods of modelling the dynamic behaviour of these track forms have been developed from the measurements, which can be used in calculation models for both bridge noise and also for rolling noise.

The prediction of wheel wear is a significant issue in railway vehicles. It is correlated with safety against derailment, economy, ride comfort, and planning of maintenance interventions, and it can result in delay, and costs if it is not predicted and controlled in an effective way. However, the prediction of wheel and rail wear is still a great challenge for railway systems. Therefore, the main aim of this thesis is to develop a method for predicting wheel wear using artificial neural networks. Initial tests were carried out using a pin-on-disc machine and this data was used to establish how wear can be measured using an Alicona profilometer. A new method has been developed for detailed wheel wear and rail wear measurements using ‘Replica’ material which was applied to the wheel and rail surfaces of the test rig to make a copy of both surfaces. The replica samples were scanned using an optical profilometer and the results were processed to establish wheel wear and rail wear. The effect of load, and yaw angle on wheel wear and rail wear were examined. The effect of dry, wet, lubricated, and sanded conditions on wheel wear and rail wear were also investigated. A Nonlinear Autoregressive model with eXogenous input neural network (NARXNN) was developed to predict the wheel and rail wear for the twin disc rig experiments. The NARXNN was used to predict wheel wear and rail wear under deferent surface conditions such as dry, wet, lubricated, and sanded conditions. The neural network model was developed to predict wheel wear in case of changing parameters such as speed and suspension parameters. VAMPIRE vehicle dynamic software was used to produce the vehicle performance data to train, validate, and test the neural network. Three types of neural network were developed to predict the wheel wear: NARXNN, backpropagation neural network (BPNN), and radial basis function neural network (RBFNN). The wheel wear was calculated using an energy dissipation approach and contact position on straight track. The work is focused on wheel wear and the neural network prediction of rail wear was only carried out in connection with the twin disk wear tests. This thesis examines the effect of neural network parameters such as spread, goal, maximum number of neurons, and number of neurons to add between displays on wheel wear prediction. The neural network simulation results were implemented using the Matlab program. The percentage error for wheel and rail wear prediction was calculated. Also, the accuracy of wheel and rail wear prediction using the neural network was investigated and assessed in terms of mean absolute percentage error (MAPE). The results reveal that the neural network can be used efficiently to predict wheel and rail wear. Further work could include rail wear and prediction on a curved track.

Sustainability is a broad concept which embodies social, economic and environmental concerns, including the possible consequences of greenhouse gas (GHG) emissions and climate change, and related means of mitigation and adaptation. The reduction of energy consumption and emissions are key objectives which need to be achieved if some of these concerns are to be addressed. As well as being an important component of sustainability in other sectors, a good transport system needs to be sustainable in its own right. Energy consumption and GHG emissions are important issues within the transport sector; in the European Union (EU), for example, transport is directly responsible for between 25 and 30 percent of all carbon dioxide (CO2) emissions, and the inclusion of indirect (Scope 2 and Scope 3) GHG emissions may increase this proportion further. If reduction targets are to be met, it may be necessary to encourage behavioural change, including modal shift from those modes of transport which are comparatively highly polluting, towards those modes which pollute less. Rail is potentially a suitable target for such modal shift from road transport (notably the private car for passenger travel) and, in some case, from short-haul and domestic aviation. However, modal comparisons are often based on average data, and are reliant on a number of assumptions. There are likely to be some circumstances where modal shift towards rail makes more sense than others, but the use of average data does not enable policy makers to be discerning. It should also be noted that many modal comparisons are also based purely on operational energy consumption and emissions, and neglect to take the whole life-cycle in to account. Embedded energy and emissions from the construction of vehicles and infrastructure can be quite significant, as can the energy consumption and emissions from vehicle idling in the case of public transport modes. After considering the concept of environmental sustainability, this research begins by reviewing existing energy consumption and emissions data for vehicle operation, where it is noted that data for cars in Europe are quite comprehensive. Manufacturers are obliged to publish fuel consumption and emissions data for each model of car they sell, although the type approval tests do not reflect real-world performance. Studies are reviewed which suggest that the gap between the tests and the real-world has been widening in recent years. The gap appears to be independent of the size of vehicle, but is larger for hybrid vehicles than it is for those powered solely by a petrol or diesel internal combustion engine. Data for trains are less comprehensive, and that data which are available are often based on a limited empirical sample, or simulated data for which a number of assumptions have been made. Sometimes, the details of the measurements taken or simulation parameters used are unclear. As a result, published data for a particular type of train in the literature are sometimes found to vary significantly. In order to make more informed comparisons between rail and other modes, two large empirical datasets have been analysed. Two UK Train Operating Companies (TOCs) have also made data from energy metering systems on-board their electric trains available, which have been used to analyse the actual energy consumption of different trains over a number of different routes. The sample size is far larger than that found in literature to date, and it has been possible to consider variation between routes and service types. The v basic principles of simulating the energy consumption (and related emissions) of a train have also been illustrated, and a software tool has been developed for Arup so that it can now make some estimate of operational energy consumption and emissions for a given train over a given route. The aforementioned empirical data have also been used to validate the tool and suggest some appropriate simulation parameters. A review of existing literature concerning whole life-cycle analysis has been undertaken. It is clear that life-cycle costs vary significantly but in general, the overall life-cycle costs of rail appear to be higher than those for any other mode. The biggest additional factors appear to be the embedded carbon and energy in the infrastructure, particularly for a system comprising a lot of bridges, tunnels and large underground stations. For the vehicles themselves, trains typically have a longer lifespan than cars, which reduces the embedded carbon and energy as functions of time. When comparisons are made between modes, passenger-km is a metric which is often chosen, because it helps account for some of the fundamental di�erences between modes, including the fact that public transport modes usually use vehicles which are much bigger than the private car. In order to make comparisons on this basis, however, something about the load factor must be known. The sensitivity to load factor is demonstrated, and the earlier empirical data analysis is used to illustrate the benefits of longer trains. A discussion then follows about the potential pitfalls of making comparisons purely on a per passenger-km basis. This thesis ends by summarising some of the �ndings. Some consideration is given towards the future and the fact that technological developments are being made in Sustainability is a broad concept which embodies social, economic and environmental concerns, including the possible consequences of greenhouse gas (GHG) emissions and climate change, and related means of mitigation and adaptation. The reduction of energy consumption and emissions are key objectives which need to be achieved if some of these concerns are to be addressed. As well as being an important component of sustainability in other sectors, a good transport system needs to be sustainable in its own right. Energy consumption and GHG emissions are important issues within the transport sector; in the European Union (EU), for example, transport is directly responsible for between 25 and 30 percent of all carbon dioxide (CO2) emissions, and the inclusion of indirect (Scope 2 and Scope 3) GHG emissions may increase this proportion further. If reduction targets are to be met, it may be necessary to encourage behavioural change, including modal shift from those modes of transport which are comparatively highly polluting, towards those modes which pollute less. Rail is potentially a suitable target for such modal shift from road transport (notably the private car for passenger travel) and, in some case, from short-haul and domestic aviation. However, modal comparisons are often based on average data, and are reliant on a number of assumptions. There are likely to be some circumstances where modal shift towards rail makes more sense than others, but the use of average data does not enable policy makers to be discerning. It should also be noted that many modal comparisons are also based purely on operational energy consumption and emissions, and neglect to take the whole life-cycle in to account. Embedded energy and emissions from the construction of vehicles and infrastructure can be quite significant, as can the energy consumption and emissions from vehicle idling in the case of public transport modes. After considering the concept of environmental sustainability, this research begins by reviewing existing energy consumption and emissions data for vehicle operation, where it is noted that data for cars in Europe are quite comprehensive. Manufacturers are obliged to publish fuel consumption and emissions data for each model of car they sell, although the type approval tests do not re ect real-world performance. Studies are reviewed which suggest that the gap between the tests and the real-world has been widening in recent years. / The gap appears to be independent of the size of vehicle, but is larger for hybrid vehicles than it is for those powered solely by a petrol or diesel internal combustion engine. Data for trains are less comprehensive, and that data which are available are often based on a limited empirical sample, or simulated data for which a number of assumptions have been made. Sometimes, the details of the measurements taken or simulation parameters used are unclear. As a result, published data for a particular type of train in the literature are sometimes found to vary significantly. In order to make more informed comparisons between rail and other modes, two large empirical datasets have been analysed. Two UK Train Operating Companies (TOCs) have also made data from energy metering systems on-board their electric trains available, which have been used to analyse the actual energy consumption of different trains over a number of different routes. This thesis ends by summarising some of the findings. Some consideration is given towards the future and the fact that technological developments are being made in both the motor and the rail industries.

Memoria para optar al título de Ingeniera Civil en Computación / Actualmente, existen millones de publicaciones de investigadores en distintas áreas de las Ciencias de la Computación, y estas continúan aumentando día a día. En los perfiles de cada investigador del área en sitios web como DBLP o Google Scholar, se puede encontrar un listado con sus publicaciones. Sin embargo, con esta información por sí sola es difícil captar cuáles son los tópicos de interés de cada investigador a simple vista, y podría ser necesario en un ámbito de colaboración entre académicos o entre académicos y estudiantes. Este trabajo busca facilitar la información resumida de los tópicos de investigación de académicos de Ciencias de la Computación mediante la generación de visualizaciones como nubes de palabras, o tag clouds, a partir de las palabras y frases clave mencionadas en las publicaciones encontradas en repositorios bibliográficos online, como los mencionados anteriormente. El sistema desarrollado en esta memoria consiste en una herramienta que permite la creación de tag clouds para perfiles de DBLP. Esta herramienta se encarga de la obtención de las publicaciones encontradas en el perfil, la extracción de potenciales keywords y la selección de las keywords más relevantes según cuatro modelos de ordenamiento. Por cada uno de estos modelos se crea una variante de tag cloud. Además, se crea un sitio web que permite el uso de la herramienta para cualquier usuario. El trabajo se enfoca principalmente en la investigación de modelos de learning to rank y la comparación de su desempeño en la tarea de definir las keywords más relevantes para un investigador de Ciencias de la Computación. Dado que existen tres enfoques distintos para resolver la tarea de ordenamiento, se utilizan cuatro modelos de learning to rank, teniendo al menos uno por cada enfoque. Estos son regresión lineal, RankSVM, LambdaMART y AdaRank. De las evaluaciones a las tag clouds creadas por la herramienta se observa que no habría una preferencia absoluta por un método por sobre los demás, sino que varía según cada persona, pero en la mayoría de los casos se le asigna el puntaje máximo a al menos una de las tag clouds generadas. Esto podría deberse a que los modelos tienden a diferir en su enfoque, en algunos casos seleccionando keywords más técnicas y en otros más genéricas. De esta forma la apreciación de un método por sobre el otro se ve afectada por las preferencias de cada uno. De esto se concluye la importancia de dar la posibilidad de elegir a los usuarios entre distintas variantes.

The mutualistic associations between Arbuscular mycorrhizal (AM) fungi and plant roots are ancient and ubiquitous across the plant kingdom, where AM fungi provide Phosphorus, Nitrogen, and water to the plant, and receive photosynthetically fixed Carbon in the form of fatty acids and sugars in return. Moreover, AM fungi are associated with increased plant resistance to both abiotic and biotic stressors such as drought and viral pathogens. Frequently used in agriculture, AM fungi are observed to increase crop yields and decrease chemical fertilizer needs for many economically important plant species. The potential to increase AM fungal effectiveness remains a driving force for current research. To determine their role in establishing and/or supporting AM symbiosis, we propose a reverse genetic study of two genes in the model legume Medicago truncatula. Based on RNA sequencing data indicating increased expression during AM symbiosis, we selected one gene that encodes for NAC TF-like protein, which belongs to a large family of plant transcription factors primarily involved in regulating the secretion of defence hormones. The second gene selected, PALM1, was recently discovered to play a role in the regulation of the trifoliate leaf structure of M. truncatula. We hypothesize that the genes under study play mechanistic roles in regulating AM fungal symbiosis and that we will observe a difference between the colonization rates of corresponding gene mutants and control groups. Firstly, we explored the involvement of the PALM1 and NAC TF genes by examining the root developmental phenotype of Medicago truncatula mutants. Secondly, we employed symbiosis assays to investigate the colonization rates of the genes in question. Results indicated that the NAC TF gene had no consistent role in the AM symbiosis, while the PALM1 gene revealed promising results, where significant increases in colonization rates were observed in PALM1 mutants throughout repeated experiments. Future research involves using this study to help pursue more effective ways to use AM fungi symbiosis in sustainable agro ecosystems.

Il est reconnu, depuis une centaine d’années, que des désordres de la coagulation, regroupés sous le terme de coagulopathies, sont souvent associés au développement néoplasique. Pendant de nombreuses années, ces coagulopathies furent souvent reconnues comme une simple conséquence du développement du cancer. D’ailleurs, pour les cliniciens, l’apparition de ces anomalies sanguines constitue souvent le premier signe clinique d’un cancer occulte. Toutefois, l’étude approfondie du lien existant entre le système hémostatique et le cancer indique que différents facteurs hémostatiques vont interagir avec soit l’environnement tumoral ou soit la tumeur elle-même et influencer le développement du cancer. Au cours de nos travaux, nous avons porté une attention particulière à deux protéines jouant un rôle primordial dans l’hémostase. Le facteur tissulaire (TF) et l’inhibiteur du facteur tissulaire (TFPI) peuvent jouer des rôles pro- ou anti-néoplasique, et ce indépendamment de leurs fonctions hémostatiques normales. Dans le premier volet de cette thèse, nous avons étudié les propriétés antiangiogéniques de TFPI. L’angiogenèse, soit la formation de nouveaux vaisseaux sanguins à partir du réseau pré-existant, est reconnue comme étant une étape clée du développement tumoral. D’après nos travaux, le TFPI peut inhiber la formation de structures de type capillaire des cellules endothéliales (CEs) de la veine ombilicale humaine (HUVEC), et ce à une IC 50 de 5 nM, soit la concentration physiologique de l’inhibiteur. De plus, le TFPI bloque la migration des cellules endothéliales lorsque ces dernières sont stimulées par la sphingosine-1-phosphate (S1P), une molécule relâchée lors de l’activation des plaquettes sanguines. Cette inhibition de la migration cellulaire s’explique par l’effet du TFPI sur l’adhésion des CEs. En effet, TFPI inhibe la phosphorylation de deux protéines clées participant à la formation des complexes d’adhésion focales soit FAK (focal adhesion kinase) et PAX (paxilin). L’inhibition de ces deux protéines suggère qu’il y ait une réorganisation des complexes focaux, pouvant expliquer la perte d’adhérence. Finalement, des études de microscopie confocale démontrent que les cellules traitées au TFPI changent de morphologie au niveau du cytosquelette d’actine provoquant une désorganisation des structures migratoires (pseudopodes). Les effets du TFPI au niveau de la migration, de l’adhésion et de la morphologie cellulaire sont strictement spécifiques aux cellules endothéliales humaines, puisque aucun n’effet n’est observé en traitant des cellules cancéreuses de glioblastomes (GB) humains, qui sont normalement des tumeurs hautement vascularisées. En résumé, cette première étude démontre que le TFPI est un inhibiteur de l’angiogenèse. Dans le second volet de cette thèse, nous nous sommes intéressés aux différents rôles de TF, le principal activateur de la coagulation. Cette protéine est également impliquée dans le développement néoplasique et notamment celui des médulloblastomes (MB) chez l’enfant via des fonctions hémostatiques et non-hémostatiques. Nos travaux démontrent que l’expression de TF est induite par la voie de signalisation de HGF (hepatocyte growth factor) et de son récepteur Met. Cet effet de HGF/Met semble spécifique aux MB puisque HGF ne peut stimuler l’expression de TF au niveau des cellules cancéreuses de glioblastomes. TF, exprimé à la surface des cellules médulloblastiques (DAOY), est responsable de l’activité pro-thrombogénique de ces cellules, ainsi qu’un acteur important de la migration de ces cellules en réponse au facteur VIIa (FVIIa). De plus, en étudiant 18 spécimens cliniques de MB, nous avons établi un lien entre l’intensité d’expression de TF et de Met. L’importance de cette corrélation est également suggérée par l’observation que les cellules exprimant les plus forts taux de TF et de Met sont également les plus agressives en termes d’index de prolifération et de dissémination métastatiques. En résumé, ces travaux représentent le point de départ pour la mise au point de TF comme un marqueur diagnostique clinique dans les cas de tumeurs du cerveau pédiatriques. De plus, l’élucidation de la voie de signalisation moléculaire responsable de l’expression de TF permet de mieux comprendre la biologie et le fonctionnement de ces tumeurs et de relier le profil d’expression de TF aux phénotypes agressifs de la maladie. Il est reconnu que HGF peut également jouer un rôle protecteur contre l’apoptose. Dans le troisième volet de cette thèse, nous avons remarqué que cette protection est corrélée à l’expression de TF. En réduisant à néant l’expression de TF à l’aide de la technologie des ARN silencieux (siRNA), nous démontrons que HGF ne protège plus les cellules contre l’apoptose. Donc, TF médie l’activité anti-apoptotique de HGF. TF assume cette protection en inactivant la phosphorylation de p53 sur la sérine 15, empêchant ainsi la translocation de p53 au noyau. Finalement, l’expression de TF et son interaction avec le FVIIa, au niveau des cellules médulloblastiques favorise la survie de ces dernières et ce même si elles sont soumises à de fortes concentrations de médicaments couramment utilisées en cliniques. Ce troisième et dernier volet démontre l’implication de TF en tant que facteur impliqué dans la survie des cellules cancéreuses, favorisant ainsi le développement de la tumeur. Dans son ensemble, cette thèse vise à démontrer que les facteurs impliqués normalement dans des fonctions hémostatiques (TFPI et TF) peuvent contribuer à réguler le développement tumoral. Tout système physiologique et pathologique est dépendant d’un équilibre entre activateur et inhibiteur et la participation de TF et de TFPI à la régulation du développement néoplasique illustre bien cette balance délicate. Par sa contribution anti- ou pro-néoplasique le système hémostatique constitue beaucoup plus qu’une simple conséquence du cancer; il fait partie par l’action de TF des stratégies élaborées par les cellules cancéreuses pour assurer leur croissance, leur déplacement et leur survie, alors que TFPI tente de limiter la croissance tumorale en diminuant la vascularisation. / For more then a century now, haemostatic disorders, also refered as coagulopathies, are recognized to be associated to neoplasia development. At first, these coagulopathies were often seen as the first clinical symptom of an occult malignancy and as such they were foreseen as merely consequence of cancer. However recent studies allowed to determine that a molecular link between haemostasis and cancer exists. Haemostatic protein were found to interact with the tumor environment or the tumor itself. In this thesis, we look at two important haemostatic proteins involved in anti- or pro-tumoral activities. These proteins we looked at were, tissue factor (TF), which as been identified as the main trigger of blood coagulation an its natural inhibitor which is named tissue factor pathway inhibitor (TFPI). In our investigations we focus on the non-haemostatic function of these proteins. First of all, we studied the effect of TFPI on angiogenesis. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a critical component of cancer. In this first paper we demonstrate that, in addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. At physiological concentration, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells. Met, the only known receptor for hepatocyte growth factor (HGF) is a member of the receptor tyrosine kinases (RTKs) family and recently this RTK emerged as one important contributor to human neoplasia. In physiologic and pathologic conditions, Met trigger various cellular functions related to cell proliferation and migration, inhibition of apoptosis and also regulate a genetic program leading to coagulation. Since medulloblastoma (MB) express high level of tissue factor (TF), the main initiator of blood coagulation, we therefore wanted to know if there was a link between Met and TF expression in these pediatric tumors. In this work we demonstrate that stimulation of MB cells in vitro with HGF lead to overt expression of TF. These data correlate with analysis of clinical MB tumors specimens, were tumors expressing high level of Met also express high level of TF. This HGF/Met/TF pathway seems specific to MB only since no effect was observed on glioblastoma cells. TF expression in MB leads to a pro-coagulation state which could initiate thrombosis and the formation of a provisional fibrin matrix. Furthermore, TF can also mediate non-haemostatic function such as cell migration, thus helping cancer cells to form distant metastasis. Taken together these results suggest that HGF/Met pathway is responsible for TF expression in MB and explained how TF participate to tumor aggressiveness. In the third part of this thesis we look at the effect of TF on cell survival as a huge body of evidence suggest a role for TF and its natural ligand FVIIa on cells death (apoptosis) protection. In this study, we show that HGF-induce anti-apoptotic effect on DAOY MB cells is correlated to TF expression. Using TF siRNA we completely reversed this anti-apoptosis activity. TF is responsible of p53 inactivation, thus allowing DAOY to resist to very high concentrations of the chemotherapeutic drug Etoposide. These finding highlights the importance of understanding the molecular biology of MB, to achieve a better clinical management and also find new therapeutic targets. Overall, this thesis demonstrates that normal haemostatic protein such as TF and TFPI are also involved in tumor development. The different activities of TF and TFPI on cancer reflected the fact that every physiological or pathological system is dependent of a balance between activator and inhibitor. Furthermore the molecular link described in this thesis between haemostasis and cancer constitutes an argument against the fact that the haemostatic system is simply a consequence of cancer. Instead we show that TF is part of a strategy designed to improve tumor expansion while TFPI try to restrain the tumor growth by limiting tumor angiogenesis.

Il est reconnu, depuis une centaine d’années, que des désordres de la coagulation, regroupés sous le terme de coagulopathies, sont souvent associés au développement néoplasique. Pendant de nombreuses années, ces coagulopathies furent souvent reconnues comme une simple conséquence du développement du cancer. D’ailleurs, pour les cliniciens, l’apparition de ces anomalies sanguines constitue souvent le premier signe clinique d’un cancer occulte. Toutefois, l’étude approfondie du lien existant entre le système hémostatique et le cancer indique que différents facteurs hémostatiques vont interagir avec soit l’environnement tumoral ou soit la tumeur elle-même et influencer le développement du cancer. Au cours de nos travaux, nous avons porté une attention particulière à deux protéines jouant un rôle primordial dans l’hémostase. Le facteur tissulaire (TF) et l’inhibiteur du facteur tissulaire (TFPI) peuvent jouer des rôles pro- ou anti-néoplasique, et ce indépendamment de leurs fonctions hémostatiques normales. Dans le premier volet de cette thèse, nous avons étudié les propriétés antiangiogéniques de TFPI. L’angiogenèse, soit la formation de nouveaux vaisseaux sanguins à partir du réseau pré-existant, est reconnue comme étant une étape clée du développement tumoral. D’après nos travaux, le TFPI peut inhiber la formation de structures de type capillaire des cellules endothéliales (CEs) de la veine ombilicale humaine (HUVEC), et ce à une IC 50 de 5 nM, soit la concentration physiologique de l’inhibiteur. De plus, le TFPI bloque la migration des cellules endothéliales lorsque ces dernières sont stimulées par la sphingosine-1-phosphate (S1P), une molécule relâchée lors de l’activation des plaquettes sanguines. Cette inhibition de la migration cellulaire s’explique par l’effet du TFPI sur l’adhésion des CEs. En effet, TFPI inhibe la phosphorylation de deux protéines clées participant à la formation des complexes d’adhésion focales soit FAK (focal adhesion kinase) et PAX (paxilin). L’inhibition de ces deux protéines suggère qu’il y ait une réorganisation des complexes focaux, pouvant expliquer la perte d’adhérence. Finalement, des études de microscopie confocale démontrent que les cellules traitées au TFPI changent de morphologie au niveau du cytosquelette d’actine provoquant une désorganisation des structures migratoires (pseudopodes). Les effets du TFPI au niveau de la migration, de l’adhésion et de la morphologie cellulaire sont strictement spécifiques aux cellules endothéliales humaines, puisque aucun n’effet n’est observé en traitant des cellules cancéreuses de glioblastomes (GB) humains, qui sont normalement des tumeurs hautement vascularisées. En résumé, cette première étude démontre que le TFPI est un inhibiteur de l’angiogenèse. Dans le second volet de cette thèse, nous nous sommes intéressés aux différents rôles de TF, le principal activateur de la coagulation. Cette protéine est également impliquée dans le développement néoplasique et notamment celui des médulloblastomes (MB) chez l’enfant via des fonctions hémostatiques et non-hémostatiques. Nos travaux démontrent que l’expression de TF est induite par la voie de signalisation de HGF (hepatocyte growth factor) et de son récepteur Met. Cet effet de HGF/Met semble spécifique aux MB puisque HGF ne peut stimuler l’expression de TF au niveau des cellules cancéreuses de glioblastomes. TF, exprimé à la surface des cellules médulloblastiques (DAOY), est responsable de l’activité pro-thrombogénique de ces cellules, ainsi qu’un acteur important de la migration de ces cellules en réponse au facteur VIIa (FVIIa). De plus, en étudiant 18 spécimens cliniques de MB, nous avons établi un lien entre l’intensité d’expression de TF et de Met. L’importance de cette corrélation est également suggérée par l’observation que les cellules exprimant les plus forts taux de TF et de Met sont également les plus agressives en termes d’index de prolifération et de dissémination métastatiques. En résumé, ces travaux représentent le point de départ pour la mise au point de TF comme un marqueur diagnostique clinique dans les cas de tumeurs du cerveau pédiatriques. De plus, l’élucidation de la voie de signalisation moléculaire responsable de l’expression de TF permet de mieux comprendre la biologie et le fonctionnement de ces tumeurs et de relier le profil d’expression de TF aux phénotypes agressifs de la maladie. Il est reconnu que HGF peut également jouer un rôle protecteur contre l’apoptose. Dans le troisième volet de cette thèse, nous avons remarqué que cette protection est corrélée à l’expression de TF. En réduisant à néant l’expression de TF à l’aide de la technologie des ARN silencieux (siRNA), nous démontrons que HGF ne protège plus les cellules contre l’apoptose. Donc, TF médie l’activité anti-apoptotique de HGF. TF assume cette protection en inactivant la phosphorylation de p53 sur la sérine 15, empêchant ainsi la translocation de p53 au noyau. Finalement, l’expression de TF et son interaction avec le FVIIa, au niveau des cellules médulloblastiques favorise la survie de ces dernières et ce même si elles sont soumises à de fortes concentrations de médicaments couramment utilisées en cliniques. Ce troisième et dernier volet démontre l’implication de TF en tant que facteur impliqué dans la survie des cellules cancéreuses, favorisant ainsi le développement de la tumeur. Dans son ensemble, cette thèse vise à démontrer que les facteurs impliqués normalement dans des fonctions hémostatiques (TFPI et TF) peuvent contribuer à réguler le développement tumoral. Tout système physiologique et pathologique est dépendant d’un équilibre entre activateur et inhibiteur et la participation de TF et de TFPI à la régulation du développement néoplasique illustre bien cette balance délicate. Par sa contribution anti- ou pro-néoplasique le système hémostatique constitue beaucoup plus qu’une simple conséquence du cancer; il fait partie par l’action de TF des stratégies élaborées par les cellules cancéreuses pour assurer leur croissance, leur déplacement et leur survie, alors que TFPI tente de limiter la croissance tumorale en diminuant la vascularisation. / For more then a century now, haemostatic disorders, also refered as coagulopathies, are recognized to be associated to neoplasia development. At first, these coagulopathies were often seen as the first clinical symptom of an occult malignancy and as such they were foreseen as merely consequence of cancer. However recent studies allowed to determine that a molecular link between haemostasis and cancer exists. Haemostatic protein were found to interact with the tumor environment or the tumor itself. In this thesis, we look at two important haemostatic proteins involved in anti- or pro-tumoral activities. These proteins we looked at were, tissue factor (TF), which as been identified as the main trigger of blood coagulation an its natural inhibitor which is named tissue factor pathway inhibitor (TFPI). In our investigations we focus on the non-haemostatic function of these proteins. First of all, we studied the effect of TFPI on angiogenesis. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a critical component of cancer. In this first paper we demonstrate that, in addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. At physiological concentration, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells. Met, the only known receptor for hepatocyte growth factor (HGF) is a member of the receptor tyrosine kinases (RTKs) family and recently this RTK emerged as one important contributor to human neoplasia. In physiologic and pathologic conditions, Met trigger various cellular functions related to cell proliferation and migration, inhibition of apoptosis and also regulate a genetic program leading to coagulation. Since medulloblastoma (MB) express high level of tissue factor (TF), the main initiator of blood coagulation, we therefore wanted to know if there was a link between Met and TF expression in these pediatric tumors. In this work we demonstrate that stimulation of MB cells in vitro with HGF lead to overt expression of TF. These data correlate with analysis of clinical MB tumors specimens, were tumors expressing high level of Met also express high level of TF. This HGF/Met/TF pathway seems specific to MB only since no effect was observed on glioblastoma cells. TF expression in MB leads to a pro-coagulation state which could initiate thrombosis and the formation of a provisional fibrin matrix. Furthermore, TF can also mediate non-haemostatic function such as cell migration, thus helping cancer cells to form distant metastasis. Taken together these results suggest that HGF/Met pathway is responsible for TF expression in MB and explained how TF participate to tumor aggressiveness. In the third part of this thesis we look at the effect of TF on cell survival as a huge body of evidence suggest a role for TF and its natural ligand FVIIa on cells death (apoptosis) protection. In this study, we show that HGF-induce anti-apoptotic effect on DAOY MB cells is correlated to TF expression. Using TF siRNA we completely reversed this anti-apoptosis activity. TF is responsible of p53 inactivation, thus allowing DAOY to resist to very high concentrations of the chemotherapeutic drug Etoposide. These finding highlights the importance of understanding the molecular biology of MB, to achieve a better clinical management and also find new therapeutic targets. Overall, this thesis demonstrates that normal haemostatic protein such as TF and TFPI are also involved in tumor development. The different activities of TF and TFPI on cancer reflected the fact that every physiological or pathological system is dependent of a balance between activator and inhibitor. Furthermore the molecular link described in this thesis between haemostasis and cancer constitutes an argument against the fact that the haemostatic system is simply a consequence of cancer. Instead we show that TF is part of a strategy designed to improve tumor expansion while TFPI try to restrain the tumor growth by limiting tumor angiogenesis.

This thesis deals with the recreational function of free time for students of the Faculty of Theology of the University of South Bohemia. The thesis is an analysis of the literature on the matter, stating kinds of recreation and brief historical overview in the context of free time. It is an analysis of recreation and active lifestyles including previous research studies. The central section is defined by the concept and function of free time period described in university education, recreational activities and leisure time of students, stating research studies. One whole chapter in the end of the theoretical part is focused on student from Faculty of Theology of the University of South Bohemia with the analysis of gender problem and gender statistics at the University of South Bohemia. The main goal of the research is to determine with the method of non-standardized questionnaire, how students the Faculty of Theology of the University of South Bohemiao use leisure time for recreation, pertinently to find proposals to improve the recreational function of free time. The data obtained from non-standardized questionnaire are compared by gender, kind of study and individual fields of study the Faculty of Theology of the University of South Bohemia, the main goal is to make an evaluation and recommendations for practice.

EPHB2, an ephrin receptor (EPH) from receptor tyrosine kinase (RTK) family, is one of the substrates for tissue factor (TF) - coagulation factor VIIa (FVIIa) complex and it is cleaved in its ectodomain. EPHB2 cleavage is important for ephrin receptor (EPH) - ephrin ligand (EFN) signaling and cell repulsion. TF has been reported to be overexpressed in different cancer types such as breast and colorectal cancer (CRC). Furthermore, EPHB2 R155C mutation, at the TF/FVIIa-mediated cleavage site, has been identified as one of the somatic mutation sites in human metastatic CRC. Therefore, the aim of the present work was to characterize the EPHB2 R155C mutation and its effect on the cleavage by TF/FVIIa on EPHB2 in context to CRC. We generated overexpression cell models for EPHB2 wild type (wt) and R155C mutant in human CRC DLD-1 cell line for in vitro compartmentalization assay analysis to demonstrate repulsion event in EPH-EFN signaling. Whereas low endogenous TF expression led to incomplete cleavage of EPHB2 wt protein, stable overexpression of TF resulted in complete cleavage. Moreover, overexpression of TF resulted in reduced compartmentalization in EPHB2 wt cells after FVIIa treatment. Transient expression of TF in EPHB2 wt and R155C cells showed no clear difference in EPHB2 cleavage. Interestingly, it was difficult to obtain similar stable overexpression level of TF in EPHB2 R155C cells compared to EPHB2 wt cells. This may lead to further research in context to the role of TF/FVIIa-mediated EPHB2 cleavage in CRC by the generation of TF overexpression cell lines using lentiviral transduction.