Ce travail de thèse concerne l'étude et l'évaluation de l'extraction et de la séparation de molécules d'intérêt à partir des résidus de colza. L'impact des différents traitements (broyage, champs électriques pulsés, décharges électriques de hautes tensions et ultrasons) sur l'amélioration de l'extraction des polyphénols et des protéines à partir des tiges de colza a été comparé. Ces traitements permettent d'endommager les membranes et/ou parois cellulaires de manière mécanique, électrique ou acoustique, facilitant ainsi la libération des composés intracellulaires vers le milieu extérieur. La cinétique d'extraction, le rendement en polyphenols totaux et en protéines et la consommation énergétique sont principalement étudiés. De plus, l'efficacité des traitements étudiés pour l'extraction des polyphénols et protéines, en fonction du degré de maturité de la plante a été étudiée. Les méthodes testées pour la séparation des extractibles (polyphénols et protéines) concernent la coagulation, la filtration membranaire ainsi que le couplage de ces deux méthodes afin de réduire la consommation de solvants organiques et la rétention des polyphénols au cours de séparation. La séparation des extractibles a été évaluée par le biais de l'analyse de pureté et du taux de rétention. Enfin, des effets positifs du traitement électrique sur l'étape de séparation des extractibles tels que l'augmentation du flux du perméat et la diminution du colmatage ont été remarqués. / This thesis work concerns the study and the evaluation of the extraction and the separation of valuable compounds from rapeseed residues. The impact of different treatments (grinding, pulsed electric fields, high voltage electrical discharges and ultrasound) on the enhancement of the extraction of polyphenols and proteins from rapeseed stems was compared. These treatments can damage cell membranes and 1 or cell walls mechanically, electrically or acoustically, thus facilitating the release of intracellular compounds to the surroundings. The extraction kinetics, yield of total polyphenols and proteins and energy consumption were mainly studied. In addition, influences of plant maturity on the efficiency of studied treatments for the extraction of polyphenols and proteins have been studied. The methods tested for the separation of extractives (polyphenols and proteins) include coagulation, membrane filtration and the combination of these two methods in order to reduce the consumption of organic solvents and the retention ofpolyphenols during separation. The separation of extractives was evaluated by means of analysis of purity and relative removal. Finally, positive effects of electrical treatment on the extractive separation step such as the increase ofpermeate flux and the decrease of membrane fouling have been observed.

L'objectif principal de cette thèse est l'évaluation de la technologie de chauffage par micro-ondes et son applicabilité dans l'étape de densification, dans le cadre d’un procédé de recyclage des déchets nucléaires à très longue vie et ensuite le suivi du procédé de frittage de l'oxyde de cérium, simulant de l’oxyde de plutonium. Dans ce travail, nous avons développé un système permettant de déterminer les propriétés diélectriques de l'oxyde de cérium et avons fait une étude comparative entre le frittage par chauffage micro-ondes dans une cavité monomode et le frittage conventionnel dans un dilatomètre classique pour deux poudres de cérine: l’une de taille micrométrique, la seconde de taille nanométrique. En outre, nous avons effectué des simulations numériques sur la base d’un modèle couplant électromagnétisme et transfert de chaleur. Ces travaux ont montré l’effet de la taille d’une particule sphérique modèle sur le champ électrique (E) dans la particule et autour d’elle. Dans le cadre d’empilements modèles, nous avons montré que la présence d’un cou entre ces particules, leur orientation par rapport au champ E et le nombre de particules constitutives de cet empilement jouent un rôle déterminant sur l’intensité du champ E, ce qui a des conséquences sur le chauffage des particules. / The main objective of this thesis is the evaluation of the heating by microwave technology and its applicability in the densification step, as a part of nuclear long life wastes recycling process and then the following up of the sintering of cerium oxide, a non-radioactive simulant of plutonium oxide. In this work, we developed a system for determining the dielectric properties of cerium oxide and made a comparative study between the sintering by microwave heating in a single-mode cavity and the conventional sintering in a dilatometer for two different powders of ceria: the first one has a micrometric particle size and the second has a nanometric one. In addition, we performed several numerical simulations on the basis of a model coupling electromagnetics and heat transfer. In these works, we have shown the effect of the size of a spherical model particle on the electric field (E) inside and around the particle. In the framework of a packing model of particles, the presence of a neck between these particles, their orientation relative to E field, and the number of these particles showed a decisive role in the intensity of the E field which has a direct impact on the heating of the particles.

High voltage (EHV and UHV) transmission facilitates transfer of large amount of power over long distances. However, due to the inherent geometry, the line and substation hardware of EHV and UHV class generate high electric fields, which results in local ionisation of air called corona discharges. Apart from producing audible noise in the form of frying or hissing sound, corona produces significant electromagnetic interferences in the radio range. The limit for this corona generated Radio Interference (RI) has been stipulated by international standards, which are strictly to be followed. In line and substation hardware, corona control rings are generally employed to limit or avoid corona. Standard dimensions of corona rings are not available for EHV and UHV class. In most of the cases, their design is based on either a trial and error method or based on empirical extrapolation. Only in certain specific cases, the dimensioning of the rings is carried out using electric field calculations. In any of these approaches, the unavoidable surface abrasions, which can lead to corona, are not considered. There are also efforts to account for nominal surface irregularity by using a surface roughness factor, which is highly heuristic. In order to address this practically relevant problem, the present work was taken up. The intended exercise requires accurate field computation and a suitable criterion for checking corona onset. For the first part, the Surface Charge Simulation Method is adopted with newly proposed sub-modelling technique. The surface of the toroid is discretised into curvilinear patches with linear approximation for the surface charge density. Owing to its high accuracy, Galerkin’s method of moments formulation is employed. The problem of singularity encountered in the numerical approach is handled using a method based on Duffy’s transformation. The developed codes have also been validated with standard geometries. After a survey of relevant literature the ‘Critical Avalanche Criteria’ is chosen for its simplicity and applicability to the problem. Through a detailed simulation, the effect of avalanche space charge in reducing the corona onset voltage is found to be around 1.5% and hence it is not considered further. For utilities not interested in a detailed calculation procedure for dimensioning of corona rings, design curves are developed for circular corona rings of both 400 kV and 765 kV class with surface roughness factor in the range 0.8 – 1. In the second part of the work, a methodology for dimensioning is developed wherein the inevitable surface abrasion in the form of minute protrusions can be accounted. It is first shown that even though considerable field intensification occurs at the protrusions, such localised modification need not lead to corona. It is shown that by varying the minor radius of the corona ring, it is possible to get a design where the prescribed surface abrasion does not lead to corona onset. In summary, the present work has successfully developed a reliable methodology for the design of corona rings with prescribed surface abrasions. It involved development of an efficient field computation technique for handling minute surface protrusions and use of appropriate criteria for assessing corona inception. It has also provided design curves for EHV and UHV class corona rings with surface roughness factor specified in the range 0.8 – 1.0.

Society’s dependence on electronic and electrical systems has increased rapidly over the past few decades, and people are relying more and more on these gadgets in their daily life because of the efficiency in operation which these systems can offer. This has revolutionized many areas of electrical and electronics engineering including power sector, telecommunication sector, transportation and many other allied areas. With progress in time, the sophistication in the systems also increased. Also as the systems size reduced from micro level to nano level, the compactness of the systems increased. This paved the way for development in the digital electronics leading to new and efficient IC 0s that came into existence. Power sector also faced a resurge in its technology. Most of the analog meters are now replaced by digital meters. The increased sophistication and compactness in the digital system technology made it susceptible to electromagnetic interference especially from High Power Electromagnetic Sources. Communication, data processing, sensors, and similar electronic devices are vital parts of the modern technological environment. Damage or failures in these devices could lead to technical or financial disasters as well as injuries or the loss of life. Electromagnetic Interference (EMI) can be explained as any malicious generation of electromagnetic energy introducing noise or signals into electric and electronic systems, thus disrupting, confusing or damaging these systems. The disturbance may interrupt, obstruct, or otherwise degrade or limit the effective performance of the circuit. These effects can range from a simple degradation of data to a total loss of data. The source may be any object, artificial or natural, that carries rapidly changing electrical currents, such as an electrical circuit. The sources of electromagnetic interference can be either unintentional or intentional. The sources producing electromagnetic interference can be of different power levels, different frequency of operation and of different field strength. One such classification of these sources are the High Power Electromagnetic Sources (HPEM) High Power Electromagnetic environment refers to sources producing very high peak electromagnetic fields at very high power levels. These power levels coupled with the extremely high magnitude of the fields are sufficient to cause disastrous effects on the electrical and electronic systems. There has been a lot of developments in the field of the source technology of HPEM sources so that they are now one of the strongest sources of electromagnetic interference. High Power Electromagnetic environment refers to the sources producing very high peak electromagnetic fields at very high power levels. These power levels coupled with the extremely high magnitude of the fields are sufficient to cause disastrous effects on the electrical and electronic systems. HPEM environments are categorized based on the source characteristics such as the peak electric field, often called threat level, frequency coverage or bandwidth, average power density and energy content. The sources of electromagnetic interference can be either unintentional or intentional. Some examples of unintentional sources are the increased use of electromagnetic spectrum which generates disturbance to various systems operating in that frequency band, poor design of systems without taking care of other systems present nearby as well as lightning. Intentional sources are High altitude Electromagnetic Pulse (HEMP) or Nuclear Electromagnetic Pulse (NEMP) due to nuclear detonations, Ultra Wide Band (UWB) field from Impulse Radiating Antennas (IRA), Nar-row band fields like those coming from High Power Microwaves (HPM), High Intensity Radio Frequency (HIRF) sources. Of these the lightning is natural and all other sources are man-made. The significant progress in the Intentional High-Power Electromagnetic (HPEM) sources and antenna technologies and the easy access to simple HPEM systems for anyone entail the need to determine the susceptibility of electronic equipment as well as coupling of these fields with systems such as cables (buried as well as aerial), airborne vehicle etc. to these types of threats. Buried cables are widely used in the communication and power sectors due to their efficient functioning in urban cities and towns. These cables are more prone to electromagnetic interferences from HPEM sources. The buried communication cables or even the buried data cables are connected to sensitive equipments and hence even a slight rise in the voltage or the current at the terminals of the equipments can become a serious problem for the smooth operation of the system. In the first part of the thesis the effect of the electromagnetic field due to these sources on the cables laid underground has been studied. The second part of this thesis deals with the study of the interaction of the EM field from the above mentioned HPEM sources with an airborne vehicle. Airborne vehicle and its payload are extremely expensive so that any destruction to these as a result of the voltages and currents induced on the vehicle on account of the incoming HPEM fields can be quite undesirable. The incoming electromagnetic fields will illuminate the vehicle along its axis which results in the induction of currents and voltages. These currents and voltages will get coupled to the internal control circuits that are extremely sensitive. If the induced voltage/ current magnitude happen to be above the damage threshold level of these circuits then it will result in either a malfunction of the circuit or a permanent damage of it, with both of them being detrimental to the success of the mission. This will even result in the abortion of the mission or possible degradation of the vehicle performance. Hence it is worthwhile to see what will be the influence of an incoming HPEM electromagnetic field on the airborne vehicle with and without the presence of an exhaust plume. In this work, the HPEM sources considered are NEMP, IRA and HPM. The electromagnetic fields produced by the EMP can induce large voltage and current transients in electrical and electronic circuits which can lead to a possible malfunction or permanent damage of the systems. The electric field at the earth 0s surface can be modelled as a double exponential pulse as per the IEC standard 61000-2-9. The NEMP field incident on the earth’s surface is considered as that coming from a source at a distance far away from the earth’s surface; hence a plane wave approximation has been used. Impulse radiating antennas are the ones that are used as the major source of ultra wide band radiation. These are highly powerful antennas that use a pulsed power source as the input and this power source is conditioned to get an extremely sharp rise time pulse. These antennas are very high power antennas that are capable of producing a significant electromagnetic field. Impulse radiating antenna is a paraboloidal reflector and hence is an aperture antenna. Initially the radiated field due to this aperture needs to be found out at any observation point from the antenna. In this thesis, the aperture distribution method is used to accurately determine the field due to the aperture. In this method the field reflected from the surface of the reflector is first found on an imaginary plane through the focal point of the reflector that is normal to the axis of the reflector, by using the principles of geometrical optics, which then is extended to the observation point. The IRA considered for the present work is the one of the most powerful IRA as per the published literature available in the open domain. This has an input voltage of 1.025 MV. The far field electric field measured at the boresight (at r =85 m) being equal to 62 kV/m, and the uncorrected pulse rise time (10%-90%) is 180 ps for this IRA. HPM sources are usually electromagnetic radiators having a reflector with a horn antenna kept at their focal point for excitation. HPM sources generally operate in single mode or at tens or hundreds of Hz repetition rates. Many HPM radiators are developed in the world each with their own peculiar geometry and power levels. In the present thesis, a single waveguide (WR-975) fed HPM antenna assembly has been studied. The chosen waveguide has a cut-o_ frequency of 1 GHz and a power level of 10 GW. The wavelength associated with the waveguide is 0.3 m. The field pattern shows a definite peak in its response when the frequency is 1 GHz, the cut-off frequency of the waveguide. The electric field coming out of the HPEM sources travel through the medium that is either air alone or a combination of air and soil respectively depending upon whether the circuit on which the coupling is analysed is an airborne vehicle or an underground cable. The media plays a major role in the coupling, as the field magnitude is influenced by the characteristic properties of the media. As height increases the magnitude of the electric field decreases for all types of sources and also the time before which the field waveform starts is increased. The electric field in the soil is decided by the soil properties such as its conductivity and permittivity. The soil is modelled in frequency domain and the high frequency behaviour of soils is considered with its conductivity and permittivity taken as functions of frequency, as the incident field has high frequency components. A soil medium can be electromagnetically viewed as a four component dielectric mixture consisting of soil particles, air voids, bound water, and free water. When electric field is incident on the soil, it gets polarized. This is as a result of a wide variety of processes, including polarization of electrons in the orbits around atoms, distortion of molecules, reorientation of water molecules, accumulation of charge at interfaces, and electrochemical reactions. Whatever is the HPEM source, an increase in the soil conductivity results in an increased attenuation of the field. Also there is a significant loss of high frequency components in the GHz range in the field due to the selective absorption by the soil. This effect causes the percentage attenuation to be maximum for HPM and minimum for NEMP and IRA lying in between these two extremities. Increase in permittivity of the soil causes attenuation of the electric field for all HPEM sources. This is due to the relaxation mechanisms in the soil due to atomic- or molecular-scale resonances. The coupling of the electromagnetic fields due to HPEM sources is considered in the first phase. Two cables are considered (i) buried shielded and (ii) buried shielded twisted pair cables. The results are arrived at using the Enhanced Transmission Line model. The induced current is more for a shielded cable than a twisted pair cable of the same configuration. The induced current magnitude depends upon the type of the HPEM source, the depth of burial of the cable and the point on the cable where the current/ voltage is computed. Current is maximum at the centre of the cable for a matched termination and the voltage is the minimum at this point. The ratio of the induced current in the inner conductor with respect to the shield current of a shielded cable is the least for an HPM, and maximum for NEMP. This is due to the fact that higher frequencies are absorbed more by the shield of the cable. This affects HPM induced current the maximum and NEMP the least because of the presence of the lower frequency components in NEMP. Induced current in the twisted pair cable depends upon the number of pairs of the cable and the pitching of the cable. The electromagnetic field from the HPEM sources propagates with less attenuation in air due to the lower resistance this medium offers for electromagnetic wave propagation. Hence any system in air, be it electrical or electronic, will be under the strong illumination by these electromagnetic fields. As the second part of this thesis, the influence of the electromagnetic fields from all the three HPEM sources on an airborne vehicle in flight is analysed. For this part of study, the Electromagnetic (EM) fields radiated by all the three sources at different heights from the earth 0s surface have been computed. The coupling study has been done for the case of a vehicle with plume as well as without plume. For the second case, the electromagnetic modelling of the plume has been done taking into consideration its conductivity, which in turn depends on the different ionic species present in the plume. The species of the exhaust plume depends upon the chemical reactions taking place in the combustion chamber of the nozzle of the vehicle. The presence of the alkali metals as impurity in the airborne vehicle propellant will generate considerable ion particles such as Na+, Cl in addition to e- in the plume mixture during combustion which makes the plume electrically conducting. But it does not influence the pressure, temperature and velocity of the plume. After the nozzle throat, the exhaust plume regains the supersonic speed, so the flow of the exhaust plume is assumed as compressible flow in the second region. The electrons have high collision frequency, high number density, high plasma frequency and lower molecular mass and hence the highly mobile electrons dominate the heavy ion particle in the computation of the electrical conductivity of the plume. The plume conductivity decreases marginally from the axis till a distance equal to the nozzle radius but the peak value increases sharply towards the exit plane edge of the nozzle radius. The induced current is computed using Method of Moments. The induced current depends upon the type of interference source, its characteristics, whether the plume is present or not and the type of the plume. The HPM induces maximum current in the vehicle because of the fact that the plume has a tendency to become more conductive at these frequencies. The induced currents due to the EM fields from IRA and NEMP comes after the HPM. The presence of the plume enhances the magnitude of the induced current. If the plume is homogeneous then the current induced in it is more.

This work studies the influence of field effect on large area 2 dimensional ribbons of fluids. A fluid of choice is confined in the channel of a metal-insulator-channel-insulator-metal architecture and is subjected to constant (d.c) or alternating (a.c) fields (de-pending on the application) along with a pressure drive flow. A general fluid would be composed of molecules having certain polarizability and be a dispersion of non-ionic and ionic particulates. The field effect response under pressure driven flow for this fluid would result in electrophoresis, electro osmosis, dielectrophoresis, dipole-dipole interaction and inverse electro osmosis phenomena. Using some of these phenomena we study applications related to desalination and energy harvesting with saline water as the ex-ample fluid for the former case, and solution processed poly vinyldene fluoride (PVDF) for the latter case. The geometrical features of \large area" and the \ribbon shape" can be taken advantage of to influence the design and performance for both applications. With regards to desalination, it is shown via experiments and theoretical models that the presence of alternating electric fields aid in ion separation along the flow when the saline water is subjected to laminar flow. Moreover, the power consumption is low due to the presence of the insulator. An average of 30% ion removal efficiency and 15% throughput is observed in the systems fabricated. Both performance parameters are discussion can be improved upon with larger channel lengths. The \2-D ribbon" and alternating field effect aid in achieving this by patterning the randomly distributed ions in the bulk into a smooth sheet charge and then repelling this sheet charge back into the bulk. The electric field exhibited by this sheet charge helps trap more ion sheets near the interface, thereby converting a surface ion trapping phenomena (when d.c is used) to a bulk phenomena and thereby improving efficiency. With regards to energy harvesting, a solution of PVDF in methyl ethyl ketone and 1-methyl-2-pyrollidone is confined to the \2-D ribbon" geometry and subject to high d.c fields. This aids in combining the fabrication, patterning and poling process for PVDF into one setup. Since the shape of the ribbon is defined by the shape of the channel, the ribbons (straight or serrated) can be used to sense forces of various magnitudes. More importantly experiments and theoretical models are studied for energy harvesting. Since the ribbon geometry defines the resonant frequency, large PVDF ribbon can be used to harvest energy from low frequency vibrations. Experiments show that up to 60 microwatt power can be harvested at 200 Hz and is sufficient to supplement the power for ICs.

Mit einem medianen Überleben von 14 bis 16 Monaten und einer 5-Jahres-Überlebensrate von weniger als 5 % zählt das Glioblastoma multiforme (GBM) zu den aggressivsten Tumoren des zentralen Nervensystems (Cloughesy et al., 2014; Batash et al., 2017; Guberina et al., 2020). Das GBM, auch als WHO-Grad IV-Astrozytom bezeichnet, ist mit > 50 % aller glialen Tumoren der häufigste maligne hirneigene Tumor (Ohgaki und Kleihues, 2005). Aufgrund ihrer infausten Prognose ist eine Weiterentwicklung und Optimierung der aktuellen Leitlinientherapie sowie die Entwicklung neuartiger Therapiekonzepte für Primärtumore und Rezidive unentbehrlich. Im Rahmen dieser Arbeit wurden neuartige Therapieansätze, wie elektrische Wechselfelder (tumor treating fields, TTFields) und der Aurorakinaseinhibitor AZD1152 sowie die konventionelle, in der Leitlinie des Primärtumors verankerte Radiotherapie eingesetzt. Während eine Strahlentherapie in erster Linie durch die Induktion von DNA-Einzel- und Doppelstrangbrüchen wirkt, beruht der Wirkmechanismus der TTFields auf eine Störung der Dipol-gesteuerten Schritte während der Zellteilung. Dies führt folglich zu einer Arretierung des Zellzyklus in der G2/M- und G1/S-Phase. Sofern eine Reparatur an den checkpoints nicht möglich ist, erfolgt die Überleitung der Zelle in die Apoptose (Suzuki et al., 2003; Wilson et al., 2014; Fontana et al., 2015; Gerelchuluun et al., 2015). Die TTFields kamen mittels des InovitroTM-Systems zum Einsatz, die insbesondere inhibierend auf die M-Phase des Zellzyklus wirken (Gutin und Wong, 2012; Saria und Kesari, 2016). Für das Glioblastoma multiforme wurde dabei eine spezifische Frequenz von 200 kHz und eine Feldintensität von 1,7 V/cm bestimmt, welche das außerhalb des Zielgebiets liegende Gewebe schont (Kirson et al., 2009; Fabian et al., 2019). Für Primärtumore eines Glioblastoma multiforme konnte in der EF-14-Studie bereits ein signifikant verlängertes Überleben durch TTFields bestätigt werden, während für das Rezidiv in der EF-11-Studie lediglich eine Verbesserung der Lebensqualität erreicht wurde jedoch keine Verlängerung der Überlebenszeit (Stupp et al., 2012; Stupp et al., 2017). Ein vielversprechender Therapieansatz scheint außerdem der Einsatz des Aurora B-Kinase-Inhibitors AZD1152 zu sein. Als enzymatischer Teil des chromosomale passenger complex (CPC) liegt die Hauptaufgabe der Aurora B-Kinase in der Kontrolle der Mitose des Zellzyklus (Vader et al., 2006). Resultierend aus der Aufhebung des genannten Kontrollmechanismus mittels AZD1152 (Barasertib™) kommt es zum Anstieg polyploider Zellen, wodurch eine Überleitung in die Apoptose erfolgt (Zekri et al., 2016). Schlussfolgernd erscheint in Anbetracht der Einzeleffekte von Radiotherapie, TTFields und Aurora B-Kinase-Inhibierung deren kombinierter Einsatz wesentlich bedeutsam, wodurch der vorliegenden Arbeit die Hypothesen eines überwiegenden Effekts der Dreifachkombination im Vergleich zu der Einzeltherapie und den jeweiligen Zweifachkombinationsbehandlungen zugrunde liegen. Für die drei in dieser Arbeit eingesetzten Primärkulturen eines Glioblastoma multiforme Rezidivs konnte für die Dreifachkombinationstherapie gegenüber den Einzelbehandlungen ein hoch bis höchst signifikant additiv-zytotoxischer Effekt nachgewiesen werden. Im Mittel gelang eine Reduktion der Lebendzellzahl auf 20 – 34 % vitaler Zellen. Auch in Bezug auf die einzelnen Zweifachkombinationen wurden signifikante, hoch signifikante sowie ein höchst signifikantes Ergebnis für die Dreifachkombinationstherapie erzielt. Lediglich für die TTFields/AZD1152-Kombinationsbehandlung der Primärkultur HT18328-3 traf dies nicht zu. Mit Hilfe der konfokalen Laser-Scanning-Mikroskopie wurden ergänzend qualitative, zellmorphologische Änderungen visualisiert. Während sich in den Einzelbehandlungen sowie den Zweifachkombinationen Veränderungen der Zell- und Kerngröße sowie eine Kernfragmentierung andeuteten, waren diese Effekte in der Dreifachkombination deutlicher ausgeprägt. Die bereits quantitativ detektierten synergistisch zytotoxischen Effekte konnten durch lichtmikroskopische Bilder verifiziert werden. Langfristiges Ziel dieser Arbeit ist, die Kombinationstherapie im Rahmen von klinischen Studien zu testen. Jedoch sollte, aufgrund der insgesamt hohen inter- und intratumoralen Heterogenität des Glioblastoma multiforme im Vorfeld zur Etablierung des klinischen Einsatzes das Verhalten weiterer Primärkulturen untersucht werden. Ebenso erscheint die Berücksichtigung der vorausgehenden Behandlung der Patienten sowie des Ploidiegrades der Primärkultur als relevant, um ein unterschiedliches Therapieansprechen sowie mögliche Resistenzmechanismen nachzuvollziehen. Ferner sollte ein neoadjuvanter Einsatz des AZD1152 weiter verifiziert werden, denn eine Verbesserung der Radiosensibilität, resultierend in einem gesteigerten Therapieansprechen, konnte bereits aufgezeigt werden (Tao et al., 2009). Zur Minimierung der systemischen Nebenwirkungen des AZD1152 (Barasertib™) wäre die Etablierung einer gezielten, lokalen Anwendung im Sinne einer intraoperativen oder minimalinvasiven Applikation zielführend.:1 Einleitung 1.1 Glioblastoma multiforme: Definition, Ätiologie, Inzidenz 1.1.1 Symptome und Diagnostik 1.2 Molekulare Klassifizierung 1.2.1 Unterteilung in primäre und sekundäre Glioblastome mittels des IDH-Status 1.2.2 Molekulare Marker primärer Glioblastome 1.2.4 Die Methylguanin-Methyltransferase (MGMT) 1.3 Konventionelle Therapie 1.3.1 Leitlinie Primärtumor – Leitlinie Rezidiv 1.3.2 Radiotherapie 1.4 Neuartige Therapiekonzepte 1.4.1 Biologischer Hintergrund 1.4.2 Aurorakinase-Inhibitoren 1.4.3 Tumor Treating Fields (TTFields) 1.5 Zielstellung 2 Material und Methoden 2.1 Material 2.1.1 Antikörper 2.1.2 Chemikalien 2.1.3 Geräte 2.1.4 Lösungen 2.1.5 Medien 2.1.6 Kits 2.1.7 Primärkulturen 2.1.8 Software 2.1.9 Statistik 2.1.10 Verbrauchsmaterialien 2.2 Methoden 2.2.1 Zellkultivierung allgemein 2.2.2 Passagieren adhärenter Zellen . 2.2.3 Kultivierung von primärem Patientenmaterial 2.2.4 Kryokonservierung und Rekultivierung 2.2.5 Bestimmung der Lebendzellzahl – Neubauer-Zählkammer 2.2.6 Durchflusszytometrische Analyse 2.2.7 Bestimmung der Lebendzellzahl mittels PI 2.2.8 Bestimmung des DNA-Gehalt/Ploidiegrades mittels PI 2.2.9 Durchflusszytometrische Immunphänotypisierung von Glioblastomzellen 2.2.10 Beschichtung von Glascoverslips 2.2.11 Bestrahlung mittels Röntgensystem 2.2.12 Titration der Bestrahlungsdosis 2.2.13 Titration einer effektiven Aurora B-Kinase-Inhibitorkonzentration 2.2.14 In vitro-Applikation der TTFields 2.2.15 Konfokale Laser-Scanning-Mikroskopie 3 Ergebnisse 3.1 Wahl der Kontrollgruppe 3.2 Typisierung der verwendeten Primärkulturen 3.2.1 Befunde der Pathologie des UKD 3.2.2 Immunphänotypisierung 3.3 Titration der AZD1152-Konzentration 3.3.1 Titration der AZD1152-Konzentration an der Primärkultur HT16360-1 3.3.2 Titration der AZD1152-Konzentration an der Primärkultur HT18328-3 3.3.3 Titration der AZD1152-Konzentration an der Primärkultur HT18816 3.4 Dosistitration der Radiotherapie 3.4.1 Titration der Bestrahlungsdosis an HT16360-1 3.4.2 Titration der Bestrahlungsdosis an HT18328-3 3.4.3 Titration der Bestrahlungsdosis an HT18816 3.5 Kombinationstherapie mit Radiotherapie, TTFields und AZD1152 3.5.1 Quantitativer Effekt der Kombinationstherapie anhand der Lebendzellzahl 3.5.2 Zytoreduktiver Effekt der Kombinationstherapie 3.5.3 Quantitativer Effekt der Kombinationstherapie anhand des Ploidiegrades 3.5.4 Qualitativer Effekt der Kombinationstherapie 4 Diskussion 4.1 In vitro-Charakterisierung der Primärkulturen 4.2 Radiotherapie . 4.3 Neuartige Behandlungsoptionen 4.3.1 TTFields 4.3.2 Aurora B-Kinase-Inhibitor AZD1152 4.4 Kombinierte Behandlungsmethoden – Zwei- und Dreifachtherapie 5 Zusammenfassung Abstract Literaturverzeichnis Tabellenverzeichnis Abbildungsverzeichnis Abkürzungsverzeichnis Anhang / With a median survival of 14 to 16 months and a 5-year survival rate of less than 5 %, glioblastoma multiforme (GBM) is one of the most aggressive tumours of the central nervous system (Cloughesy et al., 2014; Batash et al., 2017; Guberina et al., 2020). GBM, also known as WHO grade IV astrocytoma, is the most common malignant brain tumor with > 50% of all glial tumors (Ohgaki und Kleihues, 2005). Due to its dismal prognosis, further development and optimisation of the current guideline therapy as well as the development of novel therapeutic concepts for primary tumours and recurrences is indispensable. Within the framework of this work, novel therapeutic approaches such as alternating electric fields (tumor treating fields, TTFields) and the aurorakinase inhibitor AZD1152 as well as conventional radiotherapy anchored in the guideline of the primary tumor were applied. While radiotherapy primarily works by the induction of DNA single and double strand breaks, the mechanism of action of TTFields is based on a disruption of the dipole-controlled steps during cell division. Consequently, this leads to a locking of the cell cycle in the G2/M and G1/S phase. If repair at the checkpoints is not possible, the cell is transferred to apoptosis (Suzuki et al., 2003; Wilson et al., 2014; Fontana et al., 2015; Gerelchuluun et al., 2015). The TTFields were used by means of the InovitroTM system, which has a particularly inhibitory effect on the M-phase of the cell cycle (Gutin und Wong, 2012; Saria und Kesari, 2016). For glioblastoma multiforme, a specific frequency of 200 kHz and a field intensity of 1.7 V/cm was determined, which spares the tissue outside the target area (Kirson et al., 2009; Fabian et al., 2019). For primary tumours of glioblastoma multiforme a significantly prolonged survival could already be confirmed by TTFields in the EF 14 study, whereas for recurrent tumours only an improvement in quality of life was achieved in the EF 11 study (Stupp et al., 2012; Stupp et al., 2017). The use of the Aurora B kinase inhibitor AZD1152 also appears to be a promising therapeutic approach. As an enzymatic part of the chromosomal passenger complex (CPC), the main task of the aurora B-kinase is to control cell cycle mitosis (Vader et al., 2006). As a result of the removal of the above-mentioned control mechanism by means of AZD1152 (BarasertibTM), there is an increase in polyploid cells, which leads to a transition to apoptosis (Zekri et al., 2016). In conclusion, considering the single effects of radiotherapy, TTFields and Aurora B-kinase inhibition, their combined use seems to be of considerable importance. Therefore, the present study is based on the hypotheses of a predominant effect of the triple combination compared to the single therapy and the respective dual combination treatments. For the three primary cultures of a glioblastoma multiforme recurrence used in this work, a high to highly significant additive cytotoxic effect could be demonstrated for the triple combination therapy compared to the single treatments. On average, a reduction in the number of living cells to 20 – 34 % vital cells was achieved. Significant, high significant and highly significant results were also achieved with regarding to the individual dual combination treatments. Only for the TTFields/AZD1152 combination treatment of the primary culture HT18328-3 this was not true. Confocal laser scanning microscopy was used to visualise qualitative, cell morphological changes. While changes in cell and core size as well as nucleus fragmentation were indicated in the single treatments as well as in the dual combination treatments, these effects were more pronounced in the triple combination. The already quantitatively detected synergistic cytotoxic effects could be verified by light microscopic images. The long-term goal of this work is to test the combination therapy in clinical trials. However, due to the overall high inter- and intratumoral heterogeneity of glioblastoma multiforme, the behaviour of further primary cultures should be investigated in advance of establishing clinical use. In addition, consideration of the previous treatment of the patients as well as the degree of ploidy of the primary culture seems to be relevant to understand a different response to therapy and possible resistance mechanisms. Furthermore, a neoadjuvant use of AZD1152 should be further verified, as an improvement in radiosensitivity resulting in an increased response to therapy has already been demonstrated (Tao et al., 2009). In order to minimize the systemic side effects of AZD1152 (BarasertibTM), the establishment of a targeted, local application in the sense of an intraoperative or minimally invasive application would be beneficial.:1 Einleitung 1.1 Glioblastoma multiforme: Definition, Ätiologie, Inzidenz 1.1.1 Symptome und Diagnostik 1.2 Molekulare Klassifizierung 1.2.1 Unterteilung in primäre und sekundäre Glioblastome mittels des IDH-Status 1.2.2 Molekulare Marker primärer Glioblastome 1.2.4 Die Methylguanin-Methyltransferase (MGMT) 1.3 Konventionelle Therapie 1.3.1 Leitlinie Primärtumor – Leitlinie Rezidiv 1.3.2 Radiotherapie 1.4 Neuartige Therapiekonzepte 1.4.1 Biologischer Hintergrund 1.4.2 Aurorakinase-Inhibitoren 1.4.3 Tumor Treating Fields (TTFields) 1.5 Zielstellung 2 Material und Methoden 2.1 Material 2.1.1 Antikörper 2.1.2 Chemikalien 2.1.3 Geräte 2.1.4 Lösungen 2.1.5 Medien 2.1.6 Kits 2.1.7 Primärkulturen 2.1.8 Software 2.1.9 Statistik 2.1.10 Verbrauchsmaterialien 2.2 Methoden 2.2.1 Zellkultivierung allgemein 2.2.2 Passagieren adhärenter Zellen . 2.2.3 Kultivierung von primärem Patientenmaterial 2.2.4 Kryokonservierung und Rekultivierung 2.2.5 Bestimmung der Lebendzellzahl – Neubauer-Zählkammer 2.2.6 Durchflusszytometrische Analyse 2.2.7 Bestimmung der Lebendzellzahl mittels PI 2.2.8 Bestimmung des DNA-Gehalt/Ploidiegrades mittels PI 2.2.9 Durchflusszytometrische Immunphänotypisierung von Glioblastomzellen 2.2.10 Beschichtung von Glascoverslips 2.2.11 Bestrahlung mittels Röntgensystem 2.2.12 Titration der Bestrahlungsdosis 2.2.13 Titration einer effektiven Aurora B-Kinase-Inhibitorkonzentration 2.2.14 In vitro-Applikation der TTFields 2.2.15 Konfokale Laser-Scanning-Mikroskopie 3 Ergebnisse 3.1 Wahl der Kontrollgruppe 3.2 Typisierung der verwendeten Primärkulturen 3.2.1 Befunde der Pathologie des UKD 3.2.2 Immunphänotypisierung 3.3 Titration der AZD1152-Konzentration 3.3.1 Titration der AZD1152-Konzentration an der Primärkultur HT16360-1 3.3.2 Titration der AZD1152-Konzentration an der Primärkultur HT18328-3 3.3.3 Titration der AZD1152-Konzentration an der Primärkultur HT18816 3.4 Dosistitration der Radiotherapie 3.4.1 Titration der Bestrahlungsdosis an HT16360-1 3.4.2 Titration der Bestrahlungsdosis an HT18328-3 3.4.3 Titration der Bestrahlungsdosis an HT18816 3.5 Kombinationstherapie mit Radiotherapie, TTFields und AZD1152 3.5.1 Quantitativer Effekt der Kombinationstherapie anhand der Lebendzellzahl 3.5.2 Zytoreduktiver Effekt der Kombinationstherapie 3.5.3 Quantitativer Effekt der Kombinationstherapie anhand des Ploidiegrades 3.5.4 Qualitativer Effekt der Kombinationstherapie 4 Diskussion 4.1 In vitro-Charakterisierung der Primärkulturen 4.2 Radiotherapie . 4.3 Neuartige Behandlungsoptionen 4.3.1 TTFields 4.3.2 Aurora B-Kinase-Inhibitor AZD1152 4.4 Kombinierte Behandlungsmethoden – Zwei- und Dreifachtherapie 5 Zusammenfassung Abstract Literaturverzeichnis Tabellenverzeichnis Abbildungsverzeichnis Abkürzungsverzeichnis Anhang

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Eine bevorzugte Zellquelle für den Einsatz im Tissue Engineering sind mesenchymale Stammzellen (MSZ). Diese besitzen, neben einer hohen Proliferationsrate, die Fähigkeit, sich in verschiedene Zellen des mesodermen Ursprungs und in die entsprechenden Gewebetypen zu entwickeln. Um ein funktionales Gewebe zu erhalten ist es Ziel, sich bereits in vitro den in vivo Bedingungen anzunähern. Hierbei spielen neben der dreidimensionalen Struktur der Scaffolds auch die biochemische Mikroumgebung der Zellen in Form der unlöslichen extrazellulären Matrix (EZM) und den löslichen Mediatorproteinen wie Wachstums- und Differenzierungsfaktoren, sowie die physikalische Stimulation der Zellen eine wichtige Rolle. Während sich gegenwärtige Untersuchungen im TE vorwiegend mit den alleinigen Einflussfaktoren beschäftigen, verfolgt die vorliegende Arbeit das Ziel, die Auswirkungen kombinierter Stimuli durch Verwendung einer artifiziellen EZM, bestehend aus definierten Komponenten der nativen EZM, und physikalischer Stimuli durch elektrische Felder zu untersuchen. Letzteres erfolgte mit einem innerhalb der Arbeitsgruppe neu entwickelten System, dass die Stimulation von Zellen mit ausschließlich elektrischen Feldern, ohne störende Nebeneinflüsse, erlaubt.

Ce travail de doctorat traite l’optimisation des méthodes d’extraction des composés phénoliques à partir des sous-produits de la viticulture et de la viniculture, à savoir les sarments de vigne et les marcs de raisins. Plusieurs technologies innovantes sont appliquées et comparées : l’extraction accélérée par solvant (EAS), les décharges électriques de haute-tension (DEHT), les ultrasons (US) et les champs électriques pulsés (CEP). Les extractions solide-liquide faites sur les sarments ont montré que, parmi les solvants étudiés, l’eau est le moins efficace. L’ajout de la β-cyclodextrine dans l’eau améliore le procédé d’extraction mais est moins efficace que les mélanges hydroéthanoliques. L’extraction en milieu alcalin donne le meilleur rendement en composés phénoliques. L’intensification de l’extraction des composés phénoliques des sarments est possible grâce aux nouvelles technologies d’extraction. L’efficacité des méthodes testées est la moindre avec les US, moyenne avec les CEP pour atteindre le meilleur rendement phénolique avec les DEHT. La filtrabilité de ces extraits est d’autant plus lente que leur composition est complexe. L’ultrafiltration membranaire permet une très bonne purification et concentration des composés phénoliques. L’étude des mécanismes d’action des DEHT a permis d’identifier les phénomènes favorisant l’extraction des composés phénoliques à partir des sarments. Un effet mécanique des DEHT, capable de fragmenter les sarments, est en majorité responsable de cette amélioration. Le procédé énergivore du broyage pourra alors être omis. Un effet électrique contribuant également à l’intensification du procédé d’extraction est démontré. La formation de peroxyde d’hydrogène durant le traitement par DEHT est quantifiée mais ne semble pas altérer les composés phénoliques qui sont des molécules à capacité antiradicalaire élevée. Quant aux études portées sur les marcs de raisins, la variation simultanée de plusieurs paramètres opératoires a permis l’optimisation de l’extraction aqueuse et hydroéthanolique des composés phénoliques en ayant recours à la méthodologie de surface de réponse (MSR). Le passage d’un milieu aqueux à un milieu hydroéthanolique a permis d’améliorer nettement le procédé d’extraction solide-liquide des composés phénoliques et l’utilisation de l’EAS a permis l’augmentation du rendement en composés phénoliques jusqu’à trois fois par rapport à l’optimum obtenu en milieu hydroéthanolique. / This study deals with the optimization of the extraction methods of phenolic compounds from viticulture and viniculture by-products, namely vine shoots and grape pomace. Several innovative technologies were tested and compared: high voltage electrical discharges (HVED), accelerated solvent extraction (ASE), ultrasounds (US) and pulsed electric fields (PEF). The solid-liquid extraction conducted on vine shoots showed that, amongst the studied solvents, water is the least effective. The addition of the β-cyclodextrin to water improves the extraction process but remains less effective than that with hydroethanolic mixtures. The extraction in alkaline medium gives the highest phenolic compound extraction yields. The intensification of phenolic compound extraction from vine shoots was possible thanks to new extraction technologies. The effectiveness of the tested methods was the least with US, followed by PEF to accomplish the highest phenolic yield with HVED. The filterability of the extracts was slower when their composition was complex, and the membrane technology allowed a good purification and concentration of phenolic compounds. The reason behind the high effectiveness of HVED was investigated. The action mechanisms of HVED were studied in details. A mechanical effect of HVED provoked vine shoots fragmentation and particle size reduction. This was the main phenomenon responsible for the intensification of the extraction process. It also suggested that a grinding pretreatment would not be necessary prior to HVED, which considerably diminishes the energy input of the overall process. The presence of a non-mechanical effect and its contribution in the efficiency of HVED were also shown. The formation of hydrogen peroxide during the treatment was observed. However it did not seem to alter vine shoot phenolic compounds since these demonstrated a high radical scavenging capacity. As for the studies conducted on grape pomace, the simultaneous variation of several operating parameters allowed the aqueous and hydroethanolic optimization of phenolic compound extraction from these byproducts by response surface methodology (RSM). The passage from an aqueous to a hydroethanolic medium clearly improved the solid-liquid extraction of phenolic compounds from grape pomace. The use of ASE further increased the phenolic compound yield up to three times as compared to the optimum obtained with a hydroethanolic solvent.

Ce travail de recherche porte sur l’intensification de l’extraction des polyphénols à partir des écorces de chêne pédonculé et d’épicéa commun par des technologies innovantes : les champs électriques pulsés (CEP), les ultrasons (US) et les micro-ondes (MO). Ces prétraitements permettent l’amélioration de l’extraction par endommagement des membranes et/ou parois cellulaires. L’effet de chacune de ces techniques (CEP, US et MO) sur l’extraction des polyphénols a été mis en évidence à travers une étude d’optimisation quantitative et qualitative : suivi du rendement des polyphénols, de l’activité antioxydante et caractérisation chimiques des composés extraits.Une étude comparative des prétraitements (CEP, US et MO) couplés à une diffusion en milieu hydro-alcoolique alcalin, a permis de mieux comprendre les mécanismes mis en jeu lors d’un traitement d’un tissu fibreux. L’étude a prouvé que l’efficacité du procédé est indépendante de la famille d’arbre à laquelle appartiennent les écorces (résineux ou feuillus). De plus, pour valoriser les écorces de bois, l’application d’un prétraitement par CEP (20 kV/cm, 200 impulsions, 3,2 kJ/g bois) suivi d’une diffusion semble être une alternative aux ultrasons, micro-ondes et à l’extraction avec des écorces préalablement broyées. En effet, grâce à l’action électrique et mécanique des CEP, ce prétraitement permet d’obtenir un extrait riche en polyphénols (10,5 g EAG/100g MS pour l’épicéa soit 83 % d’efficacité et 5 g EAG/100g MS pour le chêne soit 82 % d’efficacité). / This research work focuses on the intensification of polyphenols extraction from Quercus robur and Norway spruce barks using innovative technologies : pulsed electric field (PEF), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE). These pre-treatments enhance the release of intracellular compounds by damaging the membrane and/or cell wall. The effect of each of these technologies (PEF, UAE and MAE) on polyphenols extraction has been highlighted in a quantitative and qualitative optimization analysis by evaluating the polyphenols concentration, antioxidant activity and by the chemical characterization of the extracted compounds.A comparative study of pre-treatments (PEF, UAE and MAE) coupled with an extraction step in an alkaline hydro-alcoholic medium allowed us to better understand the involved phenomena. The induced process efficiency is independent of the tree family to which the bark belongs (coniferous and hardwood). For bark valorization, the application of PEF treatment (20 kV/cm, 200 pulses, 3.2 kJ/g bark) followed by a diffusion step seems to be an alternative to ultrasound and microwave assisted extractions and to the extraction from grinded barks. In fact, the electrical and mechanical effect of PEF treatment allow to obtain an extract rich in polyphenols (10.5 g GAE/100g DM and an efficiency of 83 % for Norway spruce and 5 g GAE /100g DM and an efficiency of 82 % for Quercus robur bark).