Für das Tissue Engineering von Knochen werden poröse dreidimensionale Substrate (Scaffolds) als Zellträger benötigt, die in der vorliegenden Arbeit über keramische Technologie hergestellt wurden. Neben dem strukturierten und getrockneten Verbundwerkstoff (Grünkörper) und der Sinterkeramik wurde auch der Zwischenzustand nach Ausheizen der organischen Phase (Braunkörper) evaluiert. Bei der Herstellung blieb die Architektur der parallel orientierten Kanalporen, die über den Sol-Gel-Prozess der gerichteten ionotropen Gelbildung des Alginates erzeugt wurde, in allen Materialzuständen erhalten. Die Herstellungstechnologie wurde derart optimiert, dass die neuartigen anisotropen Scaffolds allen prinzipiell gestellten Forderungen für das Tissue Engineering entsprachen – sie waren porös mit weithin einstellbarer Porengröße, sterilisierbar, gut handhabbar unter Zellkulturbedingungen, biokompatibel und degradabel. Der unerwartete Favorit der Biomaterialentwicklung, der Braunkörper – eine nanokristalline, poröse Hydroxylapatit-Biokeramik – lag in einer ersten in vivo-Studie nach 4 Wochen integriert im Knochen vor. Die beobachtete Knochenneubildung deutete auf eine osteokonduktive Wirkung des Materials hin. Die in der vorliegenden Arbeit untersuchten Technologien und Biomaterialien bieten eine Basis für weitere Forschung und motivieren zur Weiterentwicklung und Nutzung als Scaffold für das Tissue Engineering oder Knochenersatzmaterial unter Verwendung der interessanten Architektur.

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Multi-metallic aerogels have recently emerged as a novel and promising class of unsupported electrocatalyst materials due to their high catalytic activity and improved durability for various electrochemical reactions. Aerogels can be prepared by a spontaneous one-step gelation process, where the chemical co-reduction of metal precursors and the prompt formation of the nanochain-consisting hydrogels, as a preliminary stage for the preparation of aerogels take place. However, detailed knowledge about the homogeneity and chemical distribution of these three-dimensional Pd-Pt aerogels at the nano-scale as well as at the macro-scale is still unclear to date. Therefore, we used a combination of spectroscopic and microscopic techniques to obtain a better insight into the structure and elemental distribution of the various Pd-rich Pd-Pt aerogels prepared by the spontaneous one-step gelation process. Synchrotron-based extended X-ray absorption fine structure (EXAFS) spectroscopy and high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) in combination with energy-dispersive X-ray spectroscopy (EDX) were employed in this work to uncover the structural architecture and chemical composition of the various Pd-rich Pd-Pt aerogels over a broad length range. The Pd80Pt20, Pd60Pt40 and Pd50Pt50 aerogels showed heterogeneity in the chemical distribution of the Pt and Pd atoms inside the macroscopic nanochain-network. Features of monometallic clusters were not detected by EXAFS or STEM-EDX, indicating alloyed nanoparticles. However, the local chemical composition of the Pd-Pt alloys strongly varied along the nanochains and thus within a single aerogel. To determine the electrochemically active surface area (ECSA) of the Pd-Pt aerogels for applications in electrocatalysis, we used the electrochemical CO stripping method. Due to high porosity and extended network structure, the resulting values of the ECSA for the Pd-Pt aerogels were higher than that for a commercially available unsupported Pt black catalyst. We show that the Pd-Pt aerogels possess a high utilization of catalytically active centers for electrocatalytic applications based on the nanostructured bimetallic framework. Knowledge about the homogeneity and chemical distribution of the bimetallic aerogels can help to further optimize their preparation by the spontaneous one-step gelation process and to tune their electrocatalytic reactivity.

Multi-metallic aerogels have recently emerged as a novel and promising class of unsupported electrocatalyst materials due to their high catalytic activity and improved durability for various electrochemical reactions. Aerogels can be prepared by a spontaneous one-step gelation process, where the chemical co-reduction of metal precursors and the prompt formation of the nanochain-consisting hydrogels, as a preliminary stage for the preparation of aerogels take place. However, detailed knowledge about the homogeneity and chemical distribution of these three-dimensional Pd-Pt aerogels at the nano-scale as well as at the macro-scale is still unclear to date. Therefore, we used a combination of spectroscopic and microscopic techniques to obtain a better insight into the structure and elemental distribution of the various Pd-rich Pd-Pt aerogels prepared by the spontaneous one-step gelation process. Synchrotron-based extended X-ray absorption fine structure (EXAFS) spectroscopy and high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) in combination with energy-dispersive X-ray spectroscopy (EDX) were employed in this work to uncover the structural architecture and chemical composition of the various Pd-rich Pd-Pt aerogels over a broad length range. The Pd80Pt20, Pd60Pt40 and Pd50Pt50 aerogels showed heterogeneity in the chemical distribution of the Pt and Pd atoms inside the macroscopic nanochain-network. Features of monometallic clusters were not detected by EXAFS or STEM-EDX, indicating alloyed nanoparticles. However, the local chemical composition of the Pd-Pt alloys strongly varied along the nanochains and thus within a single aerogel. To determine the electrochemically active surface area (ECSA) of the Pd-Pt aerogels for applications in electrocatalysis, we used the electrochemical CO stripping method. Due to high porosity and extended network structure, the resulting values of the ECSA for the Pd-Pt aerogels were higher than that for a commercially available unsupported Pt black catalyst. We show that the Pd-Pt aerogels possess a high utilization of catalytically active centers for electrocatalytic applications based on the nanostructured bimetallic framework. Knowledge about the homogeneity and chemical distribution of the bimetallic aerogels can help to further optimize their preparation by the spontaneous one-step gelation process and to tune their electrocatalytic reactivity.

El continuo af'an por reducir la cantidad de act'¿nidos minoritarios (MA) procedentes del combustible quemado en los reactores de agua ligera (Light Water Reactor, LWR) y de esa forma reducir la radiotoxicidad, ha llevado a desarrollar nuevos conceptos de combustible nuclear. El nuevo combustible por empaquetamiento de esferas (Sphere-Pac, SP) ofrece la oportunidad de reintroducir los MA en una matriz y quemarlos en un reactor r'apido de neutrones, donde se facilitan ciclos mu'ltiples por transmutaci'on de elemen- tos. Este combustible se puede utilizar tambi'en en un sistema subcr'¿tico r'apido de neutrones, es decir, un sistema nuclear accionado por un acelera- dor de part'¿culas (Accelerator Driven System, ADS), donde la subcriticidad (seguridad de parada del reactor) permite utilizar combustibles con mayor contenido de MA que en un reactor normal, reduciendo eficazmente en un solo paso la radiotoxicidad. El combustible SP se produce a partir de una soluci'on base (formada por metales y elementos qu'¿micos) mediante un proceso de gelificaci'on in- terna. Este proceso garantiza una buena homogeneidad del producto final y un riesgo de contaminaci'on mucho menor si se compara con la fabricaci'on cl'asica de pellets (combustible comprimido), puesto que se evita el uso de prensas y amoladoras. La gelificaci'on interna es una reacci'on qu'¿mica acu- osa que se produce al calentar la soluci'on hasta 80 ± 5¿ C. Cuando se realiza el proceso por calentamiento electromagn'etico, se observan algunas venta- jas con respecto al calentamiento tradicional por conducci'on (contacto de la muestra con aceite de silicio precalentado): se evita la etapa de reciclado del aceite y de los disolventes org'anicos necesarios para eliminar el aceite de la superficie de las part'¿culas producidas. En la unidad de gelificaci'on in- terna por microondas (Microwave Internal Gelation, MIG), las microondas representan una alternativa mucho m'as simple y segura: el calentamiento volum'etrico sin contacto facilita la producci'on a distancia del combustible en celdas calientes y adem'as reduce los residuos de l'¿quido contaminado. Esta tesis se enmarca dentro del proyecto Platform for Innovative Nu- clear FuEls (PINE), que tiene como objetivo fundamental la producci'on de combustible SP por MIG. En el sistema MIG, el tiempo de calentamiento es muy corto (del orden de decenas de milisegundos), por lo que se deben optimizar los par'ametros que contribuyen al calentamiento por microondas y es necesario conocer en profundidad la interacci'on entre las microondas y las muestras. En la primera parte de este trabajo se investiga un modelo t'ermico basado en diferencias finitas en el dominio del tiempo (FDTD), el cual es capaz de determinar, en cada instante durante el proceso de calentamiento, el comportamiento t'ermico de un punto definido dentro del material que se calienta. Adem'as se presenta una descripci'on detallada de los par'ametros m'as relevantes del modelo, incluyendo las condiciones de contorno (entre ellas la convecci'on). Por otra parte, se implementa anal'¿ticamente y se valida con diferentes t'ecnicas: una basada en teor'¿a de la f'¿sica, otra basada en la herramienta de ecuaciones diferenciales parciales (PDEtools) y la u'ltima basada en ejemplos encontrados en la literatura. En segundo lugar, se investigan los posibles disen¿os de cavidades de microondas para su aplicaci'on en MIG. Tanto las cavidades (selecci'on de los modos, frecuencia de resonancia, factores de calidad, etc.) como su posterior caracterizaci'on, se detallan con el objetivo de especificar el acoplamiento de energ'¿a. Los mecanismos de transferencia de energ'¿a de las cavidades se explican utilizando el m'etodo de perturbaci'on, con el que adem'as se analizan las p'erdidas de la cavidad cuando se coloca una muestra diel'ectrica en su interior. Con el modelo de transferencia de energ'¿a desar- rollado, se obtiene la tasa de generaci'on de calor por microondas, que se aplica al modelo t'ermico FDTD mencionado anteriormente. Los resultados anal'¿ticos demuestran la viabilidad de producir esferas gelificadas por MIG. Seguidamente se introducen los principales par'ametros relacionados con el calentamiento de un material por microondas, es decir, las propiedades diel'ectricas. Se desarrolla un nuevo procedimiento que permite medir estas propiedades en gotas que caen libremente a trav'es de una cavidad de mi- croondas. Se presenta el montaje experimental, cuya viabilidad se prueba a trav'es de diferentes experimentos. Las propiedades diel'ectricas medidas se incluyen en los modelos (perturbacional y t'ermico) con la intenci'on de determinar la potencia absorbida por la sustancia (en forma de gotas) y la temperatura que alcanza. En la u'ltima parte se presenta la implementaci'on del sistema MIG apli- cada al proyecto PINE, fundamental para la pr'actica de calentamiento (basado en frecuencias altas) dentro del laboratorio. Las propiedades de cada dispositivo se evaluan para realizar un estudio de potencia antes del ensamblaje del sistema MIG. De esa forma se evitan fallos al poner el sis- tema en funcionamiento. Adem'as se aportan las t'ecnicas experimentales y los resultados. La producci'on con 'exito de esferas gelificadas demuestra, sin duda, el uso favorable de las microondas en la producci'on de combustible SP por gelificaci'on interna. / In the continuous aim to reduce the amount of minor actinides (MA) from the spent fuel of Light Water Reactors (LWR) and therefore reduce its radiotoxicity (radioactive toxicity), new nuclear fuel concepts have been developed. Sphere-Pac (SP) fuel gives the opportunity to reintroduce the MA in a fuel matrix and to burn them in a fast reactor, which facilitates a multi-cycle because of its breeding feature, or in a subcritical fast system, i.e. an Accelerator Driven System (ADS) where its sub-criticality allows higher MA contents than a normal fast reactor reducing efficiently the radiotoxicity in one step. SP fuel is produced from the base solution (already containing all the elements) by internal gelation, which guarantees a good material homo- geneity and a lower contamination risk compared to the classical pellet fabrication, avoiding presses and grinding machines. The internal gelation is an aqueous chemical reaction occurring when the solution is heated up to 80 ± 5¿C. When performing the internal gelation process with electro- magnetic heating, some advantages appear with respect to the traditionally heating through conduction by contact of the sample with hot silicon oil: the recycling step of the oil and the organic solvents necessary to clean the particles from oil are avoided. In the Microwave Internal Gelation (MIG) unit, the microwaves represent a much simpler and safer alternative: the contactless volumetric heating facilitates the remote production of the fuel in hot cells and furthermore reduces the contaminated liquid waste. The fuel related project called Platform for Innovative Nuclear FuEls (PINE), in which this thesis is embedded, aims for the production of SP- fuel by MIG. In the MIG system, the heating time is very short (in the order of tens of milliseconds), therefore the microwave heating parameters have to be optimized and a good knowledge of the interaction between the microwaves and the samples must be achieved. In the first part of this dissertation a finite difference time domain (FDTD) thermal model capable to determine over each instant about the thermal behaviour of a definite point inside a material during heat process- ing is investigated. A detailed overview of the most relevant parameters on the model including the boundary conditions (e.g. convection) is pre- sented. Furthermore, the model is analytically implemented and validated with different techniques: a theoretical based physically validation, a par- tial differential equations (PDEtools) based validation and a validation with examples from the literature. Secondly, possible microwave cavity designs for MIG are researched. The cavities (selection of modes, resonant frequency, Q-factor, etc.) and its subsequent characterization for the coupling of energy are explained. Furthermore, the power transfer mechanisms of the cavities are explained using the perturbation method to analyse the losses when a dielectric sam- ple is placed inside a cavity. The developed power transfer model delivers the microwave heat generation rate which is applied to the FDTD thermal model mentioned in the previous paragraph. The analytical results provide a positive impression about the feasibility of producing gelated spheres by MIG. Next, the main parameters dealing with the heating of a material by microwaves are introduced. A new procedure that enables the measure- ment of dielectric properties of aqueous droplets freely falling through a microwave cavity is developed. The experimental setup is presented and several experiments prove its feasibility. The measured dielectric properties are afterwards included in the perturbation and thermal models with the main intention of determining the absorbed power by the material in form of drops and the reached temperature. In the last part the MIG system for the laboratory practice of the high frequency heating applied to the PINE project is implemented. Each device is characterized for a power study precedent to the MIG system assembly, avoiding then failures when putting the system into operation. In addition, the experimental techniques and the results are reported. Successful pro- duction of gelated spheres shows the favourable usage of microwave for the production of SP-fuel by internal gelation. / Cabanes Sempere, M. (2013). Innovative production of nuclear fuel by microwave internal gelation [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31641 / Alfresco

Für das Tissue Engineering von Knochen werden poröse dreidimensionale Substrate (Scaffolds) als Zellträger benötigt, die in der vorliegenden Arbeit über keramische Technologie hergestellt wurden. Neben dem strukturierten und getrockneten Verbundwerkstoff (Grünkörper) und der Sinterkeramik wurde auch der Zwischenzustand nach Ausheizen der organischen Phase (Braunkörper) evaluiert. Bei der Herstellung blieb die Architektur der parallel orientierten Kanalporen, die über den Sol-Gel-Prozess der gerichteten ionotropen Gelbildung des Alginates erzeugt wurde, in allen Materialzuständen erhalten. Die Herstellungstechnologie wurde derart optimiert, dass die neuartigen anisotropen Scaffolds allen prinzipiell gestellten Forderungen für das Tissue Engineering entsprachen – sie waren porös mit weithin einstellbarer Porengröße, sterilisierbar, gut handhabbar unter Zellkulturbedingungen, biokompatibel und degradabel. Der unerwartete Favorit der Biomaterialentwicklung, der Braunkörper – eine nanokristalline, poröse Hydroxylapatit-Biokeramik – lag in einer ersten in vivo-Studie nach 4 Wochen integriert im Knochen vor. Die beobachtete Knochenneubildung deutete auf eine osteokonduktive Wirkung des Materials hin. Die in der vorliegenden Arbeit untersuchten Technologien und Biomaterialien bieten eine Basis für weitere Forschung und motivieren zur Weiterentwicklung und Nutzung als Scaffold für das Tissue Engineering oder Knochenersatzmaterial unter Verwendung der interessanten Architektur.

Organisms are frequently exposed to fluctuating environmental conditions and might consequently experience stress. Environmental stress can damage cellular components, which can threaten especially single-celled organisms, such as yeast, as they cannot escape. To survive, cells mount protective stress responses, which serve to preserve cellular components and architecture. Recent findings in yeast show that the stress response upon energy depletion stress involves a gelation of the cytoplasm due to macromolecular protein assembly, characterized by drastic changes in cytoplasmic material properties. Remarkably, the stress-induced cytoplasmic gelation is protective, raising the question whether this could be a common strategy of cells to cope with severe stress. I hypothesized that protein aggregation induced by another common stress, severe heat shock, might cause a similar cytoplasmic gelation in yeast. Furthermore, I hypothesized that the reversibility of cytoplasmic gelation is provided by molecular chaperones, which are known regulators of protein aggregation. In this thesis, I therefore aimed to characterize the changes in the material properties of the cytoplasm upon severe heat shock as well as their underlying causes and how molecular chaperones affect these changes. To characterize heat-induced changes in the material properties of the cytoplasm, I monitored Schizosaccharomyces pombe cells during recovery from severe heat shock using a combination of cell mechanical assays, time-lapse microscopy and single-particle tracking. I found that the cells entered a prolonged growth arrested state upon stress, which coincided with significant cell stiffening and a long-range motion arrest of lipid droplets in the cytoplasm, while smaller cytoplasmic nanoparticles remained mostly mobile. At the same time, a significant fraction of proteins aggregated in the cytoplasm, forming insoluble inclusions such as heat shock granules. After stress cessation, the observed changes were reversed as stiffened cells softened and lipid droplets resumed long-range motion. Cell softening and lipid droplet motion recovery coincided with protein disaggregation. These processes could be delayed by impairing protein disaggregation through genetic perturbation of the molecular chaperone Hsp104, which functions as a protein disaggregase. In contrast, no influence on protein disaggregation or heat-induced cytoplasmic material property changes was detected for the small heat shock protein Hsp16. These results suggest that the cytoplasm gels upon severe heat shock due to protein aggregation and is refluidized during recovery with the help of Hsp104. Remarkably, cells resumed growth only after refluidization of the cytoplasm, suggesting that reversible cytoplasmic gelation may contribute to regulation of the heat-induced growth arrest. In addition, cytoplasmic gelation could potentially preserve cellular architecture during heat shock. Overall, the results from my thesis work indicate that reversible cytoplasmic gelation due to macromolecular protein assembly may be a universal cellular response to severe stress which is associated with a stress-protective growth arrest. A likely stress-specific part of this response is the chaperone-dependent refluidization of the cytoplasm, which might explain the prolonged growth arrest seen upon severe heat shock as compared to other stresses and might allow more time for the repair of heat-induced damage.:Abstract Zusammenfassung Table of contents Figure index List of abbreviations 1 Introduction 1.1 Heat shock affects cellular function and fitness 1.1.1 Cells respond to stress in phases 1.1.2 Heat shock threatens cellular homeostasis and structural integrity 1.1.3 Stress severity determines detrimental effects of heat shock 1.1.4 Heat stress causes protein aggregation 1.1.5 Heat shock granules are functional aggregates in yeast 1.2 The heat shock response protects cellular fitness 1.2.1 Cells change transcription to adapt to stress 1.2.2 Molecular chaperones are important in stress protection 1.2.3 Hsp104 is a protein disaggregase chaperone 1.2.4 Small heat shock proteins modulate protein aggregation 1.2.5 Stress severity determines modules of the heat shock response 1.3 Cytoplasmic material properties change during stress 1.3.1 Cells homeostatically adapt cytoplasmic material properties during stress 1.3.2 The cytoplasm is viscoelastic 1.3.3 Is the cytoplasm a gel? 1.3.4 Stress can induce cytoplasmic gelation 1.4 Research aims 2 Materials and Methods 2.1 S. pombe strains and growth conditions 2.1.1 Growth conditions 2.1.2 Construction of S. pombe strains 2.1.3 S. pombe transformation 2.1.4 S. pombe colony PCR 2.1.5 S. pombe strains used in this thesis 2.2 Plasmids and cloning 2.2.1 Plasmids used in this thesis 2.2.2 Construction of plasmid for fluorescent GEM nanoparticle expression 2.2.3 E. coli transformation 2.2.4 Plasmid purification from E. coli 2.3 S. pombe stress treatments 2.3.1 Heat shock treatment 2.3.2 Osmoadaptation 2.4 Cell biological methods 2.4.1 Viability assay 2.4.2 Growth assay 2.5 Cell bulk mechanical assays 2.5.1 Spheroplasting assay 2.5.2 Atomic force microscopy 2.5.3 Real-time deformability cytometry 2.5.4 RT-DC sample preparation 2.5.5 RT-DC setup and measurements 2.5.6 RT-DC data evaluation 2.6 Microscopy 2.6.1 Microscopy of GEM particles 2.6.2 Fluorescence microscopy of endogenously labeled Pabp-mCherry 2.6.3 Microscopy of µNS particles 2.7 Image analysis 2.7.1 Image analysis of Pabp-mCherry in vivo fluorescence microscopy 2.7.2 Differenced brightfield image analysis 2.7.3 Kymographs 2.8 Single-particle tracking analysis 2.8.1 Particle tracking 2.8.2 Mean squared displacement analysis 2.9 Optical diffraction tomography (ODT) 2.9.1 ODT sample preparation 2.9.2 ODT optical setup and measurements 2.9.3 ODT tomogram reconstruction and quantitative analysis 2.10 Lysis and sedimentation assay 2.10.1 Lysis buffer 2.10.2 S. pombe heat shock treatment and lysis 2.10.3 Sedimentation assay 2.10.4 Protein concentration measurement 2.10.5 SDS-PAGE 2.10.6 Coomassie staining 2.10.7 Western Blot 3 Results 3.1 Physical and chemical conditions affect heat shock survival and heat-induced growth arrest of S. pombe 3.1.1 S. pombe arrests growth during severe heat shock 3.1.2 Heat-induced growth arrest is dose-responsive 3.1.3 Heat-induced growth arrest depends on experimental conditions 3.1.4 Buffer pH and energy source have a strong impact on heat shock survival 3.1.5 Osmoadaptation protects cells during heat shock 3.2 Severe heat shock induces reversible cellular stiffening 3.2.1 Cellular rounding upon cell wall removal is delayed after heat shock 3.2.2 Elastic modulus of S. pombe cells is increased after heat shock 3.2.3 Recovery from heat-induced growth arrest is preceded by cell softening 3.3 Long-range particle dynamics in cytoplasm are abolished after heat shock 3.3.1 Small particle dynamics are largely independent of heat shock treatment 3.3.2 Lipid droplets are confined in space after heat shock 3.4 Cytoplasmic crowding increases during heat shock 3.5 Heat shock induces reversible protein aggregation 3.5.1 Insoluble protein fraction is increased after heat shock 3.5.2 Heat shock granules form reversibly during heat shock 3.5.3 HSG formation and dissolution are correlated with changes in cytoplasmic long-range dynamics 3.6 Molecular chaperones modulate cytoplasmic material property changes during heat stress recovery 3.6.1 Hsp104 but not Hsp16 is required for disaggregation of heat shock granules 3.6.2 Hsp104 but not Hsp16 is required for recovery from heat-induced growth arrest 3.6.3 Hsp104 but not Hsp16 is required for recovery of cytoplasmic long-range dynamics 3.6.4 Hsp104 but not Hsp16 is required for rapid reversal of cellular stiffening which coincides with growth recovery 4 Discussion 4.1 Summary and model 4.2 Which mechanism underlies cell stiffening upon heat shock? 4.2.1 Heat-induced protein aggregation might cause cell stiffening 4.2.2 Heat-induced protein aggregation might lead to cytoplasmic gelation 4.2.3 Many factors could contribute to protein aggregation and cytoplasmic gelation 4.3 The heat-induced growth arrest state is associated with reversible cytoplasmic gelation 4.3.1 Cytoplasmic material property changes mark the severe heat-induced growth arrest state 4.3.2 Is cytoplasmic gelation a common response to severe stress? 4.4 What are the biological consequences of cytoplasmic gelation? 4.4.1 Cytoplasmic gelation might obstruct processes that require motion of large structures 4.4.2 Is cytoplasmic gelation upon heat shock protective? 4.5 Heat shock recovery involves the chaperone-mediated refluidization of the cytoplasm 4.5.1 Cytoplasmic refluidization is required for growth recovery 4.5.2 Stress tolerance is marked by enhanced reversibility of cytoplasmic gelation 4.5.3 The protein disaggregase chaperone Hsp104 regulates the reversal of heat-induced cytoplasmic material property changes 4.6 Conclusion References Acknowledgements Publications and Contributions 5 Erklärung entsprechend §5.5 der Promotionsordnung

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