Additive manufacturing in the form of 3D printing is a revolutionary technology that has developed within the last two decades. Its ability to print an object with accurate features down to the micro scale have made its use in medical devices and research feasible. A range of life-saving technologies can now go from the laboratory and into field with the application of 3D-printing. This technology can be applied to medical diagnosis of patients in at-risk populations. Living biosensors are limited by being Genetically Modified Organisms (GMOs) from being employed for medical diagnosis. However, by containing them within a 3D-printed enclosure, these technologies can serve as a vehicle to translate life-saving diagnosis technologies from the laboratory and into the field where the lower cost would allow more people to benefit from inexpensive diagnosis. Also, the GMO biosensors would be contained with a press-fit, ensuring that the living biosensors are unable to escape into the environment without user input. In addition, 3D-printing can also be applied to reduce the cost of lab-based technologies. Cell patterning technology is a target of interest for applying more cost-effective technologies, as elucidation of the variables defining cell patterning and motility may help explain the mechanics of cancer and other diseases. Through the use of a 3D-printed stamp, bacterial cells can be patterning without the use of a clean room, thus lowering the entry-barrier for researchers to explore cell patterning. With the commercialization of 3D-printing an opportunity has arisen to transition life-saving technologies into more cost-effective versions of existing technologies. This would not only allow more research into existing fields, but also to ensure that potentially life-saving technologies reach the people that need them. / Ph. D. / 3D-printing is a revolutionary technology developed within the last two decades. Its ability to print an object with accurate features down to the micro scale have made its use in medical devices and research feasible. A range of life-saving technologies can take advantage of 3Dprinting to go from bench top technologies into the field. This technology can be applied to medical diagnosis of patients in at-risk populations. Cells are able to detect and react to their environment. We can take advantage of this to design genetically modified cells for disease diagnosis. However, genetically modified cells are heavily regulated and it is thus difficult for use outside the lab. However, by containing them within a 3D-printed enclosure, these technologies can serve as vehicles to translate life-saving diagnosis technologies from the laboratory and into the field where the lower cost would allow more people to benefit from inexpensive diagnosis. Also, the genetically modified biosensors would be contained with a seal, ensuring that the genetically modified cells are unable to escape into the environment without user input. In addition, 3D-printing can also be applied to reduce the cost of lab-based technologies. Cell patterning technology is a target of interest for applying more cost-effective technologies in order to understand how cells self-pattern and move in their environment. This may help explain the mechanics of cancer and other diseases. Through the use of a 3D-printed stamp, bacterial cells can be patterned without the use of expensive facilities, thus lowering the entry-barrier for researchers to explore cell patterning. With the commercialization of 3D-printing, an opportunity has arisen to transition lifesaving technologies into more cost-effective versions of existing technologies. This would not only allow more research into existing fields, but also to ensure that potentially life-saving technologies reach the people that need them.

L’ectoderme embryonnaire devient spécifié en ectoderme non-neural, plaque neurale et bordure neurale à la fin de la gastrulation. Les cellules de bordure neurale sont les progéniteurs de la crête neurale et des placodes. La crête neurale est une population transitoire de cellules multipotentes, qui se forme au cours de la neurulation. Quand les bourrelets neuraux s’élèvent pour former le tube neural, les cellules de la crête neurale subissent une transition épithélio-mésenchymateuse, migrent dans l'ensemble du corps pour atteindre leur destination finale et se différencier. La crête neurale donne naissance à de multiples dérivés tels que les neurones et les cellules gliales du système nerveux périphérique, le cartilage et les os du visage, ou encore les mélanocytes. Des régulations complexes, impliquant de nombreuses signalisations et la transcription de gènes-clé, orchestrent ces événements. Cependant, les premières étapes menant à la formation de la crête neurale et à la spécification précoce de la bordure neurale sont encore peu comprises. Nous avons analysé le transcriptome de la crête neurale d'embryon de l'amphibien Xenopus laevis, à la recherche de nouveaux régulateurs des premières étapes de la formation de la crête neurale. Nous avons constaté que le régulateur de la glycolyse PFKFB4, est exprimé dans l’ectoderme dorsal de la jeune gastrula et dans les cellules de la crête neurale. Ici, nous démontrons que PFKFB4 régule la spécification de l’ectoderme via la voie de signalisation Akt, indépendamment de la glycolyse, démontrant ainsi la première fonction non-glycolytique des enzymes PFKFB. En outre, cette régulation est essentielle pour permettre aux progéniteurs de l'ectoderme d’être spécifiés en plaque neurale, crête neurale, placodes ou ectoderme non neural, mettant en évidence un nouveau point de contrôle de développement. De plus, nous démontrons que PFKFB4 régule des étapes ultérieures de la formation de la crête neurale. Notre travail met en évidence que les régulateurs du métabolisme cellulaire possèdent des fonctions non-métaboliques pour contrôler des étapes de développement au cours du développement embryonnaire. / Embryonic ectoderm becomes specified into non-neural ectoderm, neural plate and neural border at the end of gastrulation. Neural border cells are the progenitors of the neural crest and placodes. The neural crest is a transient population of multipotent cells, which forms during neurulation. As the neural border elevates to form the neural tube, neural crest cells undergo an epithelial to mesenchymal transition, migrate extensively into the whole body to reach their final destinations and differentiate. Neural crest gives rise to multiple derivatives such as neurons and glia, facial cartilage, bones, melanocytes and sympatho-adrenal cells. A complex interplay of signaling and transcriptional regulations orchestrates these early patterning events. However, the first steps leading to NC formation and early specification at the NB are less understood. We analysed the NC transcriptome of frog embryos, to look for novel regulators of the early steps of NC formation. We found that the well-known glycolysis regulator PFKFB4, is expressed in early gastrula dorsal ectoderm, and in neurula neural crest cells. Here, we demonstrate that PFKFB4 regulates ectoderm specification via Akt signaling independently of glycolysis, thus demonstrating the first non-glycolytic function of PFKFB enzymes. Moreover, this regulation is essential to allow ectoderm embryonic progenitors to be patterned into neural plate, neural crest, placodes and definitive ectoderm, highlighting a novel developmental checkpoint. Moreover, we also demonstrate that PFKFB4 regulates later steps of neural crest formation. Our work highlights that regulators of cell metabolism accumulate non-metabolic related functions to control developmental steps during embryonic development.

The transformation of an initially uniform population of epiblast cells into an intricately complex central nervous system (CNS) is one of the most fascinating processes during embryonic development. Presumptive neural cells are initially specified as cells of forebrain character. Studies in various vertebrates have indicated that cells of more caudal neural character, that will generate the brain stem and spinal cord, are generated through the reprogramming of these initial rostral cells. The initial regionalization of these neural progenitor cells is central to all further diversification of neuronal cell types and the subsequent formation of functional euronal circuits. The aim of this thesis has been to enhance our understanding of which stages of embryonic development that are critical for the initial rostrocaudal regionalization of neural precursor cells, and which signaling mechanisms that orchestrate this early diversification. Both human and chick embryos have the shape of a flat disc during gastrulation. At this early stage, the chick neural plate is already regionalized and cells positioned at distinct rostrocaudal levels are specified to generate cells exhibiting a gene expression profile characteristic of the forebrain, midbrain, rostral hindbrain and caudal spinal cord, respectively. In addition, the Isthmic organizer (IsO), a secondary signaling centre at the midbrain–hindbrain border that is required for the further development of this region, is also specified already at the gastrula stage. Caudal neural character is induced by signals from adjacent tissues - the primitive streak and the paraxial mesoderm. Wingless/Wnts, Fibroblastic growth factors (FGFs) and retinoids (RA) are signaling molecules that have been proposed to promote caudal embryonic development, and exhibit spatio- emporal expression patterns that coincide with early caudalizing activities. The caudalizing activity that emanates from the gastrula stage paraxial mesoderm is mediated by Wnt signals, and the induction of caudal neural character by Wnts results from a direct action on neural precursor cells. In the presence of FGF activity, graded Wnt signaling is sufficient to induce cells exhibiting caudal forebrain, midbrain and rostral hindbrain character. The discrimination between rostral hindbrain and caudal spinal cord character appear to depend on a gradient of both Wnt and FGF signals. At hindbrain and spinal cord levels the patterned generation of neural progenitor cells along the rostrocaudal axis controls the generation of different classes of motor neurons in response to diffusible Sonic hedgehog (Shh) signals. Gastrula stage Wnt signaling is also required for this subsequent generation of motor neuron subtypes characteristic of the hindbrain and spinal cord. Later, at the early somite stage, cells characteristic of the caudal hindbrain and rostral spinal cord are specified adjacent to RA producing paraxial mesoderm. Opponent RA and FGF signals appear to act on, and refine the rostrocaudal identity of the initial hindbrain and spinal cord cells induced by gastrula stage Wnt based signals. Consistently, combinatorial Wnt, FGF and/or RA signals are sufficient to reconstruct neural progenitor cells that differentiate into motor neurons characteristic of the caudal hindbrain, rostral spinal cord and caudal spinal cord, respectively, in response to Shh. / Transformationen av en initialt uniform cellpopulation till något så komplext som det centrala nervsystemet (CNS) är en av de mest fascinerande processerna under fosterutvecklingen. Anlaget till neuronala celler är initialt programmerade att generera nervceller som är typiska för den blivande hjärnan (cerebrum). Forskning på olika vertebrata modell-organsimer har klargjort att nedre regioner av CNS, hjärnstammen lillhjärnan och ryggmärgen, genereras genom reprogrammering av dessa initiala celler. Målet med avhandlingsarbetet har varit att öka förståelsen för vilka perioder under fosterutveckingen som är kritiska för den initiala induktionen av neuronala celltyper som är specifika för dessa olika regioner, samt vilka signalerings mekanismer som styr den initiala re-programmeringen. Under gastruleringen bildar anlaget till neuronala celler en, till synes uniform, platta medialt i ektodermet i både humana-, och kyckling embryon. Anlaget till neuronal vävnad är dock redan under detta tidiga utvecklingsstadie indelat i regioner. Celler inom en specifik region är programmerade att generera celler med en genexpressions-profil som är specifik för anlaget till hjärnan, de övre delarna av hjärnstammen (diencephalon, mesencephalon, metencephalon) eller den nedre delen av ryggmärgen. Även Isthmus – ett sekundärt organisations centra som bildas i konstriktionen mellan mesencephalon och metencephalon, och som behövs för den senare utvecklingen av dessa regioner – specificeras redan på gastrula stadiet. Dessa nedre neuronala celltyper induceras av signal molekyler från närliggande vävnader som t.ex. primitivstrimman och det paraxiala mesodermet. Wingless/Wnt, Fibroblast tillväxtfaktorer (FGFs) samt vitamin A metaboliter (retinoider, RA) är exempel på signalmolekyler som påverkar de nedre vävnaderna under tidig embryonal utveckling. Dessutom indikerar spatialt och temporalt reglerade genexpressionsmönster att närvaro av dessa signalerings proteiner sammanträffar med när och var nedre neuronala celltyper specificeras. Den signal aktivitet som avges från det paraxiala mesodermet i det gastrulerande embryot medieras av Wnt signalering. För induktion av nedre neuronala identiteter krävs Wnt signalering i de presumtivt neuronala cellerna. I närvaro av FGF signalerings aktivitet är det tillräckligt med en stigande gradient av Wnt signalering för att succesivt generera celler med en genexpressions profil som är specifik för diencephalon, mesencephalon och metencephalon. Distinktionen mellan, metencephalon och nedre ryggmärgs identitet verkar vara resultatet av en gradient av både Wnt och FGF signalering. När det paraxiala mesodermet börjar bilda somiter har även celler med en genexpressions-profil som är specifik för den förlängda märgen (myelencephalon) och den övre delen av ryggmärgen blivit specificerade. Dessa celltyper bildas i regioner där det närliggande paraxiala mesodermet producerar RA. En gradient av Wnt och FGF signalering ger upphov till en initial nedre celltyps identitet som krävs för att dessa celler ska kunna svara på RA signaleringen. Antagoniserande aktiviteter av RA och FGF signalering avgör vilka celler som sedermera kommer att ge upphov till förlängda märgen eller övre-, respektive, nedre ryggmärgen. Senare under utvecklingen bildas olika regionspecifika klasser av motorneuroner i bla. förlängda märgen och ryggmärgen. Den initiala, Wnt medierade, regionaliseringen av neuronala celltyper är central även för denna process. Dessutom kan olika klasser av motorneuroner, specifika för den förlängda märgen, respective övre-, och nedre ryggmärgs regionerna, rekonstrueras in vitro genom att reprogrammera naivt neuroepitel mha. en kombination av Wnt, RA och/eller FGF.

En microélectronique, l'augmentation de la densité des composants est la solution principale pour améliorer la performance des circuits. Ainsi, la taille des structures définies par la lithographie diminue à chaque changement de nœud technologique. A partir du nœud 14 nm, la lithographie optique est confrontée à la limite de résolution pour les niveaux métalliques. Pour surmonter cet obstacle, les niveaux métalliques sont conçus en deux étapes successives de patterning regroupant chacune une étape de lithographie et une étape de gravure. Cette technique, nommée double patterning, requiert une métrologie adaptée car l'alignement entre les deux étapes et les dimensions critiques sont alors directement liées. La méthode de mesure développée dans cette thèse repose sur la scattérométrie et la mesure de l'alignement par diffraction. Un code de simulation a permis d'optimiser la conception des mires de mesure. De plus, la méthode de mesure adoptée a pu être validée expérimentalement. / In microelectronics, the increase of component density is the main solution to improve circuit performance. The size of the patterns defined by lithography is reduced at each change of technology node. From the 14 nm node, optical lithography is facing the resolution limit for metal levels. In order to overcome this hurdle, metal levels are designed in two successive steps of patterning, which is composed of lithography followed by etching. This double patterning technique requires an appropriate metrology since overlay between the two steps and critical dimensions are directly linked. The developed method is based on scatterometry and overlay measurement by diffraction. Using a simulation code, the measurement targets have been designed optimally. Then the adopted method has been validated experimentally.

In dieser Arbeit werden die verschiedenen Möglichkeiten der Elektronenstrahlstrukturierung von organischen Schichten untersucht und charakterisiert. Je nach ihrer Energie und Leistung bewirkt die Interaktion der beschleunigten Strahlelektronen mit dem Material, auf das sie treffen, unterschiedliche Wechselwirkungen. Im Rahmen der durchgeführten Versuche wird demonstriert, dass diese Wirkung von lokalen, strahlchemischen Strukturveränderungen bis hin zu einem örtlich begrenzten Materialabtrag reicht. Neben den Untersuchungen einzelner organischer Schichten, werden ebenso organische Leuchtdioden (OLEDs) und deren Veränderungen unter Elektroneneinwirkung charakterisiert. Bei der Elektronenstrahlstrukturierung einer OLED mit sehr kleinen Leistungen wird sowohl die elektrische Leitfähigkeit als auch die Leuchtdichte der OLED reduziert. Dabei sind die Veränderungen in den organischen Materialien lokal stark auf den Ort der Elektroneneinwirkung begrenzt. Dies konnte genutzt werden, um eine hochauflösende Graustufenstrukturierung zu demonstrieren und ein Bild mit Strukturbreiten von 2 µm mit einem Elektronenstrahlprozess in eine weiße OLED zu schreiben. Elektronenstrahlprozesse mit höheren Leistungen bedingen eine thermische Wirkung und können so dünne organische Schichten lokal verdampfen. Mit solch einem Prozess konnte ein linien- und flächenhafter Abtrag realisiert werden, ohne die darunterliegende Elektrode zu schädigen. OLEDs haben den Vorteil, dass sie in Dünnschichttechnik hergestellt werden können und sehr kontrastreiche und farbechte Flächenlichtquellen sind. Daher bilden sie auch die Grundlage moderner Displays, an die jedoch stets wachsende Anforderungen gestellt werden. Klassischerweise werden OLED-Farbdisplays mithilfe einer strukturierten Abscheidung durch feine Metallmasken oder durch die Nutzung weißer OLEDs zusammen mit Farbfiltern hergestellt. Im Rahmen dieser Arbeit wurde ein alternatives Strukturierungskonzept entwickelt, dass die Möglichkeit bietet, ein OLED-Farbdisplay mithilfe eines Elektronenstrahlprozesses herzustellen. Das Schichtsystem der OLED bildet einen optischen Resonator, bei dem die Elektroden die Mikrokavität darstellen und die Dicke der organischen Schichten die Resonatorlänge definiert. Mittels kavitätsselektiver Modenauswahl ist es möglich, aus dem Spektrum einer weißen OLED verschiedene Farben auszukoppeln, wenn man die Resonatorlänge ändert. In der vorliegenden Arbeit wurde diese Anpassung der Resonatorlänge durch die Elektronenstrahlstrukturierung der ersten organischen Schicht vorgenommen und so rote, grüne und blaue OLEDs erzeugt und charakterisiert. Neben den grundlegenden Untersuchungen zu diesem Ansatz werden abschließend Grenzen und Möglichkeiten des Verfahrens aufgezeigt.:1 Einleitung 2 Grundlagen 2.1 Organische Leuchtdioden (OLEDs) 2.1.1 Organische Halbleiter 2.1.2 Aufbau und Funktionsweise von OLEDs 2.1.3 Elektro-optische Charakteristik 2.2 OLED-Vollfarbdisplays 2.2.1 Funktionsweise und Konzepte 2.2.2 Strukturierungsmethoden – Stand der Technik 2.3 Elektronenstrahlstrukturierung 2.3.1 Wechselwirkungen von Elektronen mit Festkörpern 2.3.2 Thermische Mikrobearbeitung 2.3.3 Nichtthermische Mikrobearbeitung 3 Zielsetzung und Lösungsansatz 3.1 Ziele dieser Arbeit 3.2 Prinzip Mikrokavität-OLED 4 Methodische Untersuchungen und Charakterisierung 4.1 OLED-Testsubstrate 4.1.1 Aufbau und Layout 4.1.2 Schichtabscheidung 4.2 Elektronenstrahlbehandlung 4.3 Analysemethoden 4.3.1 Schichtcharakterisierung 4.3.2 Elektro-optische Charakterisierung 4.3.3 FTIR-Spektroskopie 4.3.4 Photolumineszenz-Spektroskopie 5 Experimentelle Ergebnisse und Diskussion 5.1 Nichtthermische Elektronenstrahlbearbeitung von organischen Einzelschichten 5.1.1 Spektroskopische Untersuchungen 5.1.2 Elektrische Untersuchungen von Hole-Only-Devices 5.2 Nichtthermische Elektronenstrahlbearbeitung von OLEDs 5.2.1 Elektro-optische Untersuchungen 5.2.2 Hochauflösende Graustufenstrukturierung 5.2.3 Einfluss eines anschließenden Temperns 5.3 Thermische Elektronenstrahlbearbeitung 5.3.1 Thermische Elektronenstrahlstrukturierung organischer Schichten 5.3.2 Elektronenstrahlstrukturierung für Mikrokavität-OLEDs 6 Zusammenfassung und Ausblick A Technische Ergänzunge B Literaturverzeichnis C Abbildungsverzeichnis D Tabellenverzeichnis E Abkürzungsverzeichnis F Lebenslauf der Autorin G Wissenschaftliche Publikationen H Danksagung / In this work different possibilities of electron beam patterning for organic layers are investigated and characterized. Depending on the energy and power of the accelerated beam electrons, different interaction processes with the material can be initiated. Within the performed experiments it could be demonstrated that these effects range from structural chemical changes up to a localized evaporation of material. In addition to investigations of individual organic layers, organic light-emitting diodes (OLEDs) and their changes under the influence of electrons are also characterized. When OLEDs are patterned with an electron beam process with low power, both the electrical conductivity and the luminance of the OLED are reduced. The changes in the organic materials are locally strongly limited to the location of the electron penetration. This could be used to demonstrate a high-resolution grayscale patterning and to write an image with critical dimensions of 2 µm into a white OLED using an electron beam process. Electron beam processes with higher power cause a thermal effect and are able to evaporate thin organic layers locally. With such a process, a linear and areal shaped removal could be realized without damaging the underlying electrode. OLEDs have the advantage that they can be produced in thin-film technology. Furthermore they are an area light source, that has a high contrast and very good color properties. Therefore, most of the modern displays consist of OLEDs. Traditionally, OLED color displays are made by structured deposition through fine metal masks or by the use of white OLEDs together with color filters. As part of this work, an alternative structuring concept has been developed that offers the possibility of producing an OLED color display using an electron beam process. The layer system of the OLED forms an optical resonator in which the electrodes represent the microcavity and the thickness of the organic layers defines the resonator length. By means of cavity-selective mode selection, it is possible to extract different colors from the spectrum of a white OLED by changing the resonator length. In the present work, this adjustion of the resonator length was carried out by electron beam patterning of the first organic layer, thus generating and characterizing red, green and blue OLEDs. In addition to the fundamental investigations on this approach, limits and future perspectives of the method were finally pointed out.:1 Einleitung 2 Grundlagen 2.1 Organische Leuchtdioden (OLEDs) 2.1.1 Organische Halbleiter 2.1.2 Aufbau und Funktionsweise von OLEDs 2.1.3 Elektro-optische Charakteristik 2.2 OLED-Vollfarbdisplays 2.2.1 Funktionsweise und Konzepte 2.2.2 Strukturierungsmethoden – Stand der Technik 2.3 Elektronenstrahlstrukturierung 2.3.1 Wechselwirkungen von Elektronen mit Festkörpern 2.3.2 Thermische Mikrobearbeitung 2.3.3 Nichtthermische Mikrobearbeitung 3 Zielsetzung und Lösungsansatz 3.1 Ziele dieser Arbeit 3.2 Prinzip Mikrokavität-OLED 4 Methodische Untersuchungen und Charakterisierung 4.1 OLED-Testsubstrate 4.1.1 Aufbau und Layout 4.1.2 Schichtabscheidung 4.2 Elektronenstrahlbehandlung 4.3 Analysemethoden 4.3.1 Schichtcharakterisierung 4.3.2 Elektro-optische Charakterisierung 4.3.3 FTIR-Spektroskopie 4.3.4 Photolumineszenz-Spektroskopie 5 Experimentelle Ergebnisse und Diskussion 5.1 Nichtthermische Elektronenstrahlbearbeitung von organischen Einzelschichten 5.1.1 Spektroskopische Untersuchungen 5.1.2 Elektrische Untersuchungen von Hole-Only-Devices 5.2 Nichtthermische Elektronenstrahlbearbeitung von OLEDs 5.2.1 Elektro-optische Untersuchungen 5.2.2 Hochauflösende Graustufenstrukturierung 5.2.3 Einfluss eines anschließenden Temperns 5.3 Thermische Elektronenstrahlbearbeitung 5.3.1 Thermische Elektronenstrahlstrukturierung organischer Schichten 5.3.2 Elektronenstrahlstrukturierung für Mikrokavität-OLEDs 6 Zusammenfassung und Ausblick A Technische Ergänzunge B Literaturverzeichnis C Abbildungsverzeichnis D Tabellenverzeichnis E Abkürzungsverzeichnis F Lebenslauf der Autorin G Wissenschaftliche Publikationen H Danksagung

L'acide rétinoïque (AR) est un morphogène dérivé de la vitamine A, qui intervient dans le contrôle de l'organogenèse, de la prolifération et de la différenciation cellulaires chez les Chordés. Dans ce contexte, nous avons étudié les régulations spatio-temporelles de la voie de signalisation de l’AR au cours du développement de l’amphioxus, en mettant l'accent sur l’espèce européenne Branchiostoma lanceolatum.Nous avons tout d'abord inhibé ou activé la voie de signalisation de l’AR lors du développement embryonnaire en traitant des embryons d’amphioxus à des doses variables de composés pharmacologiques interférant avec le métabolisme des rétinoïdes. Grâce à l’utilisation d’outils mathématiques spécifiques, nous avons établi un schéma détaillé des effets des traitements effectués sur le développement du système nerveux central (SNC) et du pharynx chez l’amphioxus en nous basant sur l’expression de gènes marqueurs de tissus spécifiques. À l’issue de cette première analyse, nous avons par la suite étudié les effets d’une perturbation de la signalisation de l’AR à des points clés du développement chez l’amphioxus lors de la régionalisation du SNC et du pharynx. Nous avons ainsi montré que la voie de signalisation de l’AR intervient dans la régionalisation de l’axe antéro-postérieur via le contrôle des gènes hox dès le stade gastrula et jusqu’aux stades larvaires. En outre, nous avons réalisé l'étude préliminaire du gène homologue chez l’amphioxus du gène aldh1a2 des Vertébrés, et avons démontré que la régulation du niveau de synthèse de l’AR au cour du développement est conservée entre l’amphioxus et les Vertébrés. Finalement, nous avons montré que la voie de l’AR participe également à la morphogenèse caudale chez l’amphioxus, et que le mécanisme impliqué semble différent de celui proposé chez les Vertébrés où l’AR contrôle la structuration de la nageoire caudale par le ciblage des tissus mésenchymateux. / Retinoic acid (RA) is an endogenous vitamin A-derived morphogen. In this context, we studied the spatiotemporal roles of RA signaling in amphioxus development, focusing on the European amphioxus species: Branchiostoma lanceolatum. We first created excess and insufficiency models of RA signaling by exposing amphioxus embryos to series of doses of different pharmacological compounds targeting either the RA receptors or the RA metabolism machinery. By introducing the important mathematical concept of a Cartesian coordinate system founded by René Descartes, we created detailed diagrams of the concentration-dependent defects caused by RA signaling in the central nervous system (CNS) and pharynx of amphioxus by evaluating the statistical significances of tissue-specific marker gene expression in labeled embryos. This analysis yielded a very detailed description of the sensitivities of the developing amphioxus CNS and pharynx to altered RA signaling levels. Following this initial challenge, we correlated the effects of altered RA signaling levels with key amphioxus developmental stages characterized by structural transitions in CNS and pharynx. We show that hox-mediated RA signaling in axial patterning is active beyond the gastrula stage and might be maintained until at least early larval stage, with possible roles in more regionalized axis formation and organ induction. In addition, we carried out a preliminary study on a RA synthesizing gene in amphioxus, called aldh1a, a possible homolog of the vertebrate aldh1a2 gene, demonstrating that the feedback between RA signaling and RA synthesizing levels has emerged before the split of the cephalochordate and vertebrate lineages. Moreover, we are able to show that RA signaling also participates in tail fin morphogenesis in amphioxus by a mechanism that is probably not comparable to that in vertebrates, where RA modulates caudal fin patterning through targeting mesenchymal derivatives.

Les abeilles en libre vol montrent des capacités cognitives visuelles remarquables mais les bases neurales sous-jacentes ne peuvent pas être étudiées chez des insectes en vol. À l'inverse, le cerveau des abeilles immobilisées est accessible mais ne permet pas d'explorer l'apprentissage visuel. Pour dépasser cette limite, notre objectif a été d'établir un dispositif de réalité virtuelle pour pouvoir tester l'apprentissage visuel sur des abeilles attachées. Dans un premier temps, les abeilles ont été testées sur leur capacité à discriminer des couleurs en s'appuyant sur les renforcements positifs ou négatifs qui leur étaient associés. Ces expériences ont permis de mettre en évidence le rôle de la vision active dans la réalité virtuelle et l'importance de la phototaxie dans ce type de système. Grâce à ce dispositif, un apprentissage non élémentaire en réalité virtuelle, le patterning négatif, a été mis en place. Ainsi il a été montré que les abeilles étaient capables de résoudre cette tache en réalité virtuelle malgré sa complexité. Enfin, en s'appuyant sur le protocole de discrimination de couleurs, l'activation du cerveau a été étudiée au cours d'un test de mémoire des couleurs en analysant l'expression de gènes considérés comme des marqueurs de l'activité neurale. Les résultats de cette thèse, fournissant deux protocoles de réalité virtuelle solides pour étudier les apprentissages visuels élémentaires et non élémentaires, constituent une avancée considérable vers la possibilité de comprendre les bases neurales sous-jacentes à ces apprentissages chez l'abeille qu'ils soient simples ou complexes. / Free flying bees are known for their impressive visual cognition abilities, but the neural bases underlying those are poorly studied because of the difficulty to explore the brain in a flying insect. Conversely, it is possible to have access to the brain with tethered bees but, until now, no studies explored visual learning. To bypass this limitation, our aim was to establish a virtual reality device to test visual learning in tethered bees. First, bees were tested for their abilities to learn to discriminate colors according to the reinforcement associated to each of them. These experiments allowed to highlight the role of the active vision in virtual reality and the importance of phototaxis in this kind of device. Driven by these good results, we explored the non- elementary visual learning in bees and, more specifically, the negative patterning. The results suggest that bees were able to resolve this task in virtual reality despite its complexity. Finally, using the protocol of color discrimination, the brain activation has been explored during a color memory recall thanks to a quantification of immediate early genes considered as neural markers. This thesis provides two solid virtual reality protocols to study elementary and non-elementary visual learning in honey bees. This constitutes a huge advance and will allow to go further towards the understanding of the neural bases of simple and complex visual learning.

The efficiency of an organic photovoltaic cell depends mainly on its morphology where an exciton has to migrate to a p-n junction to create a photocurrent. Therefore the distance from the bulk of the cell to a junction interface should not exceed the diffusion length of the exciton. In this thesis, two novel lithographical methods, to produce specific polymer morphologies, were developed and evaluated. In the first method, called embedded annealing, self-assembled polystyrene colloids were embedded in a polydimethylsiloxane (PDMS) film and annealed under an electric field to produce a bi-polymer structure consisting of polymer columns in a thin film of PDMS. Polymer colloids were successfully assembled into two dimensional hexagonally close packed arrays. However, the annealing process was unsuccessful. The second method, imprint annealing, aimed to increase the aspect ratio (height/width) of thermally imprinted micrometer sized polystyrene features by annealing them in uniform electric fields. The results showed that the aspect ratio of imprinted features can be significantly increased, 21-fold, while maintaining the periodicity of the original imprint. This is in contrast to previous results where smooth polymer films annealed in uniform fields where the periodicity of the resulting structures cannot be independently controlled, and are highly sensitive to the electrode spacing. Feature sizes down to 1 µm and aspect ratios up to 4.5 were achieved using imprint annealing. / Verkningsgraden hos en hos en solcell beror, för givna material, framförallt på dess uppbyggnad. För att bidra till fotoströmmen måste en genererad exciton vandra till en pn-övergång. På grund av detta bör det längsta avståndet till närmaste pn-övergång i solcellen inte vara längre än excitonens diffusionslängd. I detta examensarbete testas två olika litografiska metoder för att åstadkomma en specifik filmgeometri lämpad för organiska solceller. Den första metoden, kallad embedded annealing, går ut på att bädda in spontant ordnade sfäriska polystyrenkolloider i en polydimetylsiloxan (PDMS) -film för att sedan vid förhöjd temperatur applicera ett elektiskt fält över filmen. Förhoppningen var att på detta sätt töja ut kolloiderna till pelare genom PDMS-filmen. I det första steget ordnades kolloiderna sponant i tätpackade hexagonala tvådimensionella gitter på kiselsubstrat. Experimenten lyckades inte med hjälp av elektriska fält töja ut kolloiderna. Den andra metoden, imprint annealing, syftar till att öka höjd/bredd -förhållandet och minska diametern hos präglade polystyrenstrukturer. Dessa ursprungliga topografiska stukturer skapas med hjälp av en tryckpressmetod kallad nanoimprinting. Dessa strukturer värmdes upp, och ett uniformt elekrisk fält applicerades över dem. Mina resultat visar att man med elektriska fält avsevärt kan öka höjd-breddförhållandet hos polymerstrukturer och samtidigt bevara periodiciteten hos de ursprungliga strukturerna. Detta står i kontrast mot tidigare resultat på släta filmer, där periodiciteten inte kan kontrolleras oberonde av andra parametrar. Med imprint annealing ökades höjd-breddförhållandet hos enskilda strukturer upp till 21 gånger. Diametrar ner till 1 µm och höjd/breddförhållanden upp till 4,5 uppnåddes.

Standard cells are fundamental circuit building blocks designed at very early design stages. Nanometer standard cells are prone to lithography proximity and process variations. How to design robust cells under variations plays a crucial role in the overall circuit performance and yield. This dissertation studies five related research topics in design and manufacturing co-optimization in nanometer standard cells. First, a comprehensive sensitivity metric, which seamlessly incorporates effects from device criticality, lithographic proximity, and process variations, is proposed. The dissertation develops first-order models to compute these sensitivities, and perform robust poly and active layout optimization by minimizing the total delay sensitivity to reduce the delay under the nominal process condition and by minimizing the performance gap between the fastest and the slowest delay corners. Second, a new equivalent source/drain (S/D) contact resistance model, which accurately calculates contact resistances from contact area, contact position, and contact shape, is proposed. Based on the impact of contact resistance on the saturation current, robust S/D contact layout optimization by minimizing the lithography variation as well as by maximizing the saturation current without any leakage penalty is performed. Third, this dissertation describes the first layout decomposition methods of spacer-type self-aligned double pattering (SADP) lithography for complex 2D layouts. The favored type of SADP for complex logic interconnects is a two-mask approach using a core mask and a trim mask. This dissertation describes methods for automatically choosing and optimizing the manufacturability of base core mask patterns, generating assist core patterns, and optimizing trim mask patterns to accomplish high quality layout decomposition in SADP process. Fourth, a new cell characterization methodology, which considers a random (line-edge roughness) LER variation to estimate the device performance of a sub-45nm design, is presented. The thesis systematically analyzes the random LER by taking the impact on circuit performance due to LER variation into consideration and suggests the maximum tolerance of LER to minimize the performance degradation. Finally, this dissertation proposes a design aware LER model which claims that LER is highly related to the lithographic aerial image fidelity and the neighboring geometric proximity. With a new LER model, robust LER aware poly layout optimization to minimize the leakage power is performed. / text

Durant les dernières décennies, de plus en plus de types de cellules se sont révélées capables de sonder leur environnement mécanique par l'application de forces. Ce phénomène appelé «Mecanosensing» est lié à l'adhésion et la mécanique cellulaire, et est souvent étudié grâce à l'interaction des cellules avec des substrats artificiels. Dans des études distinctes, des surfaces chimiquement structurées avec une répartition des ligands spécifiques ont montré une forte influence sur l’adhésion et la mécanique cellulaire. Cependant, la relation entre les deux phénomènes n'a pas été beaucoup explorée, en partie parce que la fonctionnalisation de substrats mous s’est révélée être un défi technique.Pour résoudre ce problème, nous avons développé une technique simple et rentable nommée «reverse contact printing», afin de fabriquer des plots de protéines sub-microniques sur un élastomère d'élasticité contrôlée, le polydiméthylsiloxane (PDMS). Mon travail de thèse a focalisé sur la standardisation et la compréhension du procédé de transfert. A l’aide de mesures de forces réalisées par AFM nous avons mesuré l’élasticité du PDMS, ainsi que les forces de cohésion et d'adhésion effectives impliquées dans le processus. Nous avons également étudié l'adhésion cellulaire avec des lymphocytes-T sur des surfaces de PDMS d'élasticité variable. Nous avons montré que contrairement à la plupart des autres types de cellules, les cellules-T s'étalent davantage sur substrat mou que sur dur. Finalement nous avons réalisé des expériences pilotes d'adhésion cellulaire sur PDMS structuré. / In the past decade, more and more types of cells have been shown to be capable of probing the mechanics of their environment by application of forces. The stiffness of the environment strongly influences a host of cellular parameters including cell adhesion and mechanics. In separate studies, the spatial distribution of ligands, modulated by chemical patterning of a target surface, has been shown to strongly influence cell adhesion and mechanics. However, the cross-talk between the two phenomena has not been much explored, partly because patterned functionalization of soft substrates is an engineering challenge. To address this issue, we have developed a simple and technique named "reverse contact printing" for fabrication of nanometric protein patches on PDMS (polydimethylsiloxane) elastomer. My PhD work consisted of deciphering the molecular mechanisms that underlie this technique. We realized that the rate of transfer crucially depended on the molecular groups on the protein and on the nature of the PDMS surface. We used atomic force microscopy (AFM) force measurements to measure PDMS elasticity as well as protein-substrate interactions to understand the molecular mechanism governing the transfer. We have identified that a successful reverse transfer is facilitated by the grafting of appropriate chemical groups on the protein, and depends on the PDMS surface treatment and elasticity. We also studied adhesion and mechanics of T lymphocytes on PDMS. We found that surprisingly T lymphocytes spread more on softer than on harder PDMS. In on-going pilot experiments, cells on patterned soft PDMS seem to exhibit different behavior as compared to cells on patterned glass.