The cell cycle is a highly orchestrated developmental process that eventually leads to the reproduction of a cell. In metazoans, it is driven by the successive activation of cyclin-dependent kinases (Cdk) and proper coordination of cell cycle transitions and processes ensure genomic stability. DNA replication takes place during S phase to faithfully duplicate a cell’s genetic material. In eukaryotes, S phase onset involves the initiation of numerous licensed replication origins across the genome and requires the activities of two protein kinases, S phase-Cdk and Cdc7. In this thesis, I present work relating to the role of the S phase-promoting kinases in DNA replication and S phase regulation. Using the cell-free system of Xenopus egg extracts, a small molecule inhibitor of Cdc7, PHA-767491, was characterised. PHA-767491 was then used to demonstrate that Cdc7 executes its activity early in S phase before the Cdk-dependent step. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike S-Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts, which rapidly reverses Cdc7-dependent phosphorylation of chromatin-bound Mcm4 and likely functionally lowers Cdc7 activity during an etoposide-induced checkpoint response. This provides a novel mechanism for regulating Cdc7 by counteracting its activity on essential replication substrates in the event of replicative stress. In the second part of the thesis, the design strategy for generating a Cdc7-conditional knockout mouse (cko) is outlined and results from the screen for a transgenic founder are presented. A Cdc7-cko mouse will be a valuable tool to further dissect Cdc7 function and regulation in mammalian cells. In the final section, S phase entry and progression in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2 was examined. Contrary to expectations, Cdk1/Cdk2 double knockout cells can enter S phase in the absence of detectable S phase-Cdk activity. S phase progression, however, was inefficient. Cdc6 and cyclin E1 proteins were found to accumulate in high levels in these cells. The exact function(s) and mechanism(s) for these observations remain to be discovered. With this work, I hope to provide additional insight into the roles and regulation of S phase kinases in eukaryotic DNA replication.

Im Rahmen der vorliegenden Arbeit wurden promesogene Arme sowie eine Bibliothek von Sternmesogenen mit Anthracen als Donor- und Anthrachinon als Akzeptorbaustein synthetisiert und untersucht. Ein Schwerpunkt der Arbeit lag auf der Synthese, dem Upscaling, der selektiven Schützung und weiteren Umsetzung der 2,6-substituierten Anthracen- und Anthrachinon-Chromophore zu den Armbausteinen. Besondere Herausforderungen ergaben sich nicht nur in der Entwicklung einer effizienten Synthesestrategie zur Gewinnung der Chromophore, sondern auch in der Wahl geeigneter Schutzgruppen. Die sternförmigen Verbindungen, die im Rahmen der vorliegenden Arbeit präpariert wurden, enthalten 1,3,5-Trihydroxybenzen (Phloroglucin) als Kerneinheit und sind Multiarmmesogene mit der kleinstmöglichen Zahl an Armen. Durch geeignete Schutzgruppenstrategien gelang neben den C3-symmetrischen Verbindungen die gezielte Darstellung der C2-symmetrischen und unsymmetrischen Verbindungen. Die Gesamtausbeuten der semiperfluorierten Verbindungen fallen deutlich geringer aus als die der ausschließlich mit Alkylketten dekorierten Derivate, da ihre Isolierung sehr anspruchsvoll ist. Alle Verbindungen bilden ausnahmslos kolumnare Phasen. Semiperfluorierte Ketten wurden eingeführt, um eine Trennung des Donors Anthracen und des Akzeptors Anthrachinon zu erreichen. Die Kolumnendurchmesser sind bei allen kolumnaren Mesophasen wesentlich kleiner als die Durchmesser der sternförmigen Konformere der Mesogene. Angelehnt an die früher untersuchten Oligobenzoatsterne werden Modelle mit gefalteten, E-förmigen Konformeren aufgestellt. So ist es möglich, die erforderliche Anzahl an Molekülen pro Elementarzelle in einer dichten, nanosegregierten Packung anzuordnen. Mit Absorptions- und Emissionsmessungen konnte dieses Modell bestätigt werden. In allen Donor- und Akzeptor-substituierten Verbindungen wird die Fluoreszenz durch Energietransferprozesse nach Förster und Dexter fast vollständig gelöscht. Restfluoreszenz wird in dem Bereich beobachtet, der nur noch den Transfer nach Dexter zulässt und ist für die Derivate am höchsten, die in den E-förmigen Konformeren Donor und Akzeptor am besten trennen können. Die Ergebnisse dieser Arbeit zeigen, dass Anthracen- und Anthrachinonderivate eine Vielzahl komplexer zwei- und dreidimensional hochgeordneter kolumnarer Strukturen ausbilden und damit hochinteressant sind als flüssigkristalline organische Halbleitermaterialien. / As part of this work promesogenic arms and a library of star-shaped mesogens with anthracene as electron donor and anthraquinone as electron acceptor unit were synthesised and characterised. The work focused first on synthesis, upscaling, selective protection and further implementation of 2,6-substituted anthracene and anthraquinone chromophores to arm derivatives. The development of an efficient synthetic strategy, but also the selection of appropriate protecting groups was particular challenging. The star-shaped molecules prepared in this work contain 1,3,5-trihydroxybenzene (Phloroglucinol) as core unit and are multiarm mesogens with the smallest possible number of arms. In addition to the synthesis of the C3-symmetrical, suitable protecting group strategies led to selective preparation of the C2-symmetrical and unsymmetrical compounds. All over yields of semiperfluorinated compounds are significant lower compared to the alkyl chain derivatives, owing to the much more demanding work-up. The liquid crystalline properties of all star-shaped target compounds were analyzed. Without exception – all materials form columnar mesophases. Semiperfluorinated chains were used to nanosegregate the donor anthracene and the acceptor anthraquinone. Column diameters of all columnar mesophases are much smaller than the diameters of star-shaped conformers of the mesogens. According to previous investigations of oligobenzoate stars, models with folded, E-shaped conformers have been suggested. This way it is possible to arrange the required number of molecules in a dense, nanosegregated structure. This model could be confirmed by absorption und emission spectroscopy. For all donor and acceptor substituted compounds fluorescence is almost completely quenched by energy transfer processes according to Förster and Dexter. Residual fluorescence, which is only observed in the range of Dexter transfer, is highest for molecules in which donor and acceptor in the E-shaped conformer possess a high probability to be separated in the columnar stacks. The results of this work show anthracene and anthraquinone derivatives forming a number of complex two- and three-dimensional high-ordered columnar structures and thus are highly interesting for liquid crystalline organic semiconducting materials.

Les aciers Dual-Phase (DP) sont des aciers à très haute résistance mécanique (AHSS) fortement utilisés pour des applications automobiles en raison de leur très bon compromis résistance mécanique/ductilité ainsi que par leur habilité à répondre aux multiples exigences industrielles (bas prix, assemblage, revêtement etc.). A l'heure actuelle, le développement d'aciers DP apparait durable pour la caisse-en-blanc des structures automobiles. La microstructure ferrite-martensite, caractéristique des aciers DP, est obtenue par un traitement thermique complexe composé de différentes étapes au cours desquelles différents mécanismes métallurgiques entrent en jeux. Les principales difficultés de production sont liées au fait (i)que les évolutions microstructurales sont influencées par les différents paramètres de traitement thermique (vitesse de chauffe, température ...), (ii) que les différentes étapes de traitement sont interconnectées et que (iii) les évolutions microstructurales peuvent se chevaucher et, part conséquent, interagir entre elles. Ces travaux de thèse ont pour objectif d'améliorer la compréhension des évolutions métallurgiques entrant en jeux lors des traitements thermiques des aciers DP, et notamment d'améliorer la compréhension de l'influence des paramètres de traitement. Les différentes évolutions métallurgiques ont été, dans un premier temps, caractérisées en couplant un ensemble de techniques expérimentales (dilatométrie, dureté, TPE ...) et grâce à un protocole assurant de décorréler les mécanismes se superposant et interconnectés. A titre d'exemple, les deux principaux mécanismes entrant en jeux lors de l'étape de recuit intercritique ont été, dans un premier temps, étudiés séparément ((1) la recristallisation a été étudié en dessous de la température de formation d'austénite et (2) la formation d'austénite a été étudié sur un acier pré-recristallisé) avant de se concentrer sur le cas des aciers laminés à froid où la recristallisation et formation d'austénite se superposent. Le projet avait également pour objectif de développer des outils de prédictions permettant de décrire les évolutions microstructurales basés sur des approches empiriques (loi de JMAK) ainsi que vers des modèles à base plus physique (mixed-mode modèle et modèle diffusif). Une attention particulière a été dédié à discuter de la fiabilité, l'adaptabilité, des forces et limitations des différentes approches développées. / Dual-Phase steels (DP) are one of the most used Advanced High Strength Steels (AHSS) for automotive applications because they present good strength/ductility compromise and they adapt to number of industrial constraints (low price, shaping, welding, coating etc.). Nowadays, the development of DP steels seems to be promising and sustainable for the body-in-white structure. The typical ferrite-martensite microstructure, characteristic of DP steels, are obtained by a thermal treatment composed of different stages during which metallurgical evolutions occur. Major difficulties of their processing are due to the fact that (i) microstructural evolution kinetics are influenced by cycle parameters (heating rate, annealing temperature etc.), (ii) different stages are interconnected and (iii) some microstructural evolutions may overlap and, therefore, interact. This PhD-work aimed at getting a better understanding of microstructural evolutions during the thermal cycle of DP steel and, namely, the influence of cycle parameters. Different microstructural evolutions occurring during the thermal cycle were first characterized coupling different experimental techniques (dilatometry, hardness, TPE etc.) and with a particular protocol in order to decorrelate overlapping and interconnected phenomena. As example, two major evolutions occurring during the intercritical annealing were first studied individually ((1) recrystallization was investigated below austenite formation temperature and (2) austenite formation was investigated on prior recrystallized steels) before investigated cold-rolled steel case where recrystallization and austenite formation overlap. The study was then attached to develop some predictive tools to describe microstructural evolutions based on phenomenological approaches (JMAK law) towards more physical based models (mixed-mode, diffusive models). A particular care was attached to discuss on model reliability, versatility, strengths and limitations.

Die vorliegende Arbeit befasst sich mit der Synthese und Untersuchung V- und brettförmiger Flüssigkristalle zur Realisierung einer biaxialen nematischen Mesophase. Es wurde erfolgreich eine Serie neuer Mesogene mit hockeyschlägerförmiger und V-förmiger Struktur synthetisiert. Zusätzlich wurden Dimere aus einem dieser hockeyschlägerförmigen Verbindungen dargestellt. Als Kernbaustein wurde Benzo[1,2-b:4,3-b']dithiophen verwendet, dessen lokales Kerndipolmoment von 1.0 Debye sich nach theoretischen Vorgaben zusätzlich zum Bindungswinkel (108.9 °) positiv auf die Bildung einer Nb-Phase auswirken soll. Überraschenderweise bilden nur die hockeyschlägerförmigen Moleküle eine uniaxiale, optisch positive nematische Mesophase aus. Alle anderen V-förmigen Verbindungen und sogar die Dimere sind ausschließlich kristallin und keine Flüssigkristalle. Die Einkristallstrukturanalyse eines hockeyschlägerförmigen Mesogens sowie eines V-förmigen Moleküls zeigt bemerkenswerte Ähnlichkeiten auf. Ein Modell des Phasenübergangs wird präsentiert, welches die Abwesenheit der nematischen Mesophase in der Familie der V-förmigen, formstabilen Mesogene mit terminalen aliphatischen Ketten erklärt. Zudem befasst sich die Arbeit mit der Synthese und der Untersuchung brettförmiger Moleküle, welche dem optimalen Seitenverhältnis von 15 : 5 : 3 mit L > B > T zur Bildung biaxialer Mesophasen, relativ nahekommen. Ein Anthrachinon-Kernbaustein wurde dabei mit Armen bestehend aus einem Oligo(phenylenethinylen)-Grundgerüst entsprechender Länge verknüpft. Es konnten verschiedene dachförmige Mesogene dargestellt werden, bei denen die Art und Anzahl der Seitenketten sowie der terminalen Ketten variiert wurde. Thermische sowie mikroskopische Untersuchungen zeigen bei allen Verbindungen eine breite nematische Mesophase. Mittels spezieller Röntgenstreuung im magnetischen Feld kann die Bildung nematischer Domänen mit SmC-artigen biaxialen Aggregaten bestätigt werden. / The present work deals with the synthesis and investigation of V- and board¬¬¬-shaped liquid crystals for the realization of a biaxial nematic mesophase. A series of new mesogens has been succesfully prepared, realizing hockey-stick, hockey-stick dimer and V-shaped molecular topologies. As a core unit, benzo[1,2-b: 4,3-b ']dithiophene was used, whose local core dipole moment of 1.0 Debye in addition to the bond angle (108.9 °) is supposed to have a positive effect on the formation of an Nb-phase according to theoretical guidelines. Surprisingly, only the hockey stick molecules form a uniaxial, optically positive nematic mesophase. All other V-shaped compounds and even the dimers are exclusively crystalline and no liquid crystals. Single crystal structure of a hockey-stick mesogen and a V-shaped molecule shows remarkable similarities. A model of the phase transition is presented, which explains the absence of the nematic mesophase in the family of V-shaped, shape-persistent mesogens with terminal aliphatic chains. In addition, the work is about the synthesis and investigation of board-shaped molecules, which are relatively close to the optimal aspect ratio of 15 : 5 : 3 with L > W > D for the formation of biaxial mesophases. An anthraquinone core building block was linked to arms consisting of an oligo(phenylenethynylene) scaffold of appropriate length. Different roof-shaped mesogens could be presented, in which the type and number of the side chains and the terminal chains as well, were varied. Thermal and microscopic studies show a broad nematic mesophase for all compounds. By means of special X-ray scattering in the magnetic field, the formation of nematic domains with SmC-like biaxial aggregates can be confirmed.

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In recent years there has been increasing interest in novel forms of surfactants. Of particular interest are gemini surfactants, which consist of two conventional surfactants joined by a spacer at the head groups, as they exhibit lower critical micelle concentrations than can be achieved by conventional surfactants. In this work, the self-assembly behaviour of several nonionic gemini surfactants with polyethylene oxide head groups (GemnEm, where n (= 20) is the number of carbons per tail and m (= 10, 15, 20 and 30) is the number of ethylene oxides per head group) were investigated. The Critical Micelle Concentrations (CMCs) were measured using a fluorescence probe technique. The CMCs are all ~2 x 10?7 M, with almost no variation with m. The CMCs are several orders of magnitude lower than conventional C12Em nonionic surfactants. The mixing behaviour of the gemini surfactants with conventional surfactants was also studied. They obeyed ideal mixing behaviour with both ionic and nonionic surfactants. Micelle morphologies were studied using Small Angle Neutron Scattering. The gemini surfactants with the larger head groups (i.e. Gem20E20 and Gem20E30) formed spherical micelles. Gem20E15 showed strong scattering at low Q, characteristic of elongated micelles. As the temperature was increased towards the cloud point, the scattering approached the Q-1 dependence predicted for infinite, straight rods. The existence of anisotropic micelles was supported by the viscosity of Gem20E15, which increases by several orders of magnitude on heating towards its cloud point. Phase behaviour was determined using Diffusive Interfacial Transport coupled to near-infrared spectroscopy. Much of the behaviour of these systems is similar to conventional nonionic surfactants. For example, Gem20E10 forms a dilute liquid isotropic phase (W) coexisting with a concentrated lamellar phase (La) at around room temperature and forms a sponge phase at higher temperatures. This is similar to the behaviour of C12E3 and C12E4. The other surfactants studied are all quite soluble in water and form liquid isotropic and hexagonal phases from room temperature. At higher concentrations Gem20E15 formed a cubic and then a lamellar phase while Gem20E20 formed a cubic phase and then an intermediate phase. This is also comparable to the phase behaviour of conventional nonionic surfactants except the intermediate phase, which is often only observed for surfactant systems with long alkyl tails.

Polystyrene behaviour in reversed phase high performance liquid chromatography was influenced mainly by the solvent system, but secondary affects were observed depending on the stationary phase. A variety of reversed phase columns were investigated using mobile phase combinations of dichlorom ethane-methanol, dichloromethane-acetonitrile, ethyl acetate-methanol and ethyl acetate-acetonitrile. Several different modes of behaviour were observed depending on the polymer solubility in the solvent system. In the dichloromethane-methanol solvent system, polymer-stationary phase interactions only occurred when the molecules had pore access. Retention of excluded polystyrene depended on the kinetics of precipitation and redissolution of the polymer. Peak splitting and band broadening occurred when the kinetics were slow and molecular weight separations were limited !o oligomers and polystyrenes lower than 5-10(4) dalton. Excellent molecular weight separations of polystyrenes were obtained using gradient elution reversed phase chromatography with a dichloromethane-acetonitrile mobile phase on C18 columns. The retention was based on polymer-stationary phase interactions regardless of the column pore size. Separations were obtained on large diameter pellicular adsorbents that were almost as good as those obtained on porous adsorbents, showing that pore access was not essential for the retention of high molecular weight polystyrenes. In the best example, the separation ranged from the monomer to 10(6) dalton in a single analysis. Very little adsorption of excluded polymers was observed on C8 or phenyl columns. Polystyrene molecular weight separations to 7-10(5) dalton were obtained in an ethyl acetate-acetonitrile solvent system on C18 columns. Adsorption was responsible for retention. When an ethyl acetate-methanol solvent system was used, no molecular weight separations were obtained because of complex peak splitting. Reversed phase chromatography was compared to size exclusion chromatography for the analysis of polydisperse polystyrenes. Similar results were obtained using both methods. However, the reversed phase method was less sensitive to concentration effects and gave better resolution.

Severe dehydration is lethal for most biological species, however there are a number of organisms which have evolved mechanisms to avoid damage during dehydration. One of these mechanisms is the accumulation of small solutes (e.g. sugars), which have been shown to preserve membranes by inhibiting deleterious phase changes at low hydration. Specifically, sugars reduce the gel to fluid phase transition temperatures of model lipid/water mixtures. However, there is debate about the precise mechanism, the resolution of which hinges on the location of the sugars. An experimental investigation into the effects of small solutes on the phase behaviour of phospholipid membranes is presented in order help identify the mechanisms by which solutes facilitate desiccation tolerance. Differential Scanning Calorimetry (DSC) was used to determine the first comprehensive phase diagram for the synthetic phospholipid DPPC over a wide range of hydration and solute molar ratios between 0.1 and 1.0 sugars per lipid. Over the same range of hydrations and solute concentrations Small Angle X-Ray Scattering (SAXS) was used to measure the structural parameters of the membrane bilayers necessary to determine both the phase of the membrane lipids and the location of the solutes. SAXS was also used to conduct the first comprehensive study of the effect of solutes on the kinetics of the fluid - gel transition of DPPC over a range of both hydration and solute concentration. Finally, contrast variation Small Angle Neutron Scattering (SANS) was used to quantitatively determine the location of the solutes. Data from these complimentary techniques are presented which show a monotonic relationship between both transition temperature and repeat spacing with respect to solute concentration. This relationship exists between solute:lipid molar ratios between 0.1 to approximately 0.5, after which higher concentrations of solute are shown to have no further effect on either the bilayer repeat spacing or transition temperature. It is proposed that the exclusion of small solutes into sugar/water micro-phases external to the bilayer can account for this behaviour. A theoretical model previously used to describe membrane phase behaviour at low hydrations is modified to account for the presence of solutes between membrane bilayers. This model is shown to be in quantitative agreement with the experimental data up until approximately 0.5 sucrose molecules per lipid, the point of solute exclusion. Once exclusion is taken into account, the model is quantitative over the whole range of sugar concentrations.