No / Biosimilar pharmaceuticals are complex biological molecules that have similar physicochemical properties to the originator therapeutic protein. They are produced by complex multi-stage processes and are not truly equivalent. Therefore, for a biosimilar to be approved for market it is important to demonstrate that the biological product is highly similar to a reference product. This includes its primary and higher order structures and its aggregation behaviour. Representative lots of both the proposed biosimilar and the reference product are analysed to understand the lot-to-lot variability of both drug substances in the manufacturing processes. Whilst it is not easy to characterise every variation of a protein structure at present additional analytical technologies need to be utilised to ensure the safety and efficacy of any potential biosimilar product. We have explored the use of Taylor dispersion analysis (TDA) to analyse such batch to batch variations in the model protein, bovine serum albumin (BSA) and compared the results to that obtained by conventional dynamic light scattering analysis (DLS). Inter and intra batch differences were evident in all grades of BSA analysed. However, the reproducibility of the TDA measurements, enabled the stability and reversibility of BSA aggregates to be more readily monitored. This demonstrates that Taylor dispersion analysis is a very sensitive technique to study higher order protein states and aggregation. The results, here, also indicate a correlation between protein purity and the physical behaviour of the samples after heat shocking. Here, the protein with the highest quoted purity resulted in a reduced increase in the measured hydrodynamic radius after heat stressing, indicating that less unfolding/aggregation had occurred. Whilst DLS was also able to observe the presence of aggregates, its bias towards larger aggregates indicated a much larger increase in hydrodynamic radii and is less sensitive to small changes in hydrodynamic radii. TDA was also able to identify low levels of larger aggregates that were not observed by DLS. Therefore, given the potential for immunogenicity effects that may result from such aggregates it is suggested that TDA may be suitable in the evaluating detailed batch to batch variability and process induced physical changes of biopharmaceuticals and biosimilars.

A range of microgels with two different functionalities were synthesized, and their utility in novel bioapplications was examined. Cationic microgels with varying properties were developed by tuning synthesis conditions. Their size and primary amine content was analyzed, and one microgel system was selected as a model construct. Its primary amine groups were conjugated to two dyes with properties favorable for utilization as contrast agents in photoacoustic imaging. The concentration of contrast agent in single particles was determined. The implications of a high local dye concentration in the generation of high intensity photoacoustic signals, are discussed. The second bioapplication involved the targeted delivery of fibrinolytics to fibrin clots, in order to bring about dissolution of abnormal thrombi. For this purpose, core/shell microgels with carboxylic acid groups in their shells were synthesized in three size ranges. Following this, their dimension based differential localization in and around porous fibrin clots was examined. Fibrin-specific peptides were then conjugated onto the shells of these particles and the conjugates were shown to demonstrate strong interactions with the fibrin clots. The microgels conjugated to the peptide with the highest binding affinity to fibrin, were observed to bring about disruption of fibrin clots, merely through interference in the dynamic interactions among clot fibers, due to the equilibrium nature of the fibrin polymer. The implications of these novel results and future studies required to facilitate a better understanding of the phenomena involved, are discussed.

Cerebral blood flow (CBF) measures are central to the investigation of ischemic strokes, spreading depressions, functional and neuronal activation. Laser Speckle Contrast Imaging (LSCI) is an optical imaging technique that has been used to obtain CBF measures in vivo at high spatial and temporal resolutions, by quantifying the localized spatial blurring of backscattered coherent light induced by blood flow. Despite being widely used for biomedical applications, LSCI's critical limitations such as its tendency to underestimate large flow changes and its inability to accurately estimate CBF through a thinned skull have not been overcome. This dissertation presents a new Multi Exposure Speckle Imaging (MESI) technique that combines a new instrument and mathematical model to overcome these limitations. Additionally, in a pilot clinical study, an adapted neurosurgical microscope was used to obtain intra-operative LSCI images of CBF in humans. The MESI instrument accurately estimates experimental constants by imaging backscattered speckles over a wide range of the camera's exposure durations. The MESI mathematical model helps account for light that has scattered from both static and moving particles. In controlled flow experiments using tissue simulating phantoms, the MESI technique was found to estimate large changes in flow accurately and the estimates of flow changes were found to be unaffected by the presence of static particles in these phantoms. In an in vivo experiment in which the middle cerebral artery in mice was occluded to induce ~100% reduction in CBF, not only was the reduction in CBF accurately estimated by the MESI technique but these estimates of CBF changes were found to be unaffected by the presence of a thinned skull. The validity of statistical models used to derive the MESI mathematical model was confirmed using in vivo dynamic light scattering (DLS) measurements of CBF in mice. The MESI technique's potential to estimate absolute values of CBF in vivo was demonstrated by comparing CBF estimates obtained using the MESI technique to DLS measurements. The MESI technique's ability to measure CBF changes quantitatively through a thinned skull makes it particularly useful in chronic and long term studies leading to the development of better, more accurate stroke models. / text

An investigation into the light scattering properties of Saharan dust grains is presented. An electrodynamic trap has been used to levitate single dust particles. By adjusting the trap parameters, partial randomisation of the particle orientation has been introduced. While levitated, the particles were illuminated by a laser, and a rotating half-wave retarder enabled selection of vertically or horizontally polarized incident light. A laser diffractometer and linear photodiode array have been used to measure intensity at scattering angles between 0.5° and 177°. Combining these measurements with Fraunhofer diffraction as calculated for a range of appropriately-sized apertures allows the calculation of the phase function and degree of linear polarization. The phase functions and degree of linear polarisation for four case study particles are presented - the phase functions are found to be featureless across most of the scattering region, with none of the halo features or rainbow peaks associated with regularly shaped particles such as hexagonal columns or spheres. Particle models comprised of large numbers of facets have been constructed to resemble the levitated particles. Utilizing Gaussian random sphere methods, increasing levels of roughness have been added to the surfaces of these models. A Geometric Optics model and a related model, Ray Tracing with Diffraction on Facets, have been modified to calculate scattering on these particle reconstructions. Scattering calculations were performed on each of these reconstructions using a range of refractive indices and two rotation regimes – one where the orientations of the reconstructed particle were limited to match those observed when the particle was levitated, and one where the orientation was not limited. Qualitative comparisons are performed on the phase functions and degree of linear polarization, where it is observed that the addition of roughness to the modelled spheroids causes the computed phase functions to increasingly resemble those from the levitated particles. Limiting the orientation of the particles does not affect the scattering noticeably. The addition of a very small absorption coefficient does not change the comparisons considerably. As the absorption coefficient is increased, however, the quality of the comparisons decreases rapidly in all cases but one. The phase functions are quantitatively compared using RMS errors, and further comparison is performed using the asymmetry parameter.

An investigation was carried out into light scattering by Gaussian rough ice crystals. Gaussian rough crystal geometries were generated using roughness parameters derived from mineral dust grains, which have been reported to be suitable proxies for rough ice crystals. Light scattering data for these geometries was computed using the discrete dipole approximation (DDA) method. Phase functions, 2D scattering patterns, degree of linear polarisation patterns and asymmetry parameters were computed for smooth, moderately rough and highly rough crystals with a variety of orientations and size parameters. A sodium fluorosilicate ice analogue crystal with three partially roughened prism facets was created using focused ion beam (FIB) milling and 2D scattering patterns were collected from it using the small ice detector (SID) 3 cloud probe. It was found that roughness reduces features in the phase function compared to scattering by smooth hexagonal prisms, particularly when the roughness features were horizontally much larger than the wavelength. However, the most effective roughness model also takes account of horizontal features whose size is closer to that of the wavelength. Horizontal features smaller than the wavelength have very little effect.

L’Optique non linéaire est un outil très puissant pour étudier les propriétés des matériaux. Dans cette thèse, nous avons utilisé deux techniques d’optique non linéaire pour l’étude des non linéarités moléculaires: la génération de seconde harmonique induite sous champ électrique (EFISH) et la diffusion harmonique de la lumière (DHL). Tout d'abord, nous avons mis en évidence la conjugaison des groupements donneurs pi- dans les complexes d’iridium cyclométallés. Nous avons également exploré une série de molécules trinucléaires organométalliques (triaryle-1, 3, 5-triazinane-2, 4, 6-triones) fonctionnalisées par des acétylures métalliques avec des complexes des métaux de transition à leur périphérie- l’hyperpolarisabilité est beaucoup plus élevée que celles des dérivés purement organiques équivalents. D'autre part une série de complexes métalliques dipolaires et octupolaires contenant des ligands 2, 2-bipyridine photochromes à base de dithiényléthène (DTE) ont été synthétisés et caractérisés. L'étude révèle une forte augmentation de Les hyperpolarisabilités après irradiation UV correspondant à la fermeture de cycle associée au DTE. Cette forte exaltation reflète bien la délocalisation du système d’électrons pi- et la formation de chromophores push-pull dans les formes fermées. Troisièmement, nous avons étudié les propriétés optiques non linéaires de complexes de bis (phtalocyaninato) lanthanide (III) en sandwich, avec 2 phthalocyanines substituées en ABAB (alternance de donneurs d'électrons et d’accepteurs d'électrons), AB3 (3 groupes de donneurs), A4 (4 groupes d’accepteurs) et B4 (aucun groupe de donneur). L’hyperpolarisabilité du 1er ordre beta- mesurées sont les plus élevées jamais enregistrées pour des molécules octupolaires. La contribution directe d’électrons f dans les ions lanthanides complexés est également observée sur l'activité non linéaire du second ordre. Enfin, des nanosphères (AuNSs) et des nanotubes d'or (AuNRs) avec différents rapports d'aspect (AR) allant de 1,7 à 3,2 nm ont été synthétisés par la méthode de radiolyse. Le signal de deuxième harmonique émis par des AuNRs est nettement supérieur à celui des nanosphères et révèle leur dépendance à l'AR. Nous avons également mélangé un dérivé du chromophore 4-diméthylamino-N-méthyl-4-stilbazolium tosylate (DAST) avec les AuNRs et observé une influence des AuNRs sur l'amélioration des propriétés ONL du DAST. Une nette augmentation de l’hyperpolarisabilité (par un facteur 8) du derive de DAST en est la preuve. / Nonlinear optics is well known to be a highly powerful tool to investigate the properties of the materials. In this thesis we used two important nonlinear optical techniques known as Electric Field Induced Second Harmonic Generation (EFISH) and Harmonic Light Scattering (HLS) to study the first hyperpolarizability of various molecular objects. Firstly, we evidenced the pi donor conjugation in cyclometallated Ir complexes. We have also explored the series of trinuclear organometallic triaryl-1, 3, 5-triazinane-2, 4, 6-triones functionalized by d6-transition metal acetylides complexes at their periphery- large hyperpolarizabilities, far higher than those of related purely organic derivatives are reported. Secondly, a series of dipolar and octupolar dithienylethene (DTE)-containing 2, 2-bipyridine ligands with different metal ions featuring two, four and six photochromic dithienylethene units have been synthesized and fully characterized. The study reveals a large increase in the hyperpolarizability after UV irradiation and subsequent formation of ring-closed isomers. This efficient enhancement clearly reflects the delocalization of the pi-electron system and the formation of strong push&pull chromophores in the closed forms. Thirdly, we have investigated NLO properties of bis (phthalocyaninato) lanthanide-(III) double-decker complexes with crosswise ABAB (phthalocyanine bearing alternating electron-donor and electron-acceptor groups), AB3 (3 donor groups), A4 (4 donor groups) and B4 (no donor groups) ligands. First-order hyperpolarizability is measured and displays the highest quadratic hyperpolarizability ever reported for an octupolar molecule. The direct contribution of f-electrons in coordinated lanthanides ions is also observed on second order nonlinear activity. Finally, gold nanospheres (AuNSs) and gold nanorods (AuNRs) with different aspect ratios (AR) ranging from 1.7 to 3.2 nm have been synthesized by Radiolysis method. Second harmonic intensity collected from AuNRs is clearly higher than that of the nanospheres and reveals their dependence on the AR. We have also mixed the chromophore 4-dimethylamino-N-methyl-4-stilbazolium tosylate (DAST) derivative with AuNRs and observed the enhancement of DAST NLO properties in the presence of AuNRs. A clear increase in the hyperpolarizability (by a factor of 8) of DAST derivatives has been evidence.

The rendering of participating media is an interesting and important problem without a simple solution. Yet even among the wide variety of participating media the clouds stand out as an especially difficult case, because of their properties that make their simulation even harder. The work presented in this thesis attempts to provide a solution to this problem, and moreover, to make the proposed method to work in interactive rendering speeds. The main design criteria in designing this method were its physical plausibility and maximal utilization of specific cloud properties which would help to balance the complex nature of clouds. As a result the proposed method builds on the well known photon mapping algorithm, but modifies it in several ways to obtain interactive and temporarily coherent results. This is further helped by designing the method in such a way which allows its implementation on contemporary GPUs, taking advantage of their massively parallel sheer computational power. We implement a prototype of the method in an application that renders a single realistic cloud in interactive framerates, and discuss possible extensions of the proposed technique that would allow its use in various practical industrial applications.

Les métallacarboranes sont des clusters anioniques constitués d'atomes de bore, de carbone et d'hydrogène avec un cation métallique pris en « sandwich » au cœur de ce cluster (Co3+ en général) et hautement stable thermiquement que chimiquement. Ces entités sont représentées trivialement par la lettre grecque « thêta » où leurs pôles sont considérés comme hydrophobes et possèdent une charge négative délocalisée qui peut être contre balancée par un proton acide. Cette forte stabilité ainsi que ces diverses propriétés leur procurent un intérêt particulier dans des applications telles que la co-extraction du Cs et Sr dans les effluents nucléaires ou bien en médecine pour leur aptitude à inhiber la protéase du VIH de par ses liaisons hydrogènes. La chimie du bore étant relativement riche, une synthèse de quelques dérivés a été réalisée en laboratoire. Il est ainsi possible de substituer spécifiquement des atomes d'hydrogène par des atomes d'iode ou de chlore ou bien de changer le métal central par un cation Fe3+ formant respectivement les anions diiodo-COSAN (I2COSAN), dichloro-COSAN (Cl2COSAN) et ferrabisdicarbollide (FESAN). Ces métallacarboranes sont aussi considérés comme une nouvelle classe de tensioactif pour lesquels une première étude de leur auto-assemblage a été réalisée. Les résultats obtenus sur l'un d'entre eux, le cobaltabisdicarbollide ou COSAN, ont montré un effet sur la tension de surface ainsi qu'une structuration spontanée de ces clusters en forme de vésicule (à partir de 0,5 mmol/L) et par interaction Coulombienne, tendent à former des micelles à plus forte concentration (après 15 mmolL). Mais ces derniers offrent davantage de surprise de par la formation de phases lyotropes en solution aqueuse. En effet, ces phases, clairement identifiées par des techniques de diffusion au rayon-X et microscopie, montrent une dépendance en température mais aussi en concentration dont un diagramme de phase a pu être établi pour le I2COSAN en particulier. Cette thèse fait ainsi l'objet de la compréhension des phénomènes contrôlant cette agrégation de ces curieux composés. / Metallacarboranes are anionic clusters composed of boron, carbon and hydrogen with a metallic cation sandwiched at the heart of this cluster (Co3+ in general) and highly stable in thermic and chemical point of view. These entities are trivially represented by the Greek letter "theta" where the poles are considered hydrophobic and have a negative charge delocalized and counter-balanced by an acidic proton. This high stability as well as the various properties gives a particular interest in applications such as the co-extraction of Cs and Sr in nuclear waste or in medicine for their ability to inhibit HIV protease by its hydrogen bonds. The chemistry of boron is relatively rich; a synthesis of some derivatives was performed in laboratory. It is possible to substitute specifically hydrogen by iodine or chlorine atoms or change the metallic heart by other atom as Fe3+, respectively forming diiodo- COSAN (I2COSAN), dichloro-COSAN (Cl2COSAN) and ferrabisdicarbollide (FESAN) anions. Metallacarboranes are also considered as a new class of surfactant where a first study of their self-assembly has been performed. The results of one of them, cobaltabisdicarbollide or COSAN, have shown an effect at the surface tension and theses clusters formed spontaneously vesicles in dilute regime (from 0.5 mmol/L) and by Coulomb interactions, they form micelles at higher concentration (after 15 mmol/L). But they offer more surprise by the formation of lyotropic phases in aqueous solution. Indeed, these phases, clearly identified by X-ray scattering techniques and microscopy, showed temperature and concentration dependence where a phase diagram was established for the I2COSAN in particular. Therefore, the thesis is focused on the understanding of the phenomena controlling the aggregation of these curious compounds.

L'interaction matière-lumière dans des milieux opaques donne lieu à des phénomènes collectifs nécessitant le couplage d'équations atomiques et d'une équation de transport. Le piégeage de la lumière dans un système atomique multi-niveaux sera étudié expérimentalement dans une vapeur froide et théoriquement avec le couplage des paramètres atomiques à une équation de diffusion. Ensuite, du gain sera ajouté dans ce nuage d'atomes froids multi niveaux. Nous montrerons théoriquement qu'un seuil laser existe dans ce type de système combinant gain et diffusion et qu'expérimentalement le gain Raman associé à de la diffusion sur une raie résonante a permis l'observation d'un laser aléatoire à atomes froids. La validité de l'équation de diffusion nécessite une non redistribution en fréquence et donc des atomes suffisamment froids pour s'affranchir de l'effet Doppler. Finalement nous étudierons le transport dans une vapeur atomique chaude (20°C-180°C) opaque. L'effet Doppler invalide la loi de Beer-Lambert pour la longueur des pas des photons entre des diffusions qui suivent alors une statistique de Lévy. / The matter-light interaction in opaque media gives rise to collective effects which may be explained by the coupling between atomic equations and light transfer equation. The trapping of light in an opaque multi-levels atomic system will be studied experimentally in a cold vapour and theoritically. Then, this vapour will be in situation with gain and amplification of light occurs. We will show that a laser threshold exists with this kind of system. Experimentally, the mixing of Raman gain and multiple scattering on a resonant line allowed the abservation a cold-atom random laser. The validity of diffusion equation needs a non frequency shift and so the temperature of atoms should be sufficiently cold to avoid Doppler effect. Finally we study the transport of light in an opaque hot atomic vapour (20°C-180°C). The Doppler effect breaks the Beer-Lambert law for photons step size distribution which is then a Levy flight statictics.

Title: Self-assembly in mixture of surfactants and stimuli-responsive polymers with complex architecture Author: Anna Bogomolova Department: Physical and Macromolecular Chemistry Supervisor: PhD. Sergey K. Filippov, IMC AS CR, v.v.i. Supervisor's e-mail address: filippov@imc.cas.cz Abstract: The issue of construction of complex multi-block copolymers is currently one of the most researched areas. It became a logic consequence of the continuous development in polymer chemistry. Nowadays, a great interest is attracted to multi- responsive block copolymers. As a rule, they consist of hydrophilic, hydrophobic and responsive blocks. That responsive block can be either thermo-sensitive or pH-sensitive as well as sensitive to some other external stimuli. In the present work, we will try to cover topic of stimuli-responsive block copolymers and their interactions with different types of surfactants. Understanding of polymer/surfactant interactions can be a crucial step for future modeling of drug/polymer or protein(DNA)/surfactant interactions. There is a great interest in the investigation of polymer-surfactant interactions. However, while the homopolymer-surfactant interactions are characterized well enough, the same interactions for block copolymers are poorly described. The main development in the latter topic...