The original work presented in this thesis describes research into Si-based luminescent materials, prepared specifically by ion implantation and rapid thermal processing of thermal oxide films. An in-depth optical characterisation, employing photoluminescence (PL) and Raman spectroscopy was complimented with electron microscopy, revealing the source of efficient room temperature PL as nano-scale silicon inclusions (Si-NCs). The evolution of the Si-NC size and density with isothermal and isochronal annealing may be described using classical thermodynamics according to a diffusion limited, Ostwald ripening process. Values for the coarsening rate and activation energy, extracted from the evolution of the Si-NC size with annealing indicate that the transport of Si atoms and precipitate formation are enhanced in ion implanted films, attributable to the presence of vacancy and interstitial defects generated during ion irradiation. The PL and Raman spectra are well correlated with the evolving Si-NC size and density according to the quantum confinement (QC) model in which samples containing larger clusters emit at longer wavelengths. However, the formation of bound exciton states within the band gap of small clusters (< 2nm), as a result of specific surface chemistries, suppresses higher energy emissions. The increase in PL intensity with annealing was exactly correlated with the increase in PL lifetime, characteristic of the removal of non-radiative defects. A dependence of the PL dynamics on emission energy, with higher energies exhibiting shorter lifetimes, further evidences the QC effect. Blue shifted emission at high excitation flux and/or low temperature is correspondent with the slower PL dynamics and preferential saturation at longer wavelengths. Raman spectra were fit using a phonon confinement model, from which Si-NC size distributions were extracted and found to compare favourably with those obtained from TEM images. Stresses in the films, determined from the Raman peak position, were used as an independent method for calculating the Si surface energy, which is very close to the literature values. A single, high temperature anneal of Si and erbium (Er) co-doped films revealed a preferential aggregation of Er at the Si-NC formation site, which is of particular importance for the photo-sensitization of Er PL around 1.5μm. The Er PL was enhanced in the presence of Si-NCs by several orders of magnitude compared with a reference SiO2:Er. Whilst broadband pumping of the Er via Si-NCs evidences a non-resonant energy transfer mechanism with an efficiency which depends on the Si-NC size, the process is limited at high excitation flux by a combination of low sensitizer (Si-NC) density and non-radiative losses. Finally the Si-NC PL intensity in phosphorus (P) co-doped films was studied and found to depend strongly on the annealing conditions and P concentration. For lower temperature treatments, a factor 2 PL enhancement, relative to an un-doped reference was obtained, attributed to the passivation of Si-NC surface defects. Higher temperature treatments resulted in the monotonic quenching of the PL with increasing P concentration, attributed to the introduction of an efficient Augerre combination channel as a result of the ionization of P-donors inside large Si-NCs. A simple statistical model predicts this behaviour and provides an incidental estimate of the Si-NC size.

The localised corrosion behaviour of various grades of Austenitic Stainless Steels has been demonstrated by optical and electron microscopy. The effect of sensitisation upon subsequent corrosive attack has been investigated. A theoretical model based upon thermodynamic and kinetic considerations has been proposed to account for the observed experimental results. / Thesis / Master of Engineering (ME)

As novel engineered nanoparticles such as single-walled carbon nanotubes (SWCNT) are extensively used in nanotechnology due to their superior properties, it becomes critical to fully understand their biodistribution and effect when accidently inhaled. There fore, development of animaging technique which allow longitudinal in vivo follow-up of SWCNT effect based on their intrinsic properties is highly desirable. Non invasive free-breathing hyperpolarized 3He lung MRI protocol was developed complementary to proton systemic MR protocol to allow monitoring SWCNT based on their intrinsic iron impurities after intrapulmonary exposition. Combined toproton lung MRI and ex vivo optical and electron microscopy at different time points, this protocol represents a powerful multimodality imaging techniques which allows a full characterization of the biodistribution and biological impacts of iron containing SWCNT. SWCNT was found to produce granulomatous and inflammatory reactions in a time and dose dependent manner with their bio persistenc eafter intrapulmonary exposition.From biological impact evaluations after intrapulmonary exposition towards biomedical applications, SWCNT hold promise for applications in nanomedicine field with their distinct architecture and their novel physicochemical properties. The biodistribution and pharmacological profile of various well-dispersed pristine and functionalized SWCNT were assessed in blood and target tissues after their intra venous administration by longitudinal in vivo susceptibility weighted MRI and their potential effect on liver metabolism by ex vivo HRMAS 1H NMR. No presence ofacute toxicological effect (variation in liver metabolism) was observed confirmed by the absence of clustering in NMR spectra using Principal Component Analysis (specific biomarkers of toxicity).

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

Un nouveau modèle de biomembrane de type liposome a été développé à partir de lipides synthétisés comportant une unité biphényle sur leur chaînes sn2 et une chaîne aliphatique sn1 de longueur et insaturation variables. L’anisotropie de susceptibilité magnétique positive de ces molécules induit une déformation en oblate de ces liposomes dits « biphényles » dans le champ magnétique B0. Cette déformation spécifique a été caractérisée par RMN des solides 31P et 2H en faisant varier différents paramètres : l’intensité de B0, l'élasticité membranaire, la température et la taille des liposomes (Helfrich, 1973). Ces vésicules déformées ont pu être observées par microscopies optiques et électroniques et la rémanence de la déformation en dehors de B0 a pu être analysée par diffusion des rayons X aux petits angles (SAXS). Enfin, les premières applications des liposomes biphényles comme nouveau modèle de biomembrane pour analyser la structure et l’orientation (par RMN des solides 15N) de peptides ou protéines membranaires, ont été étudiées. / A new model of biomembrane (liposome) was developed from synthesized lipids containing a biphenyl unit on the sn2 aliphatic chain and possessing a sn1 aliphatic chain which varies in length and unsaturation. The positive magnetic susceptibility anisotropy of these molecules induces an oblate deformation of these «biphenyls » liposomes under the magnetic field B0. This particular deformation has been characterized by 31P and 2H solid state NMR by varying different parameters: the intensity of B0, the membrane elasticity, the temperature and the size of the liposomes (Helfrich, 1973). These deformed vesicles were observed by optical and electron microscopy and the remanence of the deformation outside B0 has been analyzed by Small angles X-ray scattering (SAXS).Finally, the first applications of biphenyls liposomes as new biomembrane model to analyze the structure and orientation of membrane proteins or peptides were studied by 15N solid state NMR

En raison de leurs propriétés uniques, des nanoparticules industriellement fabriquées comme les nanotubes de carbone (NTC) ont révolutionné le domaine de la nanotechnologie. Il apparait nécessaire de développer des techniques d’investigation in vivo basées sur les propriétés intrinsèques de ces particules et permettant un suivi longitudinal pour évaluer leur risque après inhalation accidentelle par voie respiratoire. Un protocole d’IRM pulmonaire non-invasive utilisant l’hélium-3 hyper polarisé sous respiration spontanée a été développé en complément d’un protocole d’IRM systémique proton pour permettre la détection des NTC grâce à l’effet de susceptibilité magnétique induit par les impuretés de fer, associées aux nanotubes après leur exposition intra pulmonaire. Combiné avec l’IRM pulmonaire proton et des analyses en microscopie optique et électronique à différents temps d’investigation, ce protocole d’imagerie multimodale permet d’évaluer la biodistribution et l’impact biologique des NTC bruts après exposition intra pulmonaire.Une accentuation des réactions inflammatoires (granulomes multifocaux, dépôt de fibres de collagène…) avec le temps et la dose administrée a été observée.De l’évaluation de l’impact biologique des NTC après une exposition intra pulmonaire vers leurs applications biomédicales, les nanotubes de carbone avec leurs propriétés physicochimiques fascinantes et leur forme spécifique laissent entrevoir des applications potentielles en nanomédecine. La bio distribution et le profil pharmacologique des différents types de NTC ont été évalués longitudinalement par IRM et dosage dans le sang et les organes cibles après une injection intraveineuse, et leur impact biologique sur le métabolisme du foie a été examiné ex vivo par RMN haute résolution à l’angle magique (HR-MAS). Aucun signe de toxicité aiguë (variation du métabolisme du foie) n’a été observé et les analyses statistiques conduits sur les spectres RMN (tests PCA) ne montrent aucune différence entre les échantillons analysés et donc l’absence de discrimination entre les différents groupes par rapport aux animaux contrôles. / As novel engineered nanoparticles such as single-walled carbon nanotubes (SWCNT) are extensively used in nanotechnology due to their superior properties, it becomes critical to fully understand their biodistribution and effect when accidently inhaled. There fore, development of animaging technique which allow longitudinal in vivo follow-up of SWCNT effect based on their intrinsic properties is highly desirable. Non invasive free-breathing hyperpolarized 3He lung MRI protocol was developed complementary to proton systemic MR protocol to allow monitoring SWCNT based on their intrinsic iron impurities after intrapulmonary exposition. Combined toproton lung MRI and ex vivo optical and electron microscopy at different time points, this protocol represents a powerful multimodality imaging techniques which allows a full characterization of the biodistribution and biological impacts of iron containing SWCNT. SWCNT was found to produce granulomatous and inflammatory reactions in a time and dose dependent manner with their bio persistenc eafter intrapulmonary exposition.From biological impact evaluations after intrapulmonary exposition towards biomedical applications, SWCNT hold promise for applications in nanomedicine field with their distinct architecture and their novel physicochemical properties. The biodistribution and pharmacological profile of various well-dispersed pristine and functionalized SWCNT were assessed in blood and target tissues after their intra venous administration by longitudinal in vivo susceptibility weighted MRI and their potential effect on liver metabolism by ex vivo HRMAS 1H NMR. No presence ofacute toxicological effect (variation in liver metabolism) was observed confirmed by the absence of clustering in NMR spectra using Principal Component Analysis (specific biomarkers of toxicity).

Cellulose is the most abundant biopolymer on Earth as it is found in every plant cell wall; therefore, it represents one of the most promising natural resources for the fabrication of sustainable materials. In plants, cellulose is mainly used for structural integrity, however, some species organise cellulose in helicoidal nano-architectures generating strong iridescent colours. Recent research has shown that cellulose nanocrystals, CNCs, isolated from natural fibres, can spontaneously self-assemble into architectures that resemble the one producing colouration in plants. Therefore, CNCs are an ideal candidate for the development of new photonic materials that can find use to substitute conventional pigments, which are often harmful to humans and to the environment. However, various obstacles still prevent a widespread use of cellulose-based photonic structures. For instance, while the CNC films can display a wide range of colours, a precise control of the optical appearance is still difficult to achieve. The intrinsic low thermal stability and brittleness of cellulose-based films strongly limit their use as photonic pigments at the industrial scale. Moreover, it is challenging to integrate them into composites to obtain further functionality while preserving their optical response. In this thesis, I present a series of research contributions that make progress towards addressing these challenges. First, I use an external magnetic field to tune the CNC films scattering response. Then, I demonstrate how it is possible to tailor the optical appearance and the mechanical properties of the films as well as to enhance their functionality, by combining CNCs with other polymers. Finally, I study the thermal properties of CNC films to improve the retention of the helicoidal arrangement at high temperatures and to explore the potential use of this material in industrial fabrication processes, such as hot-melt extrusion.