I advanced the understanding of particle morphology and its implications for the behavior and effects of atmospheric aerosol particles. I have developed new experimental methods for the Aerosol Optical Tweezers (AOT) system and expanded the AOT’s application into studying realistic secondary organic aerosol (SOA) particle phases. The AOT is a highly accurate system developed to study individual particles in real-time for prolonged periods of time. While previous AOT studies have focused on binary or ternary chemical systems, I have investigated complex SOA, and how they interact with other chemical phases, and the surrounding gas-phase. This work has led to new insights into liquid-liquid phase separation and the resulting particle morphology, the surface tension, solubility, and volatility of SOA, and diffusion coefficients of SOA phases. I designed a new aerosol optical tweezers chamber for delivering a uniformly mixed aerosol flow to the trapped droplet’s position. I used this chamber to determine the phase-separation morphology and resulting properties of complex mixed droplets. A series of experiments using simple compounds are presented to establish my ability to use the cavity enhanced Raman spectra to distinguish between homogenous single-phase, and phase-separated core-shell or partially-engulfed morphologies. I have developed a new algorithm for the analysis of whispering gallery modes (WGMs) present in the cavity enhanced Raman spectra retrieved from droplets trapped in the AOT. My algorithm improves the computational scaling when analyzing core-shell droplets (i.e. phase-separated or biphasic droplets) in the AOT, making it computationally practical to analyze spectra collected over many hours at a few Hz. I then demonstrate for the first time the capture and analysis of SOA on a droplet suspended in an AOT. I examined three initial chemical systems of aqueous NaCl, aqueous glycerol, and squalane at ~ 75% relative humidity. For each system I added α-pinene SOA – generated directly in the AOT chamber – to the trapped droplet. The resulting morphology was always observed to be a core of the initial droplet surrounded by a shell of the added SOA. By combining my AOT observations of particle morphology with results from SOA smog chamber experiments, I conclude that the α-pinene SOA shell creates no major diffusion limitations for water, glycerol, and squalane under humid conditions. My AOT experiments highlight the prominence of phase-separated core-shell morphologies for secondary organic aerosols interacting with a range of other chemical phases. The unique analytical capabilities of the aerosol optical tweezers provide a new approach for advancing the understanding of the chemical and physical evolution of complex atmospheric particulate matter, and the important environmental impacts of aerosols on atmospheric chemistry, air quality, human health, and climate change.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23115号 / 医科博第126号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊佐 正, 教授 髙橋 良輔, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The liquid-liquid phase separation (LLPS) of biomolecules is a phenomenon which received a lot of attention in the last years because it is not only related to theformation of membraneless organelles but also to neurodegenerative diseases. Lysozyme is a globular protein that undergoes LLPS in a buffer salt system andfor that it is well investigated with several techniques like microscopy, dynamic lightscattering (DLS) or small-angle X-ray scattering. In this work we investigate the effect of temperature, solvent and sample con-centration on the diffusion coefficient, the hydrodynamic radius and the viscosity oflysozyme using a DLS setup. Furthermore, the influence of these parameters on thecluster formation is addressed. Finally, we investigate the question if the LLPS oflysozyme in a buffer environment effects the formation of dynamic clusters.

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Liquid-liquid phase separation (LLPS) is an important phenomenon in soft condensed matter that explains many properties of membraneless organelles in living cells. The research on this topic is, therefore, a field with a wide range of applications such as biopharmacy and biomaterials. In this project, we investigate the LLPS of the antibody protein Immunoglobulin G (IgG) by analyzing the liquid dynamics of IgG solutions at a wide range of temperatures with dynamic light scattering (DLS). It was found that the slow component of the autocorrelation function increases with decreasing temperature below 0 °C. This can be attributed to either the number of protein clusters increasing as the sample approaches phase separation or LLPS droplets forming in the solution. LLPS was detected through optical microscopy, visualising the droplet formation in the IgG solution. This work confirms that LLPS can be detected for bovine IgG solutions without the presence of cosolvents and without water freezing in the sample.

The effects of polymer concentration and solvents on cocrystal morphology of low solubility drugs were investigated, both of which had an impact. The melting temperatures also decreased with increasing polymer concentration. Placing the binding agent, benzene, at different interfaces induced morphological changes, such as formation of porous cocrystals. Previously liquid-liquid phase separation (LLPS) has been reported as a hindrance in the crystallisation process impeding further development. A phase diagram was constructed, and different phases were categorised into 4 types. After separation of the highly concentrated amorphous Oil Phase II, it was prone to gradual crystallisation. Crystallisation took place over 30-60 minutes; this allowed the in-situ monitoring. A novel cocrystallisation technique was developed; from (LLPS). Cocrystals of indomethacin with saccharin and nicotinamide were obtained by mixing Oil Phase II with the coformers. In-situ monitoring by spectroscopic had gradual changes in spectra; characteristic peaks increased in height and area with the formation of crystals until the reaction was complete. With crystal formation, the XRD spectra gradually had a sharper baseline due to a decrease in the amorphous indomethacin. The photoluminescence (PL) spectra displayed several peaks coupling into one large hump together with increasing intensity as the sample crystallised. There was a shift in the peak absorbance of the pure drug crystals obtained from LLPS and the indomethacin:saccharin cocrystal obtained from LLPS. Amorphous stabilisation was achieved by mixing polymer (PVP) with Oil Phase II. There were no changes to the XRD diffractogram as the sample did not undergo crystallisation.

2024 / Within the chaos of intracellular signaling pathways, scaffold proteins serve as the control boards to organize the comings and goings of a variety of signals and proteins. There are over 300 known scaffold proteins, which act as switchboards that lessen this chaos of the cellular “soup” and enable proper cellular responses. While the terms scaffold, adaptor, docking, and anchoring protein have sometimes been used interchangeably, this dissertation focuses on scaffolds as a distinct class of proteins that are central to enhancing signaling cascades, have multiple interaction domains to facilitate higher order complex formation, and are highly conserved. In addition, scaffold proteins are not simply inert or passive platforms. Rather, there are multiple examples of scaffolds that, while catalytically inactive, are dynamic in their mechanism of action. Studying these dynamic roles is critical to our understanding of signaling cascades and how signaling-related disease states occur. NF-κB Essential Modulator (aka NEMO or IKK gamma) is a scaffold protein that has a pivotal role in the NF-κB signaling pathway. In this dissertation, NEMO is examined both for its changes in activity upon mutation and the mechanical/structural effects of mutations within a highly conserved central region termed the intervening domain (IVD). The IVD of NEMO is essential for proper function of the protein and for the coordination of phosphorylation of the inhibitor IκBα in the activation of canonical NF-κB pathway. The proper structural organization of NEMO is required for cytokine-induced activation of IκB kinase (IKK), and the impact that the IVD has on this structural organization is demonstrated. Mutations within the conserved IVD core are detrimental to the activation of the canonical NF-κB pathway and reduce the ability of NEMO to form ubiquitin-induced liquid-liquid phase separated droplets in vitro. The effects of the IVD mutations also correlate with a reduced ability of NEMO to form signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD affects the stability of the full-length NEMO molecule, due to conflicting structural demands of this region on upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these findings support the hypothesis that the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational change in NEMO. These findings demonstrate the importance of conformational change for scaffold protein function and provide new information about how clinical mutations in scaffolds can affect function and can serve as targets for therapeutic intervention.

Ce travail de thèse vise à apporter des connaissances structurales et fonctionnelles sur les protéines du gluten. Pour cela, nous utilisons les concepts et méthodes de la physique des polymères et de la matière molle. Plus précisément, nous optimisons un protocole d’extraction basé sur la séparation de phases liquide-liquide. Ce dernier permet d’obtenir des isolats de protéines à différents rapports massiques gluténines/gliadines que nous étudions ensuite dans un solvant eau/éthanol 50/50 (v/v). Les résultats, montrent que les protéines se comportent comme des chaînes de polymères en solvant θ, en régime dilué et semi-dilué avec des tailles caractéristiques définis par diffusion de rayons X et de neutrons aux petits angles. De plus, 2 tailles d’objets sont distinguées en régime dilué par diffusion dynamique de la lumière: d’une part des protéines monomériques de l’ordre d’une dizaine de nanomètres associées aux  et -gliadines et à des polymères de gluténines de faibles masses molaires et d’autre part des assemblages polymériques de l’ordre de 100 nm, principalement composés de ω-gliadines et polymères de gluténines de haute masse molaire. Ces assemblages sont mis en avant par une combinaison de mesures réalisées par chromatographie d’exclusion de taille et par fractionnement par flux de forces asymétrique et permettent de rationaliser les diagrammes de phases de ces mélanges protéiques, en fonction de la température. L’étude de la dynamique de séparation de phases de ces mélanges protéiques, par diffusion de rayons X aux petits angles, montre que celle-ci est pilotée par un phénomène de décomposition spinodale. Cette décomposition peut être arrêtée lors de trempes en température profondes mais également observée à toutes les températures de trempe, pour les échantillons les plus riches en gluténines, formant un gel dès le régime monophasique, d’après leur étude par rhéologie / The aim of this thesis is to provide structural and functional knowledge on wheat gluten proteins. For that, we use the physical methods and the concept of soft matter. We optimize an extraction protocol based on a liquid-liquid phase separation. With this protocol, we obtain protein batches with different glutenin/gliadin mass ratios, which we then study in a 50/50 water/ethanol solvent (v/v). We show that proteins behave like polymer chains in θ solvent in dilute and semi-dilute regime, whose characteristic size are extracted by small angle X-ray and neutron scattering. Moreover, two sizes of objects are evidenced in dilute regime by dynamic light scattering: monomeric proteins with a size around 10 nm which can be associated to α/β, and γ-gliadins and polymeric glutenins with low molecular weight and polymeric assemblies with a size around 100 nm composed of ω-gliadins and glutenins polymers with high molecular weight. These assemblies are revealed by a combination of size exclusion chromatography and asymmetric flow field flow fractionation and allow one to rationalize the phase diagrams of the protein mixtures with temperature. The study of the dynamics of the phase separation of these protein mixtures by small angle X-ray scattering shows that the phase separation proceeds through a spinodal decomposition phenomenon. An arrested phase separation is observed for deep quenches but also at all temperature quenches for the most glutenin rich samples, which are gels in the monophasic regime, as confirmed by rheology.

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