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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23106号 / 医博第4733号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 松田 道行, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In this thesis a robust Python based software for controlling a Chlorophyll-a Pulse-Amplitude-Modulated (PAM) fluorescence microscope and analysing subsequent data has been developed and validated. The automation software, called PACMan (PAM Automation Control Manager) was made for the purpose of increasing the amount of single cell data generated per experiment. PACMan includes an autofocus algorithm and the ability to vary experimental parameters during experiments. The analysis software, called PAMalysis, processes and facilitates interpretation of PAM experimental data, printing both text files and creating graphical output. PACMan was used on two different phytoplankton species of the Symbiodiniacae family to characterize them under thermal stressors while immobilized on a microfluidic device. The heterogeneity of the phytoplanktons response to increasing thermal stress was evaluated and the best performers under heat stress have been removed using Laser Capture Microdissection for downstream cultivation. PACMan was also used to compare the response of 4 Symbiodiniacae species to increasing relaxation time between light pulses and to image the heterogeneity of response of the common eukaryotic model organism C. reinhartii to a chemical gradient of the common herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea).

Le travail de cette thèse a été consacré au développement d’un nouvelle technique SELFI (pour self-interferences, auto-interférences en anglais). Cette méthode permet d’obtenir une localisation tridimensionnelle d’émetteurs fluorescents individuels. Nous avons démontré que cela permet l'imagerie super-résolue en 3D et le suivie 3D de molécules uniques en profondeur dans des échantillons biologiques denses et complexes. La technique SELFI se base sur l'utilisation des interférences auto-référencées (également appelées « auto-interférences ») pour remonter à la localisation 3D d’un émetteur en une seule mesure. Ces interférences sont générées via l’utilisation d'un réseau de diffraction placé en sortie du microscope de fluorescence : le signal de fluorescence diffracte sur le réseau et les ordres interfèrent, après une courte propagation, sur le détecteur. Les interférences ainsi formées sont décodées numériquement pour remonter à la localisation 3D d'une molécule fluorescente au sein de l'échantillon. Une molécule unique peut ainsi être localisée avec une précision d'une dizaine de nanomètre, et cela jusqu'à une profondeur d'au moins 50µm au sein d'un échantillon biologique vivant épais (par exemple un tissu biologique).En combinant la méthode SELFI à différentes techniques de super-résolution (PALM, dSTORM et uPAINT), nous montrons que cette méthode de localisation tridimensionnelle permet de retrouver la hiérarchie et l'organisation de protéines dans des objets biologiques. En effectuant du SELFI-PALM, nous avons pu observer différentes protéines des points focaux d’adhésion (talin-C terminale et paxiline) et retrouver les différences de hauteur attendues, et ceux sur des échantillons de cellules vivantes. Ces résultats confirment la résolution accessible avec la technique SELFI (environ 25nm) même pour un faible nombre de photons collectés (environ 500 photons par molécule).Nous mettons en évidence la robustesse de la technique SELFI en reconstruisant des images de super-résolution 3D de structures denses en profondeur dans des échantillons tissulaires complexes. En effectuant du SELFI-dSTORM, nous avons observé le réseau d’actine sur des cellules cultivées en surface de la lamelle dans un premier temps, et à différentes profondeurs (25 et 50 microns) au sein de tissus artificiels dans un second temps.Du suivi 3D de particule unique a aussi été effectué sein de tissus biologiques vivants. Nous avons observé la diffusion libre de quantum dots à différentes profondeurs (jusqu’à 50 microns, limité par l’objectif utilisé) dans des tranches vivantes de cerveau.Nous avons appliqué la technique SELFI à la détection de récepteurs postsynaptiques NMDA. Cela nous a permis d'observer, sur des échantillons de neurones en culture primaire mais aussi au sein de tranches de cerveaux de rats, une différence d'organisation entre les deux sous-unités GluN2A et GluN2B de ce récepteur au glutamate.Enfin, nous avons démontré l'importance de suivre l'évolution de l'environnement des échantillons biologiques vivants lors des acquisitions permettant la détection de molécules individuelles. Grâce à l'utilisation additionnelle et simultanée de l'imagerie de phase quantitative, nous avons pu étudier la dynamique de la membrane cellulaire durant l’activation par un facteur de croissance. L'analyse corrélative entre les images de phase quantitative en lumière blanche et les détections de molécules fluorescentes uniques permet d'obtenir de nouvelles informations pertinentes sur l'échantillon étudié. / The work of this thesis was devoted to the development of a new technique SELFI (for self-interferences). This method unlocks the three-dimensional localization of individual fluorescent emitters. We have demonstrated that this allows 3D super-resolved imaging and 3D tracking of single molecules deep into dense and complex biological samples. The SELFI technique is based on the use of self-referenced interference to go back to the 3D location of a emitter in a single measurement. These interferences are generated using a diffraction grating placed at the exit of the fluorescence microscope: the fluorescence signal diffracts on the grating and, after a short propagation, the orders interfere on the detector. The formed interferences are digitally decoded to extract the 3D location of a fluorescent molecule within the sample. A single molecule can thus be localized with a precision of approximatively ten nanometers up to a depth of at least 50 µm in a thick living biological sample (for example a biological tissue).By combining the SELFI method with different super-resolution techniques (PALM, dSTORM and uPAINT), we show that this three-dimensional localization method grants the access to the hierarchy and organization of proteins in biological objects. By performing SELFI-PALM, we observed different proteins of the adhesion focal points (talin C-terminal and paxilin) and found the expected elevation differences, and those within living cell samples. These results confirm the resolution capability of the SELFI technique (about 25 nm) even for a small number of photons collected (about 500photons per molecule).We highlight the robustness of the SELFI technique by reconstructing 3D super-resolution images of dense structures at depth in complex tissue samples. By performing SELFI-dSTORM, we observed the actin network in cells grown on the surface of the coverslip at first, and at different depths (25 and 50 microns) within artificial tissues in a second time.3D single particle tracking has also been performed in living biological tissues. We observed the free diffusion of quantum dots at different depths (up to 50 microns) in living brain slices.We applied the SELFI technique to the detection of NMDA postsynaptic receptors. We observed, in primary culture of neurons but also within slices of rat brains, a difference in organization between the two subunits GluN2A and GluN2B of this glutamate receptor.Finally, we show the importance of following the evolution of the living biological sample environment during the acquisition of images leading to detections of single molecules. Thanks to the additional and simultaneous use of quantitative phase imaging, we were able to study cell membrane dynamics during the activation by a growth factor. The correlative analysis between white light quantitative phase images and single fluorescent molecule detections provides new relevant information on the sample under study.

Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...

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京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20338号 / 工博第4275号 / 新制||工||1662(附属図書館) / 京都大学大学院工学研究科社会基盤工学専攻 / (主査)教授 石田 毅, 教授 林 為人, 准教授 奈良 禎太 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Quorum sensing (QS) is a type of microbial communication used by bacteria to coordinate their behavior based on population density, regulating complex processes like biofilm formation and virulence, among other behaviors. Quorum quenching (QQ), on the other hand, disrupts this communication, usually by degradation of the QS signaling molecule. QQ offers a potential strategy for controlling bacterial behaviors linked to pathogenicity and biofouling. Despite significant advances in understanding and modeling the spatial-temporal behavior of QS, predictive modeling of QQ remains nascent, with a notable gap in the quantitative assessment of QQ's impact on QS. Here we show quantitative evaluation and characterization of the effect of QQ on QS in agar-based experiments, combined with an experimentally validated computational model. This research utilizes green fluorescence in E. coli MG 1655 as an indicator of QS activation, focusing on the degradation of Acyl-Homoserine Lactone (AHL), a key QS molecule in Gram-negative bacteria linked to pathogenicity, by the AiiA enzyme in engineered AiiA-producing Salmonella Typhimurium 14028. Our findings suggest that QQ more effectively influences QS in spatial configurations of the populations with larger interaction surfaces and shorter diffusion distances. Contrary to our initially held hypothesis, the primary effect of QQ is not a delay in QS onset but rather an attenuation of QS activity, with the area-under-the-curve of fluorescence serving as a quantitative metric. This study also introduces, to the best of our knowledge, one of the first instances of experimentally validated predictive modeling for QQ, applied to agar-based experimental setups. We posit that the quantitative experimental characterization and modeling framework presented in this research will enhance the understanding of bacterial community interactions. Enhanced comprehension of QQ and QS behaviors holds significant promise for advancing practical applications, particularly in mitigating or diminishing undesirable QS-associated activities. This is especially relevant in areas like biofouling, waste treatment, and the reduction of infections and progression of diseases in plants and animals, areas increasingly important as concerns about drug resistance in microbes and food security escalates. / Master of Science / One of the ways bacteria communicate with each other is called quorum sensing (QS), where they use chemical signals to organize and time group behavior, including forming communities encapsulated in protective layers, called biofilms, and engaging in virulent attacks against hosts. Quorum quenching (QQ) in bacteria, however, disrupts this communication system, usually by breaking down the chemical signals that bacteria use to send messages to each other. Even though QS has been studied extensively, determining how to predict and control QQ is still a nascent area of research. Here, we studied and characterized how QQ affects QS by doing experiments with bacteria populations in agar (a jelly-like substance) and applied a computational model to explain and ultimately predict the experimental observations. Engineered QS population (E. coli MG 1655) produced Acyl-Homoserine Lactone (AHL) signaling molecules, and engineered QQ bacteria (S. Tm 14028) used the Autoinducer Inactivation A (AiiA) enzyme to break down the AHL. According to our results, QQ doesn't delay the QS bacteria's group behaviors (in our case, green fluorescent signal production); it weakens the signal instead. Understanding QQ and QS better, especially through measurements and modeling, could lead to expanded methods of deterring harmful bacterial behavior, managing waste better, and stopping diseases in plants, animals, and humans, especially with the concerning rise of drug-resistant microbes and food security. One exciting possibility is using QQ to protect plants from bacterial infections. This could be a way to shield our crops without always relying on antibiotics.

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In view of the increasing global challenge of bacterial resistance, there exists an urgent need for the rationale development of antibacterial compounds with either novel or multiple mechanisms of action. Two chalcone-derivatives, F1 and F23, demonstrated MICs within the range of 16 to >512 μg/ml against two plant pathogens (P. caratovoram and C. michiganensis subsp. michiganensis) as well as important clinical bacterial species. Both compounds displayed an MIC of 32 μg/ml against quinolone-resistant S. aureus. Whilst possessing weak activities individually, each semi-synthetic agent displayed notable synergistic action with polymyxin B against S. aureus, C. violaceum, E. coli and Ps. aeruginosa, thereby recording FICs within the range of <0.093 to 2 that indicated the existence of synergism in some instance. These chalcone compounds applied with polymyxin B displayed a notable FICindex of <0.093 against the Neisseriaceae C. violaceum, and a potential noteworthy capacity to extend the spectrum of activity of the latter antibiotic to include Gram-positive S. aureus species. F1 inhibited staphylococcal replication in broth and the combination of either of both chalcone-derivatives with polymyxin B instituted a metabolic blockage in S. aureus and other bacterial species as determined through a modified MTT reduction assay. The combined agents inflicted major disruptions to the S. aureus cytoplasmic membrane bilayer as evidenced by the release of intracellular potassium as well as the influx of Sytox Green fluorescent stain. Notable levels of cell membrane potential dissipation, leakage of intracellular potassium ions and blockage of reducing enzymes activities occurred within the first 30 minutes, well in advance of significant loss in cell viability that was recorded usually after 4 – 8 hours, suggesting these activities were prerequisites to cell death. In erythrocyte lysis assay, the synergistic combinations of 128 μg/ml of either of both chalcone derivatives with 128 μg/ml polymyxin B displayed the lowest degree of haemolysis, followed by that occurring with 32 μg/ml of the chalcone-derivatives combined with 256 μg/ml of the polypeptide antibiotic. In conclusion, further structure activity modifications aimed at improving the aqueous solubility of these chalcone-derivatives as well as the antibacterial activity recorded for certain combination concentrations of polymyxin B with either of these semi-synthetic agents may be required before considerations are made for the possibility for potential external formulations. Such preparations may include antiseptic creams, lotions, ointments, as well as aerosols that can be applied with nebulizers in targeted delivery for such cases like cystic fibrosis.