ED Sciences Chimiques
Reconstitution and structure-function investigation of regulated cell death signalosomes encapsulated in protocells
by Seamoon DEB (Institut de Chimie & de Biologie des Membranes & des Nano-objets)
The defense will take place at 14h30 - L'auditorium IECB Institut européen de chimie et biologie (IECB), 2 rue Robert Escarpit, 33607 PESSAC, France
in front of the jury composed of
- Antoine LOQUET - Directeur de recherche - Chimie et Biologie des Membranes et des Nano-objets (CBMN) - Directeur de these
- Christina SIZUN - Directrice de recherche - Institut de Chimie des Substances Naturelles (ICSN) - Rapporteur
- Anja BöCKMANN - Directrice de recherche - Institut de Biologie et Chimie des Protéines (IBCP) - Rapporteur
- Sven SAUPE - Directeur de recherche - Institut de Biochimie et Génétique Cellulaires (IBGC) - CoDirecteur de these
- Alexander Kai BUELL - Full professor - Technical University of Denmark (DTU) - Examinateur
- Raimon SABATé LAGUNAS - Associate Professor - Universitat de Barcelona (University of Barcelona) - Examinateur
- Birgit HABENSTEIN - Directrice de recherche - Chimie et Biologie des Membranes et des Nano-objets (CBMN) - Examinateur
Functional amyloids are ordered protein assemblies involved in numerous biological processes, including immune signalling and regulated cell death. This thesis investigates the relationships between the structure, assembly kinetics and function of amyloid signalosomes by combining structural biology, biophysics and biomimetic compartmentalisation. The first part involved developing an approach combining the design of fluorescent protein constructs with microfluidic techniques to reconstitute an amyloid signalosome within protocells and mimic its aggregation within a confined environment.. The prion-forming domain of Het-s, a model of cell-death signalling in the fungus Podospora anserina, was encapsulated in monodisperse aqueous microdroplets generated in a fluorinated oil phase. Conditions for droplet production, stability and imaging were optimised. Their small volume, monodispersity and large-scale production enable numerous individual reactions to be monitored in parallel and provide opportunities for high-throughput analyses. Automated time-lapse imaging was used to follow the kinetics of amyloid assembly. Coupling the platform with confocal microscopy and z-stack acquisition also enabled three-dimensional reconstruction of the distribution of the assemblies within droplets. Het-s fused to a fluorescent protein or labelled with a maleimide probe remained predominantly soluble when encapsulated alone. By contrast, its co-encapsulation with unlabelled Het-s promoted the appearance of fluorescent assemblies, demonstrating co-assembly within the confined environment. The proportion of aggregate-containing droplets notably depended on the amount of unlabelled Het-s available. Solid-state NMR analyses further indicated that Het-s retained a fibrillar conformation within droplets comparable to that observed in bulk solution. The biological functionality of the encapsulated fibrils was then evaluated by transfecting [Het-s*] mycelia, corresponding to the non-prion state of P. anserina. Following regeneration, the colonies were confronted with a het-S tester strain. Like fibrils assembled in bulk solution, fibrils originating from droplets induced a barrage reaction, whereas buffer and empty-droplet controls did not. These results show that Het-s retains its prion infectivity and functional conformation following encapsulation. Beyond this model system, the platform provides a modular nanoreactor in which medium composition, solvent, concentrations and molecular partners can be varied independently. It could therefore be adapted to investigate other proteins, RNA, enzymes or small molecules and to screen factors capable of promoting or inhibiting the nucleation, growth and propagation of protein assemblies. The second part focused on the characterisation of a bacterial antiphage defence system based on a BASS amyloid motif. The effector protein and its signalling partner were produced and investigated using biochemical, microscopic and spectroscopic approaches. Solid-state NMR spectroscopy was used to examine the molecular organisation and dynamics of the fibrillar state. Cross-polarisation, INEPT and 13C-13C correlation experiments distinguished a rigid amyloid core from more mobile regions and helped identify the residues involved in assembly. These results provide a structural description of a BASS motif involved in antiviral signal transmission in bacteria and support the proposed evolutionary relationships between bacterial and fungal amyloid signalosomes. This work establishes an integrated approach for studying amyloid signalosomes across several scales, from the molecular organisation of bacterial motifs to the kinetics, three-dimensional distribution and biological function of assemblies reconstituted within biomimetic compartments. The developed platform opens the way to high-throughput investigation of more complex systems and the controlled reconstitution of signalling pathways leading to cell death.
ED Sciences de la Vie et de la Santé
Bcl-xL deamidation in platelets: mere age marker or lifespan modulator
by Joann KERVADEC (Institut de Biochimie et Génétique Cellulaires)
The defense will take place at 14h00 - Salle de conférence IBGC, Université de Bordeaux, 1 rue Camille St Saëns, Campus de Carreire, 33000 Bordeaux
in front of the jury composed of
- Laurent MAILLET - Chargé de recherche - Université de Nantes - IRSUN - Rapporteur
- Bruno MIROUX - Directeur de recherche - Université Pierre et Marie Curie et ENS - IBPC - Rapporteur
- Isabel ALVES - Directrice de recherche - Université de Bordeaux - CBMN - Examinateur
- Lucie BERGDOLL - Chargée de recherche - Université de Marseille - LISM - Examinateur
Platelets are small anucleated blood cells essential for hemostasis, with an average lifespan of approximately 10 days in humans. They undergo apoptosis, a programmed cell death regulated by the Bcl-2 protein family, with notable roles played by Bcl-xL and Bak. In healthy platelets, Bcl-xL maintains survival by inhibiting Bak, whereas apoptosis is triggered upon Bak release, leading to apoptosis execution. Although platelet apoptosis has been extensively studied, the molecular events disrupting the Bcl-xL-Bak interaction remain unclear. This PhD project investigated the role of Bcl-xL deamidation, a spontaneous post-translational modification converting Asn52 and Asn66 into Aspartate (Asp) or isoAspartate (isoAsp). We hypothesized that this process dissociates or prevents the Bcl-xL-Bak interaction and thereby initiates apoptosis. To test this hypothesis, we studied native human Bcl-xL (untagged and full-length ) at both molecular and cellular levels. Using a minimalist in vitro system combining cell-free expression of Bcl-xL with synthetic nano-membranes (nanodiscs), we dissected the two-step transition from its peripheral membrane association to transmembrane insertion. Our first discovery was that isoAsp-containing Bcl-xL exhibits impaired peripheral association compared with Asp-deamidated Bcl-xL and with Bcl-xL wild type, which in turn decreases its transmembrane insertion. We further showed that inducing Iso-Asp formation once Bcl-xL has bound membrane-inserted Bak, disrupts the Bcl-xL-Bak complex. Therefore, this work identifies two levers by which deamidation inactivates Bcl-xL. Furthermore, in collaboration with the Établissement Français du Sang, we validated Bcl-xL deamidation as a reliable biomarker of platelet age in stored platelet concentrates collected from donors. Based on all our findings, we propose a molecular mechanism for the onset of platelet apoptosis, in which isoAsp deamidation of Bcl-xL disrupts the Bcl-xL-Bak interaction, thus releasing Bak to further execute apoptosis. Beyond the original scope, we also uncovered an unsuspected role for Bak in promoting Bcl-xL membrane insertion: this process relies on cooperative interactions involving not only canonical BH3-binding, but also hydrophobic interactions of the C-terminal helices of both proteins. Furthermore, we provided a previously unrecognized effect of mutations engineered in the BH3-binding groove of Bcl-xL: not only do they result in the well-known decrease in binding to Bak, but importantly, also reduce Bcl-xL membrane insertion. This compounding factor was overlooked in the functional cellular studies reported in the literature and calls for reevaluation. Finally, during these studies, we also discovered that conditions promoting Bcl-xL deamidation also drive its redox dimerization, even though the functional unit at the membrane remains monomeric Bcl-xL. Overall, this work identifies Bcl-xL deamidation as a potential initiating event in platelet apoptosis and provides new mechanistic insights into the membrane dynamics and interaction between Bcl-xL and Bak.
ED Sociétés, Politique, Santé Publique
When the Commons Take Place. A Study of Rural “Third Places” in Nouvelle-Aquitaine (France), Exploring the Relationship Between Built Structures and Commons Practices
by Camille BRETON (Centre Emile Durkheim)
The defense will take place at 14h00 - Amphithéâtre Dénucé Université de Bordeaux, Bâtiment Q, 3ter place de la Victoire, 33000 Bordeaux
in front of the jury composed of
- Olivier CHADOIN - Professeur - Université de Bordeaux - Directeur de these
- Olivier DAVID - Professeur des universités - Université Rennes 2 - Rapporteur
- Ludivine DAMAY - Professeure - Université libre de Bruxelles - Rapporteur
- Véronique BIAU - Directrice de recherche émérite - ENSA Paris La Villette - Examinateur
- Leila KEBIR - Professeure associée - Université de Lausanne - Examinateur
- Patrice TISSANDIER - Maître de conférences - Université Bordeaux Montaigne - Examinateur
- Simon TEYSSOU - Maître de conférences - ENSA Clermont-Ferrand - Examinateur
While third places have primarily developed in major cities since the 2000s, they are now established in rural areas and contribute to revitalizing rural communities. Although difficult to define, the concept of the “third place” can be understood as a tool for social innovation at the heart of the circular economy. While the notion of “third,” closely linked to that of “commons,” remains central to scientific and political debate, the architectural dimension of “place” has received little attention. This research investigates rural third places — as opposed to their urban counterparts — from a spatial perspective of the buildings. My work is based on field research across the rural areas of Nouvelle-Aquitaine, at the intersection of social sciences and architecture : an analysis of the diversity of third places (surveys, mapping, socio-economic territorial analysis, quantitative analysis using questionnaires, site visits) complemented by three case studies (interviews, spatial analysis of buildings, participant observation) documenting situated experiences. This study aims to understand how the architecture of third places contributes to the construction of the commons in rural contexts. First, strategies of establishment are examined through the territorial, land-use and symbolic choices made by project leaders. Then I examine the spatial and aesthetic approaches that shape an architecture of the commons. Finally, field observations lead me to question the stance and methods of the builders-users of these spaces, suggesting a transgressive and renewed vision of the world of architecture.