ED Sciences Chimiques
Shape control in minimal synthetic cells via cytoskeleton reconstitution
by Bastien LAMBERT (Centre de Recherche Paul Pascal)
The defense will take place at 14h00 - Amphithéâtre CRPP Centre de Recherche Paul Pascal (CRPP) 115 Avenue du Dr Albert Schweitzer, 33600 Pessac
in front of the jury composed of
- Jean-Christophe BARET - Professeur des universités - Centre de Recherche Paul Pascal - Directeur de these
- Aurélie BERTIN - Directrice de recherche - Institut Curie, CNRS - Rapporteur
- Patrice GAURIVAUD - Ingénieur de recherche - Anses Laboratoire de Lyon - Rapporteur
- Anne LE GOFF - Maîtresse de conférences - Université de technologie de Compiègne - Examinateur
- Cécile ZAKRI - Professeure des universités - Centre de Recherche Paul Pascal - Examinateur
- Laure BEVEN - Professeure des universités - Université de Bordeaux - CoDirecteur de these
Understanding how cells maintain and adapt their shape is a central focus in biology. Indeed, the shape of a cell is intrinsically tied to its function, and is finely regulated during cell processes like division. Cytoskeletons, dynamic protein networks, play a crucial role in shaping both eukaryotic and prokaryotic cells. Because of their lack of a cell wall, bacteria of the class Mollicutes offer simple models to study the role of cytoskeleton proteins on cell shape. Among these, spiroplasmas stand out due to their helical shape and unique motility, which relies on the propagation of kinks along their bodies. Their characteristic motility and morphology are governed by their cytoskeleton, primarily composed of multiple isoforms of MreB, whose arrangement and specific role within the structure remains to be defined. My research aimed to elucidate the specific roles of these MreB isoforms, particularly in driving helicity and kink formation. I employed two complementary experimental approaches to achieve this. The first approach involved heterologously expressing Spiroplasma MreB proteins in Mycoplasma capricolum (Mcap), a related bacterium that lacks a cell wall and typically has no well-defined shape. This system provided a valuable opportunity to explore how introducing MreB isoforms might influence the shape and behavior of Mcap. By introducing various MreB isoforms, especially MreB1 and MreB5, we were able to demonstrate that these proteins can confer a helical shape. In addition, MreB5 could initiate and propagate kinks in Mcap. Cryoelectron microscopy revealed that MreB filaments associate with the plasma membrane, suggesting a direct role in membrane curvature modulation. However, despite the helical transformation and kink propagation, these MreB-expressing cells did not display efficient motility in culture broth, indicating that other unknown components from Spiroplasma are necessary for full motility. The second approach of my study focused on reconstituting the Spiroplasma cytoskeleton in vitro within synthetic microcompartments, specifically, giant unilamellar vesicles (GUVs). This method aimed to simulate the natural cellular environment, providing a more controlled platform to analyze how MreB proteins influence membrane shape and mechanics. This approach unfolded in three main stages: 1. I successfully expressed Spiroplasma MreB proteins heterologously in E. coli, developing a robust purification protocol involving a dual extraction strategy that overcame challenges related to protein aggregation. This optimization yielded high-concentration protein samples, crucial for subsequent experiments. 2. Using microfluidic techniques, I generated GUVs with precisely controlled sizes and compositions. By employing an octanol-assisted liposome assembly on custom-designed microfluidic chips, I produced GUVs that closely mimic a cellular environment, essential for studying the interactions between proteins and lipid membranes. 3. I designed a microfluidic system that enabled the simultaneous encapsulation of MreB proteins and ATP, recreating conditions conducive to studying how these proteins impact membrane structure. While initial results did not show visible membrane deformation, the encapsulated MreBs significantly influenced GUV survival, potentially by altering membrane integrity. In summary, my research demonstrated that Spiroplasma MreB isoforms, particularly MreB5, can induce helical shape and kink propagation in Mcap, although full motility requires additional, unidentified factors. I also developed and optimized protocols for the heterologous production and purification of MreB proteins, overcoming significant aggregation challenges. Moreover, my microfluidic GUV production method and the encapsulation of MreB proteins lay the groundwork for future studies, which will be critical for further dissecting bacterial morphology and exploring shape control in synthetic systems.
ED Sciences de la Vie et de la Santé
ARTIFICIAL INTELLIGENCE FOR THE CHARACTERIZATION OF 3D CULTURES IN HIGH CONTENT FLUORESCENCE MICROSCOPY
by Christer LOHK (Institut Interdisciplinaire de Neurosciences)
The defense will take place at 14h30 - Salle de Conférence IBGC UMR 5095, 146 rue Léo Saignat 33000 Bordeaux
in front of the jury composed of
- Jean-Baptiste SIBARITA - Cadre scientifique - Université de Bordeaux - Directeur de these
- Christophe ZIMMER - Full professor - Rudolf Virchow Center, University of Würzburg - Rapporteur
- Xavier DESCOMBES - Directeur de recherche - INRIA Sophia Antipolis, CNRS - Rapporteur
- Thomas WALTER - Directeur de recherche - Computational Oncology (U1331), Institut Curie - Examinateur
- Macha NIKOLSKI - Directrice de recherche - Computational Biology and Bioinformatics team, IBGC / CNRS, University of Bordeaux - CoDirecteur de these
Over the recent years, 3D cell cultures have become the gold standard in cell biology, with applications ranging from fundamental research to high-content drug screening and biomedicine. Among them, organoids offer a major advantage over traditional two-dimensional cell cultures as they accurately replicate the cellular architecture and function of a tissue of interest. 3D cell cultures have, therefore, become irreplaceable as biological models for studying disease mechanisms, normal tissue development, and screening drug responses. The main focus of this thesis project was the development of computational tools for the analysis of 3D cell culture images obtained with the light sheet microscopy-based high-content screening platform developed in our lab. This acquisition system employs disposable microfabricated chips, containing arrays of small pyramidal wells enabling the cultivation and parallel three-dimensional imaging of individual organoids. With this approach, imaging data can be acquired simultaneously from over a hundred single organoids using two complementary modalities: i) fluorescence single-objective Selective Plane Illumination Microscopy (soSPIM), which captures three-dimensional multi-channel image stacks, and ii) transmission light modality, allowing for rapid, label-free acquisition of two-dimensional images of the samples. When used in conjunction with an integrated well detection algorithm, the image acquisition process can be fully automated and run over extended periods of time. This thesis presents a toolkit of computational workflows for monitoring and quantification of drug-induced changes in spheroid and organoid cell cultures. Our proposed methods include i) a deep learning-based real-time segmentation model to rapidly localize organoid boundaries in transmission light images, ii) foundation model-based approach for volumetric segmentation of organoids in three-dimensional images, and iii) a feature extraction pipeline that integrates classical image features with deep representations from vision transformer and variational autoencoder networks to quantify morphological differences across conditions. The feature extraction strategy supports both quality control during acquisition workflow and comprehensive toolset for phenotypic profiling of organoids in fluorescence and brightfield images. Combined with the soSPIM-based culturing and imaging capabilities, these established methods provide a foundation for future large-scale, fully automated experimental pipelines targeted at predicting the response of 3D cell cultures to chemical and biological treatments.
THE CHAPERONE DNAJB6 AS A REGULATOR OF ALPHA-SYNUCLEIN AGGREGATION IN NEURONAL MODELS OF INDUCED SYNUCLEINOPATHY
by Ænora LETOURNEUR (Institut des Maladies Neurodégénératives)
The defense will take place at 14h00 - Salle de conférence Batiment CARF 146 rue Léo Saignat 33000 Bordeaux
in front of the jury composed of
- Caroline NOCCA SMET - Full professor - Département Biologie de la Faculté des Sciences et Technologies l'Université de Lille - Rapporteur
- Pierre GENEVAUX - Directeur de recherche - Laboratoire de Microbiologie et Génétique Moléculaires (LMGM) de l'Université Toulouse 3 Paul Sabatier (UT3) et du Centre National de la Recherche Scientifique (CNRS) - Rapporteur
- Elodie ANGOT - Chef de projet - ROCHE - Examinateur
- Thierry BARON - Directeur de recherche - Unité Maladies Neuro-Dégénératives - Examinateur
The protein α-synuclein self-assembles into amyloid fibrils, found in the neuronal or glial inclusion observed in synucleinopathies. In vitro studies have characterized the aggregation mechanisms of the protein, but the processes that the cell can use to modulate this aggregation are still under investigation. We chose to work in models of induced synucleinopathy, both primary cultures and in adult mice brains. We first look at a panel of molecular chaperones, which have been shown to be involved in this regulation, before focusing of DNAJB6. Indeed, we observed, with this protein, a specific relocalization of DNAJB6, condensed as punctae, to the neo-formed aggregates. This observation, combined with the studies showing DNAJB6 to have an affinity for amyloid structures, made us hypothesize that DNAJB6 had a functional role in the regulation of the induced synucleinopathy. Indeed, functional studies then allowed us to observe an inhibition of the aggregation of α-syn when DNAJB6 is overexpressed, both in primary cultures and in mice brains. We deduced that this effect was due to a selective interaction of DNAJB6 with the fibrillar α-syn, and not the monomeric one. Furthermore, we were able to show that this inhibition of the aggregation was due to the activity of DNAJB6 alone and not, as we thought in the beginning, due to its involvement in a system also containing Hsp110 and Hsc70, for which DNAJB6 is a co-chaperone. Indeed, the mutated H31Q DNAJB6, which cannot interact with Hsc70, showed the same inhibitory effect as the wild-type DNAJB6. We confirmed this point by reconstituting in vitro the system and seeing that, in contrast with DNAJB1, DNAJB6 does not induce a disaggregase activity of the system on the α-syn fibrils.
ED Sociétés, Politique, Santé Publique
Evaluation of sexually transmitted infections in an advanced combined sexual health care strategy including pre-exposure prophylaxis among female sex workers in Côte d'Ivoire
by N'Zébo Marcellin NOUAMAN (Bordeaux Population Health Research Center)
The defense will take place at 10h00 - Salle n°4, rdc BU 146 rue Léo Saignat, 33076 Bordeaux
in front of the jury composed of
- Valériane LEROY - Directrice de recherche - Université de Toulouse - Examinateur
- Charlotte CHARPENTIER - Professeure des universités - praticienne hospitalière - Université Paris Cité - Rapporteur
- Christian LAURENT - Directeur de recherche - Université de Montpellier - Rapporteur
- Didier Koumavi EKOUEVI - Chargé de recherche - Université de Bordeaux - Examinateur
Background: Sub-Saharan Africa accounts for over 40% of all sexually transmitted infections (STIs) worldwide. Female sex workers (FSWs) remain particularly vulnerable to STIs. In Côte d'Ivoire, as in other West African countries, STIs are treated using a syndromic approach, whereby diagnosis and treatment are based on the observed clinical symptoms. Due to missed opportunities for diagnosis and the risk of resistance, the WHO recommends incorporating biological screening tests where possible. Meanwhile, the introduction of pre-exposure prophylaxis (PrEP) for HIV prevention is prompting a rethink of STI management strategies as part of a wider sexual health service for key populations. This thesis aimed to assess exposure to STIs (including HIV) and describe how they are managed as part of a comprehensive sexual health service for female sex workers (FSWs) in the San Pedro region. Methods: Firstly, our work was based on data from an exploratory survey and the ANRS 12361 PrEP-CI project as part of an evaluation of the relevance and feasibility of offering PrEP to female sex workers (FSWs) in Côte d'Ivoire. We carried out a survey, including an evaluation of the incidence of HIV based on recent infection tests, to assess the appropriateness of a PrEP programme. The findings of this study then informed the establishment of a cohort to study the sexual health of FSWs using a community-based approach: the ANRS 12381 Princesse project. One of the challenges was to estimate not only the prevalence of various syndromic and biological STIs and screening coverage, but also the factors associated with syndromic STIs, and the levers and obstacles to comprehensive STI management. Results: Despite the widespread use of condoms in this port city of Côte d'Ivoire, sex workers remain a population at risk of exposure to HIV and other STIs. The HIV incidence rate in San Pedro remains high at around 3%. Our results show a high prevalence of syndromic sexually transmitted infections (STIs) and similar rates of curable bacterial STIs to previous data in this population and national estimates. However, these bacterial STI rates may be underestimated due to difficulties in taking anovaginal samples for PCR tests, such as menstruation, time constraints, pain and discomfort. Furthermore, our results demonstrate the essential nature of capacity building for doctors and its role in motivating their involvement in the improved management of STIs. Providing biological tests and resources adds real value, as it helps create a trusting relationship between doctors and the female sex worker (FSW) population, thereby increasing their access to care. Conclusion: This study confirms that FSWs, even those who have been involved in this activity for several years, remain highly exposed to HIV and other STIs, particularly those living in precarious conditions. Given this situation, appropriate prevention programmes focusing on the needs of individuals must be implemented, including new prevention tools such as PrEP, and taking into account the living and working conditions of FSWs. Another priority is developing and simplifying diagnostic tests for use at the point of care in order to reach as many vulnerable, high-risk and hard-to-reach people as possible who are not currently receiving care. National programmes must collaborate to screen for both syndromic and biological STIs, reducing the STI burden among FSWs.