Les bactériophages neutralisent les systèmes de défense et détournent les fonctions vitales de leur hôte pour favoriser leur multiplication. Les gènes de phages qui gouvernent cette prise de contrôle de l’hôte restent mal connus, pourtant leur caractérisation présente un intérêt majeur pour mettre à jour des fonctions bactériennes spécifiquement ciblées par les phages et pour concevoir de nouveaux agents antibactériens.Le phage T5 injecte son ADN dans la bactérie Escherichia coli en deux étapes. Seuls les gènes précoces codés par 8% du génome entrent dans la cellule et le transfert s’arrête. Leur expression induit la dégradation du chromosome de l’hôte et l’inactivation de ses systèmes de restriction et de réparation de l’ADN. Après quelques minutes, le reste de la molécule d’ADN est injecté, ce qui permet la production de nouveaux phages. Deux gènes précoces A1 et A2 ont été identifiés comme essentiels pour la reprise du transfert de l’ADN et A1 est également nécessaire pour induire la dégradation de l’ADN de l’hôte. A1 et A2 sont les deux seuls gènes connus pour être impliqués dans la régulation de ce système original d’infection, mais leur fonction n’a jamais été identifiée.Ma thèse porte sur la caractérisation fonctionnelle et structurale des protéines A1 et A2. J’ai purifié A1 et démontré in vitro qu’elle avait une activité DNase dépendante du manganèse. Sa structure atomique a été résolue par cryomicroscopie électronique à 3.01 Å de résolution, montrant une organisation octamérique de symétrie D4 inédite pour une DNase. Chaque monomère (61kDa) contient un domaine exonuclease dont le site actif lie deux ions Mn2+ et qui s’apparente au site catalytique des domaines exonucléases de la DNA polymerase II et des DNAses associées aux systèmes de recombinaison homologue et de réparation de l’ADN comme Mre11. En construisant différents mutants de A1, j’ai identifié certains acides aminés essentiels pour l’activité catalytique et, par des expériences de complémentation fonctionnelle, j’ai montré que cette activité était indispensable pour l’infection. L’ensemble de ces résultats suggèrent que A1 est la DNase, jusqu’ici inconnue, responsable de la dégradation massive du génome de l’hôte au tout début de l’infection. Enfin, j’ai observé que la production de A1 pendant l’infection induit une forte activité recombinase. De nombreux autres bactériophages qui n’appartiennent pas à la famille des T5virus produisent également une protéine similaire à A1 dont la fonction n’a jamais été identifiée. Ce travail est un premier pas vers la compréhension de son rôle dans le mécanisme général d‘infection par les phages. Une deuxième partie de cette thèse porte sur la caractérisation structurale de A2. Des recherches de similarité indiquent la présence d’un domaine Helix-Turn-Helix typique des régulateurs transcriptionnels. J’ai purifié A2 et montré que cette protéine de 14 kDa est un dimère en solution. La caractérisation des propriétés biochimiques de A2 a permis de débuter l’étude de sa structure par RMN.Les résultats de ma thèse ont révélé la structure originale d’une DNase de bactériophage qui contrôle la dégradation du génome bactérien et la régulation du transport de l’ADN viral au début du cycle infectieux. Ces résultats soulèvent des questions intrigantes : comment l’ADN de T5 est-il protégé de l’activité DNase de A1 ? Comment A1 et A2 interagissent-elles lors des étapes de prise de contrôle de l’hôte ? / Bacteriophages defeat bacterial defences and hijack host cell machineries to establish a favourable environment for their multiplication. Early-expressed viral genes that govern host takeover are highly diverse from one phage to another and most of them have no assigned function. They thus represent a pool of novel genes whose products potentially subvert bacterial cell vital functions and could help in designing new antibacterial strategies.T5 phage uses a unique 2-step mechanism to deliver its DNA into its host Escherichia coli. At the onset of the infection, only 8 % of the genome enter the cell before the transfer temporarily stops. Expression of the genes encoded by this DNA portion leads to host chromosome degradation and inactivation of host restriction and DNA mending systems. After a few minutes, T5 DNA transfer resumes, allowing further phage multiplication. A1 and A2 are early genes required for DNA transfer completion and A1 is also necessary to trigger host DNA degradation. A1 and A2 are the only two genes known to be involved in the regulation of this original infection system, but their function yet remains to be characterized.The objectives of this work were to characterize the function and structure of A1 and A2 proteins. I have purified the A1 protein and shown that it has a manganese-dependent DNase activity in vitro. Cryo Electron Microscopy at 3.01 Å resolution unravelled its structure, showing an octameric organization with a D4 symmetry, which is unprecedented for a DNase. Each monomer (61 kDa) carries an exonuclease domain harbouring an active site with two Mn2+ ions. This site is similar to those from the exonuclease domain of the DNA polymerase II and from DNases involved in DNA mending and recombination events like Mre11. I identified essential catalytic residues for the DNase activity and demonstrated that this activity is crucial for infection by engineering A1 mutant proteins and by doing functional complementation assays. Taken together, my results suggest that A1 could then be the elusive DNase responsible for the massive host genome degradation observed during T5 phage infection. Eventually, I uncovered a recombinase activity associated to A1 production during infection. Similar proteins to A1 with unknown functions are produced in several other bacteriophages outside of the T5virus family. This work is a first step towards understanding the role of this protein in the general mechanism of infection by bacteriophages. In a second part, I worked on the structural characterisation of A2 protein. Similarity searches revealed a helix-turn-helix domain typically found in transcriptional regulators. I purified and demonstrated the dimeric organisation of this 14-kDa protein in solution. This initial characterization of A2 has opened avenues for further NMR studies.During my Ph.D., I uncovered the structure of an original bacteriophage DNase that controls bacterial genome degradation and that regulates viral DNA transport at the beginning of the infectious cycle. These results open the intriguing question about the mechanism for T5 DNA protection from A1 DNase activity as well as about the interplay between A1 and A2 during the host takeover.

Les cryo-aimants supraconducteurs sont des sources de champs magnétiques intenses, compactes et peu gourmandes en énergie. Il existe diverses méthodes d’aimantation mais seule l’aimantation par champ magnétique pulsé (PFM) permet d’obtenir des champs excitateurs élevés sans recourir à des bobines supraconductrices refroidies.Basé sur une collaboration industrielle, ce travail a été consacré dans un premier temps à la conception d’un générateur de courant pulsé compact et évolutif générant des rampes de pulse de polarité réglage pouvant atteindre 3000A.Dans un second temps, nous avons mis en place les différents systèmes permettant l’aimantation des cryo-aimants refroidis soit à 77 K, soit dans un cryostat refroidi par un cryo-générateur.Enfin, l’aimantation par pulses de champ des cryo-aimants supraconducteurs a montré la possibilité de piéger un champ magnétique de l’ordre du tesla, réversible et reproductible. Les résultats obtenus répondent aux impératifs industriels de l’étude. Ils sont très encourageants pour le développement d’un dispositif de champ magnétique réversible à base de cryo-aimants supraconducteurs pour l’instrumentation scientifique ou pour les applications électrotechniques. / Superconducting cryomagnets are high magnetic fields sources that are both compact and energy efficient. There are various magnetization technics but only the magnetization by pulsed magnetic field (PFM) results in high excitation fields, that otherwise could only be obtained with large superconducting coils.This work was done in the framework of an industrial collaboration. In a first step, a compact and innovative pulse current generator enabling the generation of pulses with a 3000 Amps maximum intensity was designed and fabricated. Secondly, different systems for the magnetization of cryo-magnets either cooled down to 77 K or cooled in a cryostat by a cryo-generator were implemented. Finally, the pulsed field magnetization of superconducting cryo-magnets has shown that reversible and reproducible magnetic field in the one Tesla range could be generated by the set up.The results obtained are consistent with the industrial goals of this study. They are very encouraging for the development of reversible magnetic field devices based on superconducting cryo-magnets and dedicated to scientific instrumentation or for electrotechnical applications.

The aim of this thesis is to investigate applicability of regulariza- tion by Krylov subspace methods to discrete inverse problems arising in single particle analysis (SPA). We start with a smooth model formulation and describe its discretization, yielding an ill-posed inverse problem Ax ≈ b, where A is a lin- ear operator and b represents the measured noisy data. We provide theoretical background and overview of selected methods for the solution of general linear inverse problems. Then we focus on specific properties of inverse problems from SPA, and provide experimental analysis based on synthetically generated SPA datasets (experiments are performed in the Matlab enviroment). Turning to the solution of our inverse problem, we investigate in particular an approach based on iterative Hybrid LSQR with inner Tikhonov regularization. A reliable stopping criterion for the iterative part as well as parameter-choice method for the inner regularization are discussed. Providing a complete implementation of the proposed solver (in Matlab and in C++), its performance is evaluated on various SPA model datasets, considering high levels of noise and realistic distri- bution of orientations of scanning angles. Comparison to other regularization methods, including the ART method traditionally used in SPA,...

Catanoinic systems spontaneously form micelles and vesicles, which are self-assembled spherical structures made up by surfactants. In the core of the micelle a drug, or other organic substance, can be kept to stabilize it when placed in an aqueous environment. The micelle-to-vesicle transition corresponds to the moment when the drug is releases, and understanding which factors that trigger this transition is thus of great interest for the pharmaceutical industry. In this study the micelle-to-vesicle transition in water and the effect of salt were studied for the systems 95 mol% SDS/DDAB and 95 mol% SDeS/DDAB with different total concentrations. The static light scattering measurements showed that the micelle-to-vesicle transition for the system 95 mol% SDS/DDAB was shifted to lower total concentrations both when 50 mM NaBr and 100 mM NaBr were added, and that the transition was unaffected by changing the anionic surfactant from SDS to SDeS when no salt had been added. A phase separation was observed when 50 mM NaBr was added to 95 mol% SDeS/DDAB (the Krafft point was probably reached), and when 100 mM NaBr was added to the same system the sample remained opaque one week after mixing the sample and also after heating it to 40°C in a water bath. The curve for sample 95 mol% SDS/DDAB 1/8192 mM + 100 mM NaBr was oscillating implying possible defects in the vesicle membrane. The cryo-TEM images confirmed the light scattering results and additionally showed that at higher total concentrations agglomeration occurred, while whenever salt was added less vesicles seemed to appear, while both discs and broken vesicles were present suggesting that the disc structure is preferred over the spherical structure when salt is present. Also a vesicle inside another vesicle was discovered for the sample 0.95 SDS/DDAB 3.75 mM + 50 mM NaBr. The mole fraction of anionic surfactant in the aggregates (x) was calculated using a MATLAB code based on the Poisson-Boltzmann theory. The results from the calculations showed that a higher amount of SOS was needed for the system 0.95 SOS/CTAB than the amount of SDS and SDeS needed for the systems 0.95 SDS/DDAB and 0.95 SDeS/DDAB when forming aggregates, indicating that a shorter chain of the anion and the higher spontaneous curvature of the cation leads to a higher curvature. Also a larger amount of cation was needed when the tail was single than when it was double in order to form stable spherical structures. Finally, as the total concentration decreased the x value also decreased in all cases, thus the spontaneous curvature was decreased.

Background: Long-term cyclic tensile testing with equine palmar/plantar tendons have not yet been performed due to problems in fixing equine tendons securely and loading them cyclically. It is well established that the biomechanical response of tendons varies during cyclic loading over time. The aim of this study was to develop a clamping device that enables repetitive cyclic tensile testing of equine superficial digital flexor tendon for at least 60 loading cycles and for 5 min. Results: A novel cryo-clamp was developed and built. Healthy and collagenase-treated pony SDFTs were mounted in the custom-made cryo-clamp for the proximal tendon end and a special clamping device for the short pastern bone (os coronale). Simultaneously with tensile testing, we used a biplanar high-speed fluoroscopy system (FluoKin) to track tendon movement. The FluoKin system was additionally validated in precision measurements. During the cyclic tensile tests of the SDFTs, the average maximal force measured was 325 N and 953 N for a length variation of 2 and 4 % respectively. The resulting stress averaged 16 MPa and 48 MPa respectively, while the modulus of elasticity was 828 MPa and 1212 MPa respectively. Length variation of the metacarpal region was, on average, 4.87 % higher after incubation with collagenase. The precision of the FluoKin tracking was 0.0377 mm, defined as the standard deviation of pairwise intermarker distances embedded in rigid bodies. The systems accuracy was 0.0287 mm, which is the difference between the machined and mean measured distance. Conclusion: In this study, a good performing clamping technique for equine tendons under repetitive cyclic loading conditions is described. The presented cryo-clamps were tested up to 50 min duration and up to the machine maximal capacity of 10 kN. With the possibility of repetitive loading a stabilization of the time-force-curve and changes of hysteresis and creep became obvious after a dozen cycles, which underlines the necessity of repetitive cyclical testing. Furthermore, biplanar high-speed fluoroscopy seems an appropriate and highly precise measurement tool for analysis of tendon behaviour under repetitive load in equine SDFTs.

Tubulin post-translational modifications (tPTMs) are currently studied as vital, yet obscure, cytoskeletal regulators. Their regulatory function relies on the spatiotemporal control over the activity of multiple tubulin modifying enzymes that functionalize microtubules, enabling their differentiation. The cilium, one of the organelles with the richest tPTMs diversity, has been studied with determination for the last decades, allowing the interrogation of the molecular processes that give rise to its function. The inner structure of this thin organelle, the axoneme, comprises a microtubule scaffold periodically decorated with macromolecular complexes whose characterization has been achieved with pseudoatomic detail. The molecular distribution and mechanism of action of tPTMs in cilia remain elusive. Using a combination of immunolabelling and cryoelectron tomography we interrogated the molecular function two tPTMs in the axonemal context. We showed that tubulin polyglycylation spanned most of the microtubular surface and was required for axonemal dynein activity regulation and male fertility. Additionally, there was an enrichment of polyglutamylation on a single microtubule protofilament, forming a pattern complementary to that of polyglycylation, that was required for proper coupling of microtubule sliding and bending forces during ciliary beating.

Cryogenic electron microscopy (cryo-EM) is an evolving field allowing molecular visu- alizations with picometer resolutions. Images are acquired by shooting electrons through molecular samples and detecting the scattered electrons. From such data, 3D shapes of the molecules can be inversely reconstructed. Currently, describing and simulating the cryo-EM image formation is based either on naive transmittance models or complicated wave-function formalisms. In this thesis, we explore the possibility of simulating cryo-EM image formation via Monte Carlo electron tracing. We combine a delta-tracking algorithm with an elec- tron elastic differential cross-section function and Rutherford formulae to derive two Monte Carlo estimators. The derived models are implemented in a high-performance C++/CUDA environment and compared with other common models. Our particle-based simulated images show considerable similarity to the wave-based state-of-the-art multi- slice model. We also evaluate our models on class averages of real measurements. Both of our proposed models have significantly higher normalized cross-correlation scores with the measured class averages when compared to the most commonly used transmittance model. The thesis proves the viability of a particle-based Monte Carlo simulation of elec- tron microscope...

Pore forming toxins (PFTs) are a large group of proteins found mainly in bacteria with some exceptions found in animals. They bind and form pores in their target membranes and form pores, which leads to cell death. Among these are cholesterol-dependent cytolysins (CDC), which require the presence of cholesterol to bind target membranes. Mitilysin (Mly), a protein of interest in this project, belongs to the CDC group of pore forming toxins. It is produced by the bacterium Streptococcus mitis, a pathogen closely related to Streptococcus pneumoniae, found in human oral cavity, which causes several diseases such as Viridans Group Streptococcal (VGS) toxic shock syndrome and endocarditis. Mly is a homologue of the toxin Pneumolysin, which is produced by S. pneumoniae. However, the mechanism of pore formation is not well known. The purpose of this project is to understand the mechanism of CDC pore formation, focusing on the key amino acid residues that are responsible for transitioning from Mly pre-pore to pore state. The findings will aid in the design of inhibitors of pore formation as potential anti-bacterial drug candidates. The major goal of the project was to determine the 3-dimensional (3D) structure of assembled Mly pore. Mly is expressed in E.coli and purified by Ni-NTA affinity chromatography. Pore formation is confirmed by a hemolysis assay and negative stain-transmission electron microscopy. Mly pores are vitrified, analyzed and imaged in a cryo-electron microscope. 2D images were processed to generate a 3D density map. However, our Mly pore 3D map was incomplete due to lack of 2D projection angles resulting from preferred orientation of pore particles during sample preparation. To overcome this problem, we aim to use DNA origami, which requires His-tagged Mly. We were able to determine that His-tagged Mly retains its pore formation ability.

Visualization and image filtering are important parts of cryo-electron tomography analysis. ArtiaX, a plugin developed for UCSF ChimeraX, has been extended to improve the functionality of these two parts. For the visualization, a method of moving 3D surfaces to remove overlap between them has been developed and implemented. To accommodate this, a Monte Carlo approach using Poisson disc sampling for approximating volume of overlap between 3D surfaces is used, and a novel method for measuring overlap has been invented, called the Normal Projection Method, useful for measuring the depth of overlap between surfaces. For the image filtering, tomogram averaging and frequency filters have been added to the ArtiaX toolbox. / Visualisering och bildfiltrering är viktiga delar inom kryoelektrontomografianalys. ArtiaX, ett plugin utvecklat för UCSF ChimeraX, har utökats för att förbättra funktionaliteten inom dessa två områden. För visualiseringen har en metod för att flytta 3D ytor så att de inte överlappar utvecklats och implmenterats. För att skapa denna funktion används en Monte Carlo metod med Poisson disk sampling för att uppskatta volymen av 3D ytor, och normalprojektionsmetoden, en ny metod för att mäta överlappsdjup har skapts och implementerats. För att förbättra bildfiltreringen har tomogram-snittning och frekvensfilter lagts till bland verktygen i ArtiaX.

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.