The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.

L’Adénylate cyclase (CyaA), produite par B. pertussis, agent responsable de la coqueluche, est un des principaux facteurs de virulence de la bactérie. La toxine est une grande protéine multi-domaine qui est synthétisée comme un précurseur inactif, proCyaA. Ce précurseur est converti dans la forme active après une acylation spécifique. Après la sécrétion, la toxine envahir les cellules eucaryotes par un mécanisme unique qui implique la translocation de son domaine catalytique dans le cytosol des cellules eucaryotiques. Cette mécanisme est toujours pas clair et nombreuses questions restent ouvertes. Dans la présente étude, nous avons étudié les propriétés structurales et fonctionnelles des différentes espèces de (pro)CyaA en solution et inséré dans la membrane. Nous avons observé que le repliement de (pro)CyaA dans la forme monomérique dépend de la présence de calcium et de l'acylation post-traductionnelle. En outre, nous avons observé que la présence du calcium améliore fortement la stabilité de la protéine. De plus, nous avons identifié un segment hydrophobe dans CyaA, mais pas dans proCyaA, qui intervient dans les premières étapes du repliement de la protéine. L'analyse macroscopique a révélé que CyaA est plus stable et compacte par rapport à proCyaA. Nous avons aussi observé que les deux toxines sont capables de perméabiliser les membranes in vitro, mais que seulement la toxine monomérique et acyle est capable d'exercer des activités de membranes efficaces dans la cellule (hémolyse, translocation de AC et production de cAMP). Nous proposons que la toxine monomérique est la seul espèce compétent et fonctionnel. / Adenylate cyclase is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whopping cough. The toxin is a huge multi-domain protein synthesized as an inactive precursor, proCyaA, which is converted into the active form upon a specific acylation. Once secreted across the bacterial cell envelope, the toxin invades eukaryotic cells through a unique mechanism that involves the direct translocation of its catalytic domain inside the cytosol of the target cells. This mechanism is still not clear and many questions remain open. In the present study we investigated the structural and functional properties of various (pro)CyaA species in solution and upon membrane-insertion. We found that the (re)folding of CyaA into a monomeric form critically depend upon the presence of calcium and the post-translational acylation. We observed that calcium binding strongly improves the stability of the protein. Moreover we identified a hydrophobic segment in CyaA, but not in proCyaA, which is involved in the early stages of the refolding process. Macroscopic analysis showed that CyaA is more stable and compact as compared to proCyaA. We also observed that both toxins are able to permeabilize membranes in vitro, although only the monomeric and acylated toxin is able to exert efficient membrane activities in cellula (i.e., hemolysis, AC translocation and cAMP production). We propose that the monomeric species is the functional competent and active state and that the acyl chains play not only a structural role but are also essential for the functional activities of the toxin.

SWEPOS nätverks-RTK (Real Time Kinematic) är en nationell korrektionstjänst för GNSS som är flitigt använd i Sverige. En begränsning är dock att den kräver internetuppkoppling och att referensnätet endast täcker Sverige. Trimble har en global korrektionstjänst som heter Centerpoint RTX (Real Time eXtended) som inte har dessa begränsningar. Länge hade Centerpoint RTX en förhållandevis lång konvergenstid för att vara ett attraktivt alternativ vid detaljmätning, men i takt med att fler satellitsystem implementerats och referensnätets omfattning utökats har konvergenstiden förkortats. Syftet med det här arbetet är att utvärdera Centerpoint RTX för detaljmätning med avseende på lägesosäkerhet och tidsåtgång. Detta görs som en jämförelse med SWEPOS nätverks-RTK. Tre mätmiljöer valdes ut och i dessa miljöer utfördes två tester för att bestämma lägesosäkerhet och tidsåtgång. Lägesosäkerheten undersöktes genom att montera två mottagare tätt intill varandra på en speciell distansarm (eng. Lever arm). Mottagarna loggade först råa observationer i 2 timmar vilka användes för att efterberäkna referenskoordinater. Sedan kopplades mottagarna upp på respektive korrektionstjänst utan att förflyttas från sina positioner och loggade därefter 1 position i sekunden i 4 timmar. För att mäta tidsåtgången användes en mottagare som växelvis kopplades upp mot SWEPOS nätverks-RTK och Centerpoint RTX och tiden det tog att uppnå god kvalitet på mätningarna mättes. Resultaten visar att Centerpoint RTX uppnår en kvadratisk medelavvikelse (RMS) på ca 1–1,5 cm i plan i lätta och normala mätmiljöer och drygt 2 cm i plan i den svåra mätmiljön. I höjd är RMS ca 1,5 cm i den lätta mätmiljön och 3,5 cm och 4,5 cm i den normala respektive svåra mätmiljön. Centerpoint RTX påverkas mer av mätmiljön än SWEPOS nätverks-RTK och uppvisar tendenser till systematiska avvikelser i nordkoordinaten och i höjdled. Tidsåtgången är runt 30 s i lättare mätmiljöer, i den svåra mätmiljön ärtidsåtgången 83 s för Centerpoint RTX medan SWEPOS nätverks-RTK klarar 30 s i alla miljöer. Medeltalsbildning förbättrar mätningarna med Centerpoint RTX i den svåra miljön, men i de andra miljöerna är effekten liten. Centerpoint RTX kan användas för detaljmätning i god mätmiljö där kraven på lägesosäkerhet (1 sigma) är runt 2 cm i plan och 5 cm i höjd, Vid högre krav bör den systematiska avvikelsen kontrolleras mot kända punkter och eventuellt se om den kan modelleras. I svåra miljöer bör medeltalsbildning över längre tider användas för att klara ett RMS på 2 cm i plan. / SWEPOS network-RTK (Real Time Kinematic) is a national service for GNSS corrections in Sweden. It is limited by the requirement to have an internet connection and the coverage area Sweden. Trimble has a global correction service in Centerpoint RTX (Real Time eXtended) which is not limited in that way. Until a few years ago, Centerpoint RTX had too long convergence times for being attractive in land surveying, but recently these convergence times have been significantly shortened, which makes Centerpoint RTX an attractive alternative to SWEPOS network-RTK. The aim of this bachelor thesis is to assess Centerpoint RTX for land surveying applications regarding measurement uncertainty and time required. This assessment is done in a comparison with SWEPOS network-RTK. Two tests were made in three environments, one to measure uncertainty and one to measure time. The uncertainty was measured using two receivers mounted with a short distance between them on a lever arm. The receivers first logged raw observations for 2 hours which were post-processed later to compute reference coordinates. Without moving the receivers, they were connected to their correction service and started to measure positions with 1 Hz frequency for 4 hours. To measure time one receiver was used that was alternately connected to SWEPOS network-RTK and to Centerpoint RTX and the time needed to obtain high quality observations was clocked. It is found that Centerpoint RTX reaches a Root Mean Square Error (RMS) of 1–1,5 cm horizontally in the easy and normal environments and a little higherthan 2 cm in the bad environment. Vertically, the RMS is around 1,5 cm in the easy environment and 3,5 cm and 4,5 cm in the normal and bad environments respectively. Centerpoint RTX seems to be affected more by the environment and shows tendencies to systematic errors in the north component and height. The required time was around 30 s in the easier environments, but in the bad one the time was 83 s for Centerpoint RTX whilst SWEPOS network-RTK required around 30 s in all environments. Occupation time can strengthen the positions of Centerpoint RTX, especially in bad environments but this effect is small in other environments. Centerpoint RTX can be used for applications requiring standard uncertainties about 2 cm horizontal and 5 cm vertical. If there are higher requirements, a recommendation would be to check the service for systematic errors on known points and eventually model these. In bad environments longer occupation time should be used to achieve 2 cm horizontal RMS.

RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...

Video conferencing applications has reached worldwide usage in recent years by the help of the improvements in network infrastructures for public services. Media data covers a significant ratio of data traffic over IP networks. However, it is challenging to ensure a decent quality of service (QoS) on public networks in terms of video and audio quality. The main factor that may cause degradation in media playback quality is packet losses. There are various techniques available to conceal packet losses in lossy channels. According to the application needs and channel characteristics such as loss patterns and round trip times, retransmission or forward error correction techniques may be applied at application level. These two techniques have different challenges which lead to tradeoffs between them, thus one might be chosen over the others. In this thesis work, retransmission’s worst case performances under considered packet loss patterns and various round trip times are compared to performances of forward error correction schemes. In addition, implementation details with respect to the relevant RFCs are provided as an example to give a better judgement on the obtained results. Results obtained under the packet loss patterns that are generated with a simple Gilbert-Elliot 2-state model shows that forward error correction techniques are a reasonable choice of error concealing in the real-time transport protocol (RTP) stack where round trip time in the channel is greater than 200 ms. In addition, bandwidth overhead revealed by forward error correction stays higher than retransmission’s bandwidth overhead in all sample runs. In cases where round trip times are high, then the choice of forward error correction scheme is bound to the packet loss pattern. In the results section, it is obtained that ReedSolomon performs well in terms of residual packet losses, which are the packets not being recovered, and bandwidth overhead when losses occur in long bursts.

This thesis deals with the removal of noise in speech recordings that have been recorded in an MRI environment. For this purpose, the Nvidia RTX Voice technology, the VST plug-in module Noisereduce and a self-designed method of subtractive de-noising of recordings are used. A program with a simple graphical interface in Python is implemented within the work to retrieve the recordings and then de-noise them using the proposed methods. The work includes measurements in a magnetic resonance environment with two microphones. The quality of the processed recordings is tested within the program using the STOI (Short-Time Objective Intelligibility Measure) method as well as the subjective analysis method within listening tests.

This thesis deals with photorealistic rendering and real-time global illumination. Thesis contains overview of algorithms used for real-time global illumination of which the Dynamic Diffuse Global Illumination with Ray-Traced Irradiance Fields was implemented. This algorithm uses hardware accelerated ray tracing to compute global illumination in a scene. Hardware ray tracing requires use of new generation of graphics API from which Vulkan was choosen for this thesis.

FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...

Les lymphomes diffus à grandes cellules B (DLBCL) sont la forme de lymphomes non–Hodgkiniens agressifs la plus fréquente chez l’adulte, et sont très hétérogènes à la fois sur le plan biologique et clinique. Bien que plus de la moitié des patients peuvent être guéris avec le traitement standard R-CHOP (combinant la polychimiothérapie CHOP aux anti-CD20 comme le rituximab) 30 à 40 % des patients échappent ou sont réfractaires au traitement, déterminant des morbidités et mortalités importantes liées au nombre limité d’alternatives thérapeutiques. L’objectif des travaux de cette thèse était centré sur les mécanismes de résistance thérapeutique dans ces lymphomes, et plus particulièrement les résistances au rituximab. Dans une première partie de la thèse, nous avons étudié le rôle de facteurs endogènes neuropeptidiques, les neurotrophines (NTs), et l’implication de leur signalisation dans la survie des cellules tumorales et leur sensibilité à l’apoptose induite par le rituximab. Nous avons montré dans un premier temps que les cellules B tumorales des patients présentaient des taux parfois élevés de neurotrophines (NGF, BDNF) et de leurs récepteurs de haute (Trk) et de basse affinité p75NTR. Puis les résultats obtenus in vitro sur des lignées cellulaires humaines de DLBCL et in vivo (xénogreffes tumorales) ont mis en évidence l’existence d’un axe de survie BDNF/TrkB/p75NTR pouvant interférer avec l’efficacité de l’immunothérapie. Cet axe contribue à la survie des cellules tumorales et pourrait aussi participer à la résistance thérapeutique aux anti-CD20 en modulant l’expression du CD20 à la surface des exosomes. En effet ces microvésicules, sécrétées en grande quantité par les cellules B tumorales, expriment le CD20 à leur surface et seraient à ce titre impliquées dans l’échappement thérapeutique en réalisant des « récepteurs –leurres » vis-à-vis des anticorps thérapeutiques. Dans une 2e partie de cette thèse, nous avons évalué dans les DLBCL le rôle potentiel de nouvelles cibles émergentes en oncologie, les prohibitines (PHBs) et le facteur d’initiation de la traduction eIF4A. Pour cela, nous avons utilisé un ligand de ces acteurs cellulaires de la famille des flavaglines, le FL3. Nous avons montré que le FL3 présente un effet apoptotique très important in vitro sur les lignées cellulaires de DLBCL et in vivo sur les tumeurs induites chez la souris. Nos travaux ont permis d’en préciser les mécanismes moléculaires, mettant en évidence le rôle des PHBs en lien notamment avec l’activation d’Erk1/2, et la formation et l’activité du complexe eIF4F dans la survie des cellules de DLBCL. Les données préliminaires obtenues à partir des biopsies de patients montrent, de plus, que la forte expression de PHB1 pourrait avoir une valeur pronostique dans ces lymphomes. L’ensemble de nos travaux ont permis de mettre en évidence de nouvelles voies de survie et d’échappement thérapeutique dans les DLBCL, qui pourraient permettre d’identifier aussi de nouveaux marqueurs biologiques à valeur diagnostique et/ou pronostique pour le choix de thérapies ciblées. / Diffuse large B cell Lymphomas (DLBCL) are the most aggressive and heterogeneous biological and clinical form of non-Hodgkin lymphomas in adults. Although more than 50% of patients can be cured with standard therapy R-CHOP (combining the CHOP chemotherapy to the anti-CD20 as rituximab) 30 to 40% of patients exhibit primary refractory disease or relapse after initial response to therapy, determining morbidities and significant mortality related to the limited number of treatment options. The aim of this thesis was focussed on therapeutic resistances of these lymphomas, notably those of rituximab. In the first part of this thesis, we have evaluated the role of endogenous factor signaling, the neurotrophins (NTs), in DLBCL cell survival and sensitivity to the cytotoxicity of rituximab. We showed first that a high expression of neurotrophines (NGF, BDNF) and their high (Trk) and low (p75NTR) affinity receptors was often found in tumor B cells of DLBCL patients. Results obtained in vitro, on human cell lines of DLBCL, but also in vivo (xenografts) showed evidence of a survival BDNF/TrkB/p75NTR axis that can interfere with the efficacy of immunotherapy. This axis promotes survival of tumor cells and may also participate in rituximab resistance in regulating CD20 expression at the surface of exosomes. Indeed, these microvesicles, secreted in large amounts by the tumoral B cells, express the CD20 and would be involved in the therapeutic escape acting as decoy receptors upon rituximab exposure. In the second part of the thesis, we evaluated in DLBCL the potential role of new oncogenic targets, PHBs proteins and the initiation factor of translation eIF4A. To this end, we used one of their ligands, a synthetic flavagline named FL3. We showed that FL3 determines a strong apoptosis in vitro on DLBCL cell lines and in vivo on tumors induced in mice (xenografts). Our works have clarified the molecular mechanisms, demonstrating involvement of PHBs, in correlation with ERK1/2 activation, and eIF4F complex formation and activity. Preliminary data obtained in patient biopsies showed a high expression of PHB1 in tumor B cells that may be decisive for cell survival and patient outcome lymphomas.Overall, present results show evidence of new survival and rituximab escape mechanisms in DLBCL, that should allow to identify new diagnostic and prognostic biomarkers for alternative therapeutic options.

Legionella pneumophila est responsable d'une forme de pneumonie, la legionellose ou de maladie du légionnaire. Entre 2012 et 2015, les cas annuels ont grimpé de 5848 à 7069 en Europe, la France, l’Allemagne, l’Italie et l’Espagne correspondant à 69% du total. De façon inquiétante, la mortalité était de 8,2% faisant de cette maladie un réel enjeu de santé publique. Un facteur de virulence produit par cette bactérie est la protéine RtxA (~700 kDa) de la famille des protéines RTX (Repeats in ToXin) sécrétée via un système de sécrétion de type 1. Dans ce travail, in vitro, la protéase périplasmique LapG clive la partie N-terminale de RtxA au sein d'un motif di-alanine (position 108-109). La construction de mutants déficients dans l’expression de LapG et LapD a révélé une localisation de RtxA sous le contrôle de ces deux protéines, mécanisme semblable au modèle LapA décrit chez P. fluorescens. Un mutant lapG maintient RtxA à la surface de cellules, à l’opposé d’un mutant ?lapD. Nous avons identifié des systèmes homologues T1SS/LapDG dans de nombreuses espèces Legionella ainsi que d’autres gammaproteobactéries. Concernant la virulence de L. pneumophila, les mutants déficients pour le T1SS (lssBD/tolC) étaient plus altérés dans leur virulence que des mutants du système LapDG. Nous avons également montré, grâce à des expériences de compétition, que L. pneumophila semble cibler les cellules hôtes via la protéine RtxA. L’utilisation d’anticorps spécifiques anti-RtxA nous a permis de détecter RtxA à la surface des cellules hôtes, mais aussi de réduire de la virulence de L. pneumophila, suggérant un rôle important de RtxA lors du processus d’infection, bien que non limitant / Legionella pneumophila is the causative agent of a form of pneumonia called legionellosis or Legionnaires’ disease. Between 2012 and 2015, the reported European cases of legionellosis increased from 5,848 to 7,069 cases per year where France, Germany, Italy and Spain accounted for 69% of the reported cases. Worryingly, the case fatality of incidents was 8.2% making this disease a considerable health concern. One virulence factor produced by this bacterium is a large protein (~700 kDa) belonging to the RTX (Repeats in ToXin) family called RtxA secreted by the type 1 secretion system. The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (a.a. 108-109). We also show using lapG and lapD mutant strains, that RtxA release is controlled by these two proteins similar to Pseudomonas fluorescenes LapA. We observed that a strain lacking LapG protease maintains RtxA on the cell surface, while a strain lacking LapD does not exhibit cell surface RtxA. Interestingly, we identified the presence of homologous potential T1SS/LapDG systems in many Legionella species and other Gammaproteobacteria. Regarding L. pneumophila virulence, our work showed that mutants for L. pneumophila T1SS (lssBD/tolC) were more disruptive to its virulence than lapG/lapD mutants. We also hypothesize, by challenging infection, that L. pneumophila might be actively targeting its host via RtxA. Additionally, by observing rtxA mutants as well as detecting RtxA on host surface briefly after inoculation and attenuating virulence by using anti RtxA antibodies, we assume an important but not limiting role for this protein in the infection process