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Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.

Le formaldéhyde (HCHO) est un polluant majeur de l’air intérieur. L’objectif de cette thèse est de réaliser les avancées scientifiques et technologiques nécessaires à l’obtention d’une méthode analytique basée sur un dispositif microfluidique de mesure du formaldéhyde dans l’air associant précision, sélectivité, rapidité d’analyse avec pour objectif majeur une autonomie suffisante sur de longues durées, typiquement un mois. Le principe de la méthode reposait initialement sur trois étapes clés, à savoir le piégeage du formaldéhyde gazeux en solution, la réaction du formaldéhyde avec un agent dérivatif, puis la détection du produit de dérivation par colorimétrie ou fluorimétrie. La méthode a finalement évolué vers seulement deux étapes distinctes grâce à l’utilisation d’un dispositif microfluidique innovant dans lequel le piégeage et la réaction ont lieu simultanément. L’étude des performances analytiques du dispositif a permis de valider la méthode développée pendant cette thèse. / Formaldehyde (HCHO) is a major pollutant in indoor air. The objective of this work is to realize the scientific and technological advances required to obtain an analytical method based on a microfluidic device to measure air formaldehyde combining precision, selectivity, analysis speed with for major objective a sufficient autonomy on a long time, typically one month. The principle of the method was initially based on three key steps, the gaseous formaldehyde uptake in solution, the formaldehyde derivatization reaction, then the detection of reaction product by colorimetry or fluorimetry. The method has finally advanced toward only two definite steps thanks to the use of an innovative microfluidic device in which uptake and reaction take place simultaneously. The study of analytical performances of the device allows to validate the method developedduring this work.

Fluorescence imaging has been increasingly relied upon as the method of choice for many biological and medical applications. As demands for more sensitive and higher resolution imaging are ever-increasing, it is critical that photostable, and robust fluorophores capable of delivering high emission rates are available. Fluorescent silver nanoclusters offer an attractive compromise between the photostability and brightness of quantum dots and the compact versatility of organic chromophores. They have been shown to be superior in many roles, including as single molecule fluorophores and bulk multiphoton biological staining agents. The two-photon absorption cross sections are several orders of magnitude larger than commercially-available dyes, and they have demonstrated superior photostability under high intensity irradiation. In addition to the endogenous effects of the cluster, its small size of only a few atoms renders it highly susceptible to surface and environmental effects, which manifests, for example, in the observed photoinduced charge transfer between the silver cluster and oligonucleotide. This state has been shown to be highly advantageous in imaging applications, as control of this state enables better control over the time-averaged emission rate of the molecule. The mechanism of charge transfer, and the possible means by which this state can be controlled will be also be investigated in this work.

Thymidylate kinase (TMK) is a key enzyme for DNA synthesis. It occurs at the junction of the de novo and salvage pathways for the synthesis of deoxythymidine triphosphate (dTTP). Its inhibition affects cell viability, thereby making it an important target for the development of anticancer, antibacterial and antiparasitic drugs. This thesis describes the analyses of the sequence, structure and dynamics of thymidylate kinase to obtain insights into its function. Two thermophilic variants of the enzyme were chosen for our studies. The studies provide valuable insights about the active site residues and the mechanism of catalysis, which have implications in protein engineering and design of specific inhibitors. Following is a chapter-wise description of the overall layout of the thesis. Chapter 1 | Introduction: This chapter provides a brief survey of the literature on TMKs and the scope of the work presented in the thesis. TMK belongs to the nucleoside monophosphate kinase (NMPK) family of enzymes, which includes adenylate kinase (AMK), guanylate kinase (GMK), uridylate kinase (UMK) and cytidylate kinase (CMK). The NMPK family of enzymes is associated with the reversible transfer of the terminal phosphoryl group from a nucleoside triphosphate (NTP) (usually adenosine triphosphate, i.e., ATP) to a nucleoside monophosphate (NMP). The identity of the NMP substrate varies among different enzymes. NMPKs share a common Rossmann fold and are comprised of a conserved P-loop, Lid region, CORE and NMP domains. The enzymes in the NMPK family also contain structurally similar active site architecture. Besides the three signature motifs, there are other conserved residues at the active site of TMK which are involved in interactions with the substrates ATP and dTMP. Despite the overall similarity, TMKs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the residues responsible for these differences have not been studied. This thesis describes a comprehensive analysis of the sequence space of TMKs to detect the residues involved in such diversity. Subsequently, TMKs from a thermophilic archaeon (Sulfolobus tokodaii) and a hyperthermophilic bacterium (Aquifex aeolicus) were chosen for biochemical characterization and structural studies. Of these, the Sulfolobus tokodaii TMK (StTMK) has low sequence identity to the other TMKs of known three dimensional (3D) structures. Crystal structure analyses depicted the presence of some novel structural features and provided insights into the role of a conserved Arginine residue in function, which was verified through computational studies and mutagenesis experiments. Finally, the study on Aquifex aeolicus TMK resulted in multiple crystal structures of the apo form and different holo forms. These helped us to understand the mechanistic details of TMK-mediated catalysis, namely, the order of substrate binding and the reaction mechanism for phosphate transfer. Chapter 2 | Materials and Methods: This chapter provides a brief description of the procedures used to carry out the thesis work. The protein samples were purified to a high degree using column chromatography, and the purity was assessed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. Circular dichroism (CD) spectroscopy was employed to assess if the purified protein was well-folded. The pure and properly folded protein samples were used in further experiments. Differential scanning fluorimetry (DSF) was performed to determine the melting temperature of the thermophilic protein. MicroScale Thermophoresis (MST) and Surface Plasmon Resonance (SPR) were carried out to detect protein-substrate interactions. The protein samples were crystallized using the hanging drop vapour diffusion and microbatch under-oil techniques using commercially available crystallization screens, and the conditions which gave crystals were further optimized. Diffraction data, collected at either the home source or the synchrotron, were processed and scaled. Subsequently, phase information was obtained using the molecular replacement (MR) calculations. The MR solution was refined till convergence and its geometry was validated using different softwares. Finally, molecular dynamics (MD) simulations were performed to study the functionally important motions in the protein. Chapter 3 Insights into substrate specificity and oligomerization mode of Thymidylate Kinases from sequence evolution and conformational dynamics: Thymidylate kinase homologs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on thymidylate kinase from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our sequence analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs, which was also indicated in MD simulations on the protein 3D structure. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode and substrate recognition in thymidylate kinases and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors. Chapter 4 | Biochemical and Structural characterization of a thermophilic variant of thymidylate kinase: This chapter reports the biochemical characterization and crystal structure determination of thymidylate kinase from the hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. The different structures had resolution ranging between 1.60 Å and 2.40 Å. StTMK is a thermostable enzyme with a melting temperature of 85.3 °C, as observed from thermal unfolding studies. The protein exists as a dimer in solution. A coupled enzyme assay, performed using thermo-stable lactate dehydrogenase (TtLdh) and pyruvate kinase (TtPk) from Thermus thermophilus, showed that StTMK has optimum activity at 80 °C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. A novel insertion (9 residues long) is observed in the C-terminal stretch of the Lid region. However, it is relatively rigid, which may be attributed to the presence of two proline residues and a hydrogen bond with an arginine residue in the α4/α5 loop. The C-terminus of the α2 helix points away from the Lid region in StTMK to avoid steric clashes with the Lid insertion. The main chain dihedral angles of the conserved Arg in the DRX motif are in the disallowed region of the Ramachandran plot in all holo TMK structures, wherein it forms several conserved hydrogen bonds with residues in the P-loop, α4 helix and α7 helix, as well as with the phosphate groups of both the substrates. A similar feature is observed in some of the StTMK structures. However, torsion angles in the allowed region of the Ramachandran plot are observed in one chain each in two of the apo structures. Further, conformational rearrangements of this Arg and its neighboring residues at the binding site of the second substrate are observed. The functional implication of this variation is described in the next chapter (chapter 5). Chapter 5 | Role of a conserved active site Arginine residue in Thymidylate kinase: Analysis of the structures of StTMK revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Conformational heterogeneity of Arg can also be observed in some structures of Staphylococcus aureus and human TMK. However, the functional implication of this feature has not been probed before. The rearrangements of Arg93 are accompanied by related changes in the conformations of its neighbouring residues at the active site. This leads to three distinct conformational states in the dTMP-binding site, namely ‘Arg in’, ‘Arg intermediate’ and ‘Arg out’. Only the ‘Arg in’ state was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. This is hindered in the ‘Arg out’ and ‘Arg intermediate’ states. MD simulations showed that the torsion angles of the DRX Arg can sample between allowed and disallowed values in the apo-protein, with a preference for the catalytically suitable disallowed conformation in the holo-protein. Computational alanine scanning and MM/PBSA binding energy calculation further revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Our work provides the first experimental evidence for the functional importance of Arg93 and gives insight into its regulatory role in the catalytically competent placement of dTMP. Our study also has implications for the development of potent inhibitors to lock the enzyme in the catalytically non-productive state. Chapter 6 | Characterizing active site dynamics from structural studies on the Intermediates along the reaction coordinate of a hyperthermophilic Thymidylate Kinase: TMK belongs to the family of nucleoside monophosphate kinases (NMPKs), several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild type protein and mutants. Further, the enzyme was found to exhibit broad specificity towards phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes. Finally, the implications of the work described in this thesis and its future applications have been discussed in the section titled ‘Future prospects’. The work described in chapters 3 – 6 have been published in peer reviewed journals. Additionally, the author was involved in several collaborative projects which also resulted in publications (reprints attached in appendix).

Le microphytobenthos (MPB) des latitudes tempérées est dominé par les diatomées. Deux grands groupes se distinguent, les épipéliques (mobiles) des sédiments vaseux, et les épipsammiques (fixées) des sédiments sablo-vaseux. Afin de mieux comprendre la production des vasières, le MPB a été étudié par des approches du niveau physiologique au niveau écologique. Dans un premier temps, l’étude s’est focalisée sur des expérimentations en laboratoire. La réponse des différents groupes à la lumière a montré que la forme de vie et la mobilité sont en lien étroit avec la capacité de photoprotection physiologique. Ainsi, les diatomées non-mobiles présentent une meilleure photoprotection physiologique que les diatomées mobiles qui peuvent fuir les excès de lumière. Dans une deuxième partie, le travail s’est effectué sur des échantillons ramenés en laboratoire. Des profils de migrations ont été réalisés par mesure continue de la fluorescence. Il a été établi que le MPB présente un rythme de migration interne pouvant être modulé par la lumière. De plus la qualité de la lumière modifie les profils de migration. Il est communément admis que les phases de division cellulaire se dérouleraient en profondeur. La cytométrie en flux permet de vérifier cette hypothèse. Finalement les mesures effectuées en laboratoire ont été comparées à des mesures effectuées directement sur le terrain à l’échelle de la communauté. Il a ainsi pu être vérifié que la photoprotection sous lumière fluctuante est fonction de la population. Pour les populations épipéliques, la photoprotection physiologique ne varie pas au cours des fluctuations lumineuses, laissant supposer que la migration module ces fluctuations. Les populations épipsammiques, quant à elles, modifient leur réponse physiologique en fonction des fluctuations lumineuses. / Microphytobentos (MPB) from temperate latitude is mainly composed of diatoms. Those microorganisms can be separated in two groups: the epipelic one from muddy sediments (composed of mobile diatoms) and the epipsammic one from sandy-muddy sediments (composed of diatoms living attached to their substrate). In order to investigate mudflats’ primary production, the MPB compartment was studied through diverse approaches from the physiological level to the ecological one. In the first place, laboratory experiments (in vitro experiments), focusing on light reaction of epipelic and epipsammic diatoms, showed that their life form and their mobility were strongly connected to their physiological photoprotection ability. Thereby, the motionless diatoms were characterized by higher physiological photoprotection abilities than the mobile ones, which could avoid excess of light. In the second place, the fluorescence of collected samples (in vivo experiments) was measured to acquire diatoms’ migration profiles. The results pointed out an internal and light-regulated migration pattern of the MPB and furthermore highlighted the effect of light quality on migration profiles. Besides, the commonly accepted hypothesis of deep cell division phases was tested and confirmed through flow cytometry experiments. Eventually, laboratory measurements were compared to in situ ones realized at the scale of the whole community. These comparisons revealed that diatoms photoprotection in fluctuating light depended on the targeted populations. Epipelic organisms were indeed characterized by an unvarying photoprotection, diatoms migration regulating alone the effect of light fluctuations. On the contrary, motionless epipsammic populations required a light-regulated photoprotection.

This thesis was focused on a determination of nadroparin using sequential injection analysis and flow injection analysis with fluorimetric and spectrophotometric detection. The principle of determination was based on the interaction of nadroparin with phenothiazine dye. Methylene blue was used as phenothiazine dye. The determination was performed on a laboratory made FIA instrument and on the commercial SIA instrument. Optimal conditions for SIA were concentration of dye 6 ∙ 10-5 mol dm-3 , injected volume 100 µl, flowrate 1,5 ml min-1 , for FIA: concentration of dye 3 ∙10-5 mol dm-3 flowrate 2 ml ∙ min-1 , injected volume= 100 µl, for fluorimetric detection was sensivity of the detector 1000 V, Emission was measured by 2 nm and excitation wavelenght 621 nm. For spectrophotometric detection, absorbance was detected at the absorption maximum wavelength. Repeatability ranged from 2.01 to 2.85%. The limit of detection for FIA was 0.05-0.09 IU ml-1 , for SIA were limits of detection in range 0,25 - 1,63 IU ml-1 , limits of quantification in range 0,83 - 5,44 and linear range was from 0,5 - 20 IU ml-1 . The limits of detection, limits of determination and the linear range could be corrected for the SIA by the injected volume of phenothiazine dye.

The submitted thesis deals within the sensitive fluorimetric determination of scandium with a new reagent 8-Hydroxyquinoline-5-sulphonic acid. All important factors such as time, pH, reagent concentration, buffers, surfactants and selected interferents have been studied with respect to the selectivity, sensitivity, precision and detection limit on the determination by using classic fluorescence spectra and their first derivation. This reagent was also used for the determination of scandium in real waters. The new reagent was shortly compared to morin and lumogallion when used for scandium.

Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method. An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS. 23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.

Les levures Candida sont des pathogènes opportunistes responsables d’infections graves chez les patients immunodéprimés. Au cours de ce travail, nous avons développé un modèle cellulaire in vitro pour la caractérisation multiparamétrique des phénotypes d’interaction entre les levures Candida et les macrophages et les neutrophiles, principaux effecteurs de la défense anti-Candida. Il repose sur l’utilisation de marqueurs fluorescents pour le suivi quantitatif de l’interaction en cytométrie en flux et en fluorimétrie. Ce modèle a été validé par la comparaison de l’interaction de trois espèces de levures, C. albicans, C. glabrata et C. lusitaniae, avec des macrophages murins et des neutrophiles humains. Deux stratégies principales de survie des levures à la phagocytose ont été mises en évidence : par la résistance à la phagolyse et la multiplication des levures à l’intérieur des phagocytes jusqu’à leur éclatement, ou par l’évitement de la phagocytose et la multiplication des levures à l’extérieur des phagocytes. L’interprétation des données quantitatives a été confirmée par microscopie à fluorescence et vidéo-microscopie. Afin de mieux comprendre les interactions Candida-phagocytes, nous avons mis au point des outils pour l’analyse fonctionnelle de gènes chez C. lusitaniae. Une stratégie de PCR chevauchante a été développée pour l’obtention de mutants nuls de C. lusitaniae, sans étape de clonage. C’est ainsi que le gène OLE2, codant une Δ9 désaturase d’acides gras potentiellement impliquée dans la biosynthèse de la prostaglandine PGE2, a été invalidé. Le mutant ole2Δ présentait de très nets défauts de filamentation et de reproduction sexuée. Par rapport à une souche sauvage, le mutant ole2∆ était massivement phagocyté par les macrophages, et la survie des phagocytes était plus importante, ce qui suggère un rôle important des lipides insaturés et des oxylipides dans la signalisation cellulaire au cours de l’interaction Candida-phagocytes. Dans la dernière partie de notre travail, nous avons construit une banque de 10 000 mutants de C. lusitaniae par l’intégration aléatoire d’un marqueur dans le génome. Le criblage de cette banque à travers notre modèle cellulaire d’interaction permettra d’identifier de nouveaux gènes impliqués dans l’interaction avec les phagocytes afin de mieux comprendre la physiopathologie des candidoses et de trouver de nouvelles pistes thérapeutiques. / Candida species are opportunistic pathogens causing severe infectious diseases in immunocompromised patients. In this work, we developed a tool for a multi-parameter characterization of the cell interactions between the yeasts Candida and both macrophages and neutrophils, which constitute the main defense against candidiasis. It relies on the labelling of each population with specific fluorescent markers, and on the use of fluorimetry and flow cytometry to assess interactions. The tool has been validated by comparing the interactions of three yeast species C. albicans, C. glabrata and C. lusitaniae, with murine macrophages and human neutrophils. We found that yeasts use two main ways for escaping phagocytosis, which has been confirmed using video-microscopy: either (1) by surviving to phagolysis and dividing into the phagosome until phagocytes burst, or (2) by avoiding phagocytosis and dividing outside phagocytes. In order to better understand the cellular and molecular mechanisms involved in Candida-phagocytes interactions, we developed new molecular tools for the functional analysis of genes in C. lusitaniae, notably a two-step cloning-free PCR-based method for the deletion of genes. This method was successfully used for the deletion of OLE2, a gene encoding a Δ9-desaturase of fatty acids, possibly implicated in prostaglandin PGE2 biosynthesis. The ole2Δ mutant exhibited strong defects in both pseudofilamention and sexual mating. During macrophages infection, ole2Δ yeast cells were massively internalized and triggered less phagocytes cell death than the wild type strain, suggesting that unsaturated fatty acids and/or oxylipids could play a role during interaction with phagocytes. Lastly, a bank of 10,000 mutants was constructed in C. lusitaniae by the random integration of a genetic marker in the genome. The screening of this bank through our tool to analyse cellular interactions will be undertaken to gain insights into understanding of the early stages of the infectious process.