Dissertations / Theses on the topic 'Criblage ciblé'
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Caria, Giovanni. "Développement d'une stratégie de caractérisation de l'état de contamination organique des sols par criblage en masse ciblé et non ciblé." Electronic Thesis or Diss., Université de Lille (2022-....), 2022. http://www.theses.fr/2022ULILR060.
The environmental pollution by multiple organic compounds linked to human activities is a recurring problem. Environmental compartments are subject to continuous monitoring such as surface and human supply waters and outdoor and indoor air. On the other hand, agricultural soils are only controlled in the case of the spreading of organic amendments, which could contain polyaromatic hydrocarbons and polychlorobiphenyls. Research work has been carried out for several years to assess the fate of organic pollutants in agricultural soils using more efficient and sensitive analysis methods increases to the development of quadrupole mass spectrometry coupled with gas or liquid chromatography. Nevertheless, many methods are used only to a selection of a few dozen organic molecules. In addition, the possible implementation of monitoring of organic pollutants in environmental matrices requires making choices and does not allow having a very broad knowledge about all the organic pollutants.The objective of this work is to propose a methodological strategy in order to approach the organic composition of soils by target and non-target screening analysis. The methodological development, carried out in non-target analysis, can contribute to the reflection of monitoring the quality of soils. Non-target analysis can make it possible to direct the target analysis towards a selection of organic compounds of interest and to determine environmental markers.The thesis report includes the development of target analysis methods in soils on a selection of pesticides, drugs and hormones extracted by pressurized-liquid extraction (PLE) and analysed by liquid chromatography coupled with a high-resolution time-of-flight quadrupole mass spectrometer (LC-QTOF-MS). The target analysis method has been validated for pesticides in agricultural soils, and applied to a selection of forty French agricultural soils from Centre-Val de Loire region. Non-target analyses were carried out too for these 40 soils by LC-QTOF-MS after two different PLE extractions, one with acetonitrile and the other with ultrapure water. Each soil extract was injected sequentially in positive, then negative ionization according to two mass settings, one in Full scan MS and bbCID MS (suspect screening) and the other one in Full scan MS and auto MS/MS (non-target screening or NTS). The final dataset is then very substantial with 8 different data for each soil sample, of which 4 used in suspect mode and 4 in NTS mode. 825 organic compounds were detected and identified by suspect screening analysis using the TargetScreener database backed by the LC-QTOF-MS. A classification of these compounds according to the value of identification quality criteria could be established and allowed a prioritization of the study of a few dozen compounds of interest. The non-target screening analysis was carried out for the « acetonitrile » soil extracts ionized by positive and negative modes using the Metaboscape chemometric software and by querying multiple databases independent of the analysis tool. The identification could be confirmed or reinforced in NTS mode for organic compounds already identified in suspect mode.The distribution of the 40 soils in groups of homogeneous agricultural use made it possible to highlight the presence of organic compounds exclusively in one group, thus constituting a sample group marker. The work of the thesis opens up many perspectives for the exploitation of non-target analysis data obtained for the 40 French agricultural soils, studies on the quality of French soils and pharmacovigilance of representative soils of the French national territory
Cortejade, Aurélie. "Approches et outils pour l’évaluation de l’Exposome : du dosage de contaminants vers le screening non ciblé pour la caractérisation des expositions humaines environnementales." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10219/document.
These research works highlight the development of analytical methods, based on mass spectrometry, to assess the Exposome according to different strategies. A selective multiresidue method for the analysis of plastic additives and their degradation products that may be released by plastic packaging in food and beverages and thus ingested by man was developed. This method consists of a Stir Bar Sorptive Extraction with bars covered by polydimethylsiloxane derivatives, followed by an analysis by liquid chromatography coupled to tandem mass spectrometry with a triple quadrupole instrument. To detect and quantify a wide range of contaminants in contact with man in daily routine, a screening method was developed by liquid chromatography coupled to high resolution mass spectrometry with a quadrupole-time-of-flight instrument from urinary matrix. The targeted screening method validated according to FDA guidelines allows the quantification of contaminants classified according to different families, in urine without sample preparation, at concentrations of the order of ng.mL-1. This method was applied to volunteers’ urine samples. The non-targeted screening method allows issuing numerous assumptions of compound identification after MS/MS fragmentation. The implementation of this tool to measure the Exposome associated with statistical studies, contribute greatly to the understanding of the causal relationships between diseases and environmental factors
Austin, Sisley. "Criblage d’inhibiteurs de l’interaction virus/hôte [LP]PxY/Nedd4 : une cible antivirale à large spectre." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0340/document.
Broad-spectrum antiviral identification is considered as one of the major aims of theactual virology research and one strategy consists in targeting virus/host interaction. Using theAlphaScreen® technology and the adenoviral model protein VI/Nedd4-2, we performed highthroughputbiochemical screening targeting the [LP]PxY/Nedd4 interaction, a commoninteraction of different virus families. We identified candidate inhibitors from a librarycompound approved by health agencies. We tested, characterized and validated the antiviraleffect of those compounds on two very different virus families. Indeed, compounds C9(Sulconazole) and C4 (Flunarizine) decrease replication of the adenovirus, a DNA nonenvelopedvirus and the replication of the Marburg virus, an RNA enveloped virus from theFilovirus family. Taken together, those results permit us to validate the [LP]PxY/Nedd4interaction as good target for a broad spectrum antiviral and to propose the “repositioning” ofcompounds C4 and C9 as antivirals. Moreover, we have synthesized new analogues from C9showing similar effect on AdV replication compared to the original molecule (C9). Inconclusion, our work on developing new broad-spectrum antivirals highlights the possibilityto use imidazole derivatives as a new class of antiviral compounds
Tremblay-Létourneau, Maude. "La synthèse de la coiffe en tant que nouvelle cible thérapeutique potentielle." Mémoire, Université de Sherbrooke, 2012. http://hdl.handle.net/11143/6366.
Dorard, Emilie. "Etude d’une cible thérapeutique pour la maladie d’Alzheimer et mise au point de nouveaux modèles cellulaires de criblage." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB060.
Tomé, Catarina da Silveira. "Intéractions avec le ribosome et changements conformationnels de la GTPase bactérienne EngA, une cible potentielle pour de nouveaux antibiotiques." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAY033/document.
The development of new therapeutics against bacterial infections has aroused great interest over the last years in the context of drug resistance. The starting-point in the pursuit of new antibiotics for which bacterial resistance mechanisms do not exist is the identification of novel cellular targets. Genetics studies in the early 2000s have identified engA as a conserved bacterial gene whose product is a GTPase that could represent a potential drug target: it is conserved among bacteria, essential for cell survival, and absent in humans.Since EngA acts as an assembly factor for the bacterial ribosome, one of our aims was to develop an assay to screen inhibitors of the EngA-ribosome interactions. These interactions are modulated by EngA conformational changes that are in turn triggered by the binding of different nucleotides to the catalytic G-domain. As the interplay between all these events in bacteria is still not resolved, we have used a multi-technique approach to explore these questions in order to obtain useful information for the setting up of a robust screening assay.SAXS and limited proteolysis showed a conformational change occurring in solution upon addition of either di- or tri-phosphate nucleotides. While model validation analysis confirmed the GDP-bound conformation, the GTP-bound state does not match any known EngA structure. Binding studies have revealed modulation of interactions by different nucleotide-bound states. Furthermore, response to nucleotides occurs at high concentrations, suggesting that the role of EngA in promoting ribosome assembly could be monitored by the intracellular nucleotide concentration. Efforts on identifying the GTP-bound state 3D structure by crystallography have resulted in EngA structures in different crystal forms. Although all the obtained structures represent the GDP-bound state, packing analysis has revealed conserved crystal contacts that can potentially stabilise this conformation during nucleation. Specific mutations aiming at disrupting these contacts may help to promote crystallisation of alternative conformations. Cryo-EM investigation has been initiated in order to obtain the structure of the B. subtilis EngA:50S complex. So far, an electron density map at 6.4 Å resolution has been obtained and its interpretation is underway
Saccucci, Laurent. "Intérêt thérapeutique de la protéine A20 des orthopoxvirus comme cible pertinente d'apatamères peptidiques et de compsés chimiques bloquant ses intéractions essentielles à l'intérieur du complexe de réplication virale." Grenoble 1, 2009. http://www.theses.fr/2009GRE10338.
Variola virus (VARV), the etiologic agent of smallpox was responsible of the most devastating infectious disease. Because of successful preventive measures by immunization, the World Health Organization (WHO) declared global eradication of smallpox in 1980. The subsequent discontinuation of vaccination has rendered all children and many adults virtually susceptible to smallpox infection. If variola virus was used in an act of terrorism or warfare it could cause a real catastrophe. So it is essential to develop new antiviral molecules with different mechanisms of action and usable immediately in case of terrorist attack. Here we report the use of two original strategies to identify new effective anti-orthopoxvirus agents targeting specifically the viral replication complex of vaccinia virus (VACV), a valuable surrogate for the smallpox virus. First, through a yeast two-hybrid assay, we have selected peptide aptamers directed against the VACV A20 protein, a central component of the replication complex and shown to form, with the D4 protein, a processivity factor for the viral DNA polymerase. Peptide aptamers are combinatorial protein molecules designed to inhibit the function of target proteins in living cells. We have proved that one selected aptamer interacting with a central region of A20 was able to significantly inhibit viral DNA synthesis and viral production in cell culture. Second, we have performed a high-throughput screening of small molecules to isolate compounds capable of disrupting A20 interaction with either D4, an uracil DNA glycosylase or D5, a DNA-independent nucleoside triphosphatase (NTPase) that contains a helicase domain. The screen is based on an automated dual-luciferase yeast two-hybrid assay, performed in 384-well plates. Among a collection of 27,600 compounds from diverse commercial chemical libraries we have identified two potential inhibitors that exhibit antipoxvirus effect on infected cell culture. These compounds were also found to specifically inhibit DNA replication. Thus, the screening for inhibitors of protein-protein interactions within viral replication complex remains to be a promising strategy for identifying new compounds active against orthopoxvirus infections
Galmiche, Cécile. "Assemblage par chimie click de fragments d’anticorps produits en bactéries pour un criblage fonctionnel rapide in vivo." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONT3507.
Anti-tumoral monoclonal antibodies are currently produced in eukaryotic cells. For cost and time reasons, a limited number of potential candidates are selected after in vitro tests. They are produced at large scale and then tested in vivo. To test more antibodies and more rapidly, we chose to produce single chain variable fragments (scFv) in bacteria, and to couple them to the eukaryotic constant fragment (Fc) thanks to click chemistry to reconstitute immunoglobulin-like compounds. For a given cost, this enables to produce and test in vivo a larger number of clones. This independent production of fragments is also a flexible tool allowing the combination of different Fc isotypes/allotypes with different scFvs.Click chemistry is based on a specific and high-yield reaction between and azide and a cyclooctyne. Therefore, antibody fragments were functionalised on specific residues (tags) by chemical linker so that each part will contain one of these chemical moieties at their extremity. The first step consisted in introducing tags into the anti-HER2 scFv 4D5 C-terminus and human IgG1 Fc N-termini sequences. The scFvs were produced with yields higher than 100 mg/L in the E. coli cytoplasm and in vitro oxidized with copper sulfate. The Fc fragment was classically produced in human cells. Five chemical or enzymatical reactions were optimised and compared in terms of specificity and yield. The coupling between an amine and a glutamine tag catalysed by microbial transglutaminase gave the best results. The scFv fragment was thus functionalised with an azadibenzocyclooctyne and the Fc fragment with an azide at 60-70%. When mixed together, these fragments formed a (scFv)2-Fc and a scFv-Fc with global yields respectively of 10-20% and 20-30% after optimisation.After the click reaction, the scFv + Fc mix binds to the HER2 receptor on the same way as the eukaryotic (scFv)2-Fc in terms of HER2-binding and proliferation inhibition capacity. Now, it must be demonstrated that their proliferation inhibition of a HER2-positive cell line is similar. The final aim is to get a similar tumour growth inhibition on murine xenografts
Hutin, Mathilde. "Criblage de la diversité d'Oryza spp. pour l'identification de nouvelles sources de résistances dépendantes des effecteurs TAL à X. oryzae pv. oryzae, agent de la bactériose vasculaire du riz." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS164.
Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most destructive bacterial disease of rice. Xoo pathogenicity critically depends on the TAL (Transcription Activator-Like) effectors. TALs are type three effectors secreted through a type three secretion system into the eukaryotic cell where they act as transcription factors able to manipulate the host transcriptome via the induction of specific genes. DNA-binding specificity involves a unique central repeated region in the TAL effector whereby each repeat directly binds to one single nucleotide. TALs can act as major virulence effector thtough the induction of so-called susceptibility (S) genes that are essential for disease development, or act as avirulence effector by inducing so-called executor (E) resistance genes that promote host defense responses. The goal of this PhD project was to identify and characterize novel TAL-dependent resistance sources to control BLB. In rice, the best characterized S genes are those of the clade III of the sugar transporters SWEET family. The most important is OsSWEET14 as this gene is targeted at unrelated DNA boxes by four TAL effectors, which belong to strains of different lineages and geographic origins. The evolutionary convergence for the induction of SWEET14 reflects its crucial role as major determinant of rice susceptibility to Xoo. A molecular screening of the OsSWEET14 promoter was performed using the natural diversity of wild African rice in order to identify polymorphism that could affect the TAL/DNA binding and thus lead to loss of susceptibility. This work allowed the identification of xa41(t) that confers broad spectrum recessive resistance to Xoo. In a second part of my PhD project, a phenotypic screen for resistance against the Xoo African strain MAI1 of a hundred of rice accessions enabled to identify five TALs. Among them, Tal2 and Tal9 were shown to trigger resistance on the rice variety IR64 the genome of which is fully sequenced. RNAseq analysis identified a small set of resistance E gene candidates underlying potentially IR64 resistance against Xoo strain MAI1. Finally, as a third strategy we aimed within a collaborative project to investigate if PiCO39 that confers resistance of rice towards Magnaporthe oryzae could also control BLB and BLS (Bacterial leaf streak). To that end, Artificial TAL effectors (dTALe) were designed to induce specifically the M. oryzae AVR1-CO39 construct in resistant (PiCO39) and susceptible (piCO39) transgenic backgrounds. We show that the induction of AVR1-CO39 by Xoo or Xoc drastically impairs bacterial colonization in a PiCO39-dependent manner, highlighting the potential of exploiting rice blast or other resistance genes as novel strategies to control rice pathogenic Xanthomonas bacteria
Murat, Jean-Benjamin. "Etudes biochimiques, structurales et fonctionnelles du complexe MARS de Toxoplasma gondii, une nouvelle cible thérapeutique." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV031/document.
Toxoplasma gondii, a parasite of felids gut, is responsible for toxoplasmosis, a disease that can induce severe sequelae or death in the foetus or immune-depressed patients. Currently available treatments can prevent or cure most of the cases, but are at risk for side effects and cannot suppress cysts, which cause the chronic disease and are responsible for disease when the immune status is altered. Aminoacyl-tRNA synthetases (aaRS) are essential for translation, by charging tRNA with cognate aminoacids, a preliminary step of the protein synthesis process.A gene coding for a protein homologous to p43 (which interacts with a subset of aaRSs in higher eukaryotes) was identified in the genome of T. gondii. Following its epitope tagging, we show that Tg-p43 is not secreted nor exported beyond the vacuole as a cytokine, as it is for its human counterpart; however, biochemical analysis of the Tg-p43 interactome reveals four aaRSs as interacting partners, namely Methionyl-, Glutamyl-, Glutaminyl- and Tyrosyl-tRNA synthetases. This is the first description of the multi-aaRS (MARS) complex in the Apicomplexa phylum; it is strictly localized in the parasite cytoplasm. The unexpected presence of the Tyrosyl-tRNA synthetase in the complex raises several questions about how the complex is organised and assembled, and also evolved. Electronic microscopy along with size exclusion chromatography shows heterogeneity and loose structure of the complex, similarly to recent data characterizing higher eukaryotic complexes. Disruption of the complex by knocking-out of the gene Tg-p43 does not induce detectable phenotypic modification, nor alterations of the virulence and cystogenesis in a murine model.Alongside the study on the MARS complex, we used an in silico approach to screen for new compounds to inhibit T. gondii Glutaminyl-tRNA synthetase. We thus identified one parasitostatic compound that was able to significantly slow down parasite growth while having a relatively low in vitro toxicity against the human host cell. The function of the MARS in T. gondii still remains unknown; the role of Tg-p43 in the post-transcriptional control or any other biological function is probably too subtle to be measured under our experimental conditions. However, our data help to some extent to better measure the evolutionary history of the MARS family. The therapeutic side of this work, although preliminary, may serve as a base for anti-T. gondii drug discovery focusing on aaRS inhibitors, which are obviously good candidate targets
Lewis, Matthieu. "Identification de voies de résistance aux inhibiteurs de tyrosine kinase dans la leucémie myéloïde chronique par criblage CRISPR-Cas9." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0054/document.
The characterization of malignant tumour growth and the understanding of resistance mechanisms to treatment in cancer is of utmost importance for the discovery of novel “druggable” targets. Efficient genetic screening, now even more possible with the convergence of CRISPR-Cas9 gene editing technology, next-generation sequencing and bioinformatics, is an important tool for deciphering novel cellular processes, such as resistance to treatment in cancer. Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterised by the t(9;22) genetic abnormality, which encodes the driver of CML, the BCR-ABL1 fusion protein. Imatinib mesylate, a tyrosine kinase inhibitor, specifically eliminates CML cells by targeting and blocking the kinase activity of this protein, yet, as for all targeted therapies in cancer, resistance to treatment exists. In order to discover alternative BCR-ABL1 independent mechanisms of imatinib resistance, we utilized the genome-scale CRISPR knock-out library GeCKO v2 to screen for imatinib sensitising genes in vitro on K562 cells. We revealed genes that seem essential for imatinib induced cell death, such as pro-apoptotic genes (BIM, BAX) or MAPK inhibitor SPRED2. Specifically re-establishing apoptotic capabilities in BIM knock-out (KO) cells with BH3-mimetics, or inhibiting MAP-kinase signalling in SPRED2 KO cells with MEK inhibitors restores sensitivity to imatinib, overcoming resistance phenotypes. In this work, we discovered previously identified pathways (apoptosis, MAP-kinase signalling) and novel pathways that modulate response to imatinib in CML cell lines, such as the implication of the Mediator complex, mRNA processing and protein ubiquitinylation. Targeting these specific genetic lesions with combinational therapy can overcome resistance phenotypes and paves the road for the use of precision oncology
Furnon, Wilhelm. "La protéine non-structurale NS1 du virus West Nile : étude fonctionnelle et cible potentielle de nouvelles molécules antivirales." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1008/document.
Among emerging mosquito-borne viruses (arboviruses), flaviviruses like Dengue, Zika and West Nile virus (WNV) are very often involved in outbreaks. WNV causes several neuroinvasive diseases, which can be lethal, in humans and horses each year. This virus is a threat for both, human and animal public health. Furthermore, there is no human vaccine currently or any specific antiviral treatments against WNV.Among viral factors which are essential for flavivirus infection, the nonstructural glycoprotein NS1 is a multifunctional protein. The secreted form sNS1, is released in the extracellular medium from infected cells and is strongly involved in immune system dysregulation. The functions of sNS1 play roles in immune escape and, paradoxically, in pathogenesis which is observed in severe forms of the disease. Because most of this data are about Dengue Virus, we would like to study, in vitro, functional properties of the sNS1WNV during infection of epithelial, glial and neuronal mammalian cells. Based on the high sNS1 protein structure similarities among flaviviruses, our hypothesis suggests a role of sNS1WNV in neuroinvasive infections.The sNS1WNV protein doesn’t seem to modulate viral infection steps. However, it is involved in actin cytoskeleton remodeling in epithelial cells. sNS1WNV is also involved in the activation of antiviral response pathways in non-infected neuronal cells. On the other hand, by targeting sNS1 and envelope protein E of WNV, we performed a screening of aRep molecules (artificial proteins with alphahelicoïdal repeats) and isolated ligands with high affinity for these viral factors. Because this new type of molecules is able to specifically bind to sNS1 and E, they have potential to be used for the development of new diagnostic tools and antiviral therapeutic agents
Leblanc, Cécile. "Rôle de la 4'-phosphopantethéïnyl transférase PptT dans la multiplication et la persistance de Mycobacterium tuberculosis et mise en place d'un test d'activité enzymatique pour la recherche de nouveaux antituberculeux." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1930/.
Mycobacterium tuberculosis (Mtb), the etiologic agent of human tuberculosis, displays an unusual cell envelope that contains a large variety of lipids with unusual structures, some of them essential for the bacterial viability and others important for pathogenicity. The synthesis of these lipids involves multifunctional enzymes that are only functional if converted from their inactive apo-forms to their active holo-forms by the covalent attachment of a 4'-phosphopantetheinyl (P-pant) group. This modification is achieved through the action of a 4'-phosphopantetheinyl transferase named PptT, which transfers the P-pant moiety of coenzyme A to a conserved domain of these proteins. In this study, we investigated whether PptT represents a novel drug target for treating tuberculosis. The construction of pptT conditional expression mutants of M. Bovis BCG and Mtb enabled us to demonstrate that PptT is required for mycobacterial growth and survival in several environments, including those encountered in macrophages, and during the various stages of infection in the mouse model. An in vitro enzymatic assay based on the catalytic activity of PptT has been developed and adapted to high throughput screening for the search of PptT inhibitors. Finally, we found that the enzyme PptT can be inhibited by small compounds. Altogether, our findings indicate that PptT meets all the requirements for a good drug target and provide tools for the search of inhibitors against this enzyme. Besides, these results suggest that some polyketide-derived lipids may be required for Mtb persistence within the host
Truffaux, Nathalene. "Nouvelles cibles thérapeutiques dans les gliomes infiltrants du tronc cérébral de l'enfant." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T022/document.
Diffuse Intrinsic Pontine Glioma (DIPG) is a rare, unresectable and universally fatal tumor. Due to the lack of available material, no improvements have been made in the knowledge of the biology of this tumor until recent years, leaving radiotherapy as the only efficient treatment, and only transiently. Recently, the effort engaged for collecting samples in this disease at the diagnosis or at the autopsy resulted in an unprecedented number of analyses consequently improving our knowledge in DIPG. Those studies bring evidences for their differences with adult gliomas, but also with other pediatric supratentorial glioma showing specific genomic alterations. Thus, Platelet-Derived Growth Factor Receptor Alpha (PDGFRα) appeared to be one of the major target given its frequent aberrations found in those tumors. Investigating an effective drug to inhibit this pathway led us to evaluate the effect of dasatinib, which is known as a multi-targeted inhibitor. We report here the in vitro efficacy of dasatinib on new cell lines of DIPG developed from stereotaxic biopsy at diagnosis. Because therapies are largely inefficient in the clinic when they are used as a monotherapy, we bring out our interest on combining dasatinib with an inhibitor of MET, which is the 2nd most common amplified oncogene in these tumors.Additionally, an innovative strategy of pharmacological screening has been successfully tested. New drugs, potentially efficient in DIPG, have been fonctionnaly-defined, including Histone deacetylase inhibitors (HDACi), Cyclin-Dependent Kinases inhibitors (CDKi) and proteasome inhibitors as well.Finally, by using whole genome sequencing (WGS), we have been able to discover new genetic abnormalities, never encountered before in other cancers. Among those, mutations of histone H3K27M with a high frequency of 80% were found, suggesting that they have a fundamental role in tumors genesis. Moreover, ACVR1/ALK2 activating mutations have been identified as well. And this gene now represents a new target to explore. This work reports the research of new therapeutic targets through an exploratory approach using drug screening and WGS on the one hand, and on the other hand through a preclinical validation approach in terms of targeted therapies with tyrosine-kinases inhibitors
N'Guessan, Cécilia. "La phosphatase PPM9 de Plasmodium : caractérisation moléculaire et fonctionnelle, structure 3D du site catalytique et découverte de nouvelles molécules antipaludiques." Thesis, Lille, 2020. http://www.theses.fr/2020LILUS033.
Malaria today is one of the wide spread infectious diseases in the world. In 2018, 405 000 malaria deaths have been reported. RTS, S/A01 the only vaccine tested on a large scale does not fulfil its promises with a lack of efficiency. Plasmodium falciparum (Pf), the deadliest agent of malaria, has developed resistances to almost all chemotherapeutics. It is necessary to understand the biology of this parasite in order to develop new drugs. In Pf, extensive research has now been started to study the Pf kinome and to examine whether targeting kinases could represent an effective mean for the treatment of the infection, the study of its phosphatome is still under-investigated. Amino acid sequence comparative analyses of Plasmodium berghei (Pb), a rodent malaria species, revealed that 6 are Plasmodium specific. Among these phosphatases, the metalloprotein phosphatase 9 (PPM9), a Plasmodium specific serine/threonine phosphatase, was also suggested to be essential for blood stage parasites development. Besides in a high-throughput saturation mutagenesis method in Pf, PPM9 gene was also identified essential. The present project is focused on the molecular and functional characterization of the PPM9 and on the validation of this specific phosphatase as a new potential target for malaria. The gene has been cloned, annotated and expressed as a recombinant protein and its phosphatase function has been characterized. The enzymatic activity of PfPPM9 recombinant protein has been standardised using a malachite green phosphate assay kit and this activity is manganese dependant. Functional characterization was explored by conditional gene knock-out studies as well as by generating knock-in parasite lines to follow their trafficking during the parasite lifecycle (in Pf and Pb). PfPPM9 seems to be mainly localised in the parasite cytoplasm and could be exported in the cytoplasm of red blood cell. Among these studies, we employ CRISPR-Cas9 in Pf to facilitate use of the dimerisable Cre-recombinase (diCre) that is used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin-controlled manner. Finally, we solved in silico the 3D structure of PfPPM9 by homology modelling and identified a new set of potential specific inhibitors. We screened in silico ZINC15 database and ICPAL base on the 3D structure. We have tested around 80 compounds for their anti-plasmodial in vitro activity. We have highlighted 3 hits: M19, M51 and M74. M19 has a half maximal inhibitory concentration (IC50) of 3,87 μM +/- 0,25 and a unique scaffold as antimalarial compound. Besides, via NMR studies (Waterlogsy and CPMG), we have shown a specific interaction between these hits and PfPPM9. As a perspective, PPM9 interactome will be carried out to determine its target/partner proteins in the parasite. In conclusion, this study will lead to a deeper understanding of the role of PPM9 in the parasite development and the discovery of new antimalarial compounds
Lachaud, Sophie. "Régulation de l'expression de PD-L1 dans le mélanome : identification de cibles thérapeutiques au moyen d’un crible génétique pour traiter le mélanome cutané TIE1 Regulates PD-L1 Expression in Melanoma." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL065.
Cutaneous melanoma arises from melanocytes, the pigment-producing cells of the skin. is responsible for approximately 75% of deaths due to skin cancers. This tumor account for approximately 1% of cutaneous melanoma, but is responsible for 75% of deaths due to skin cancer. Its aggressiveness is coming from its highly metastatic potential and once it is disseminated, chances of survival decrease drastically. Treatment by surgery is efficient when it is diagnosed early. Though, until recently, for metastatic or unresectable melanoma, treatments were quite limited.The arrival of immunotherapies to treat advanced melanoma arouse a lot of hope. These monoclonal antibodies boost the immune system in order to kill cancer cells. The most recent and efficient are the ones targeting the programmed cell death protein 1 or its ligand (PD-1/PD-L1). The main role of PD-1 immune checkpoint is to limit the activation of engaged peripheral T cells. Indeed, the activation of T cells by the interaction of its TCR with MHC/peptide complexes leads to IFNγ; secretion. This cytokine will induce the expression of PD-L1 and PD-L2 at the plasma membrane of surrounding cells. The engagement of PD-1 with its main ligand PD-L1 leads to the inhibition of CD8+ T lymphocytes. This feedback loop attenuates T-cell responses and limits the extent of immune-mediated tissue damage that can happen with an excessive immune response. Some cancer, including melanoma which is highly immunogenic, escape the immune system by taking advantage of this mechanism to overexpress PD-L1 and inhibit effector functions and proliferation of CD8+ T lymphocytes. Immunotherapies targeting PD-1 or PD-L1 with monoclonal antibodies were then deployed to disrupt the interaction between the two partners and restore, at least partially, T-cell activity against cancer cells and drive to tumor regression. Most of the time, basal PD-L1 expression in melanoma is low and is strongly increased by IFNγ, produced by immune cells like CD8+ T lymphocytes. Thus, preventing IFNγ-induced PD-L1 expression would restore the effector functions of LT CD8+ and avoid tumor progression as anti-PD-1/PD-L1.This aim of my thesis project was to make use of a genetic screen to identify positive regulators of the expression of PD-L1 at the plasma membrane of melanoma with a genetic screen by focusing on druggable genes. This screen was based on a shRNA library targeting genes coding for a protein for which an inhibitor already exists or harboring a structure or an activity that could lead to drug development. The bioinformatic analysis led to the identification of new regulators that positively regulate PD-L1 at the plasma membrane of melanoma that could serve as therapeutic targets for melanoma treatment
Prevel, Camille. "Développement de biosenseurs fluorescents et d’inhibiteurs pour suivre et cibler CDK4/cycline D dans le mélanome." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONT3505/document.
CDK/cyclins play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting established cancer biomarkers and attractive pharmacological targets. In particular, CDK4/cyclin D, which is responsible for coordinating cell cycle progression through G1 into S phase, is a relevant target in several cancers including melanoma, associated with mutation of CDK4, cyclin D, p16INK4a and pRb.As there are no sensitive and direct approaches to probe CDK4/cyclin D activity in physiological and pathological conditions, the first goal of my thesis has consisted in engineering a fluorescent biosensor to probe this kinase in vitro and in cellulo. Once characterized and validated in vitro, the biosensor was applied to detect CDK4/cyclin D alterations in biopsies from human skin and melanoma xenografts in fluorescence-based activity assays, and in living cancer cells by fluorescence microscopy and timelapse imaging.Moreover, only few inhibitors are currently available to target CDK4/cyclin D and most of them bind the ATP pocket. As such, the second major goal of my thesis project has consisted in identifying non-ATP competitive inhibitors, either through rational design of peptides or by screening small molecule libraries. To this aim, two fluorescent biosensors were engineered which discriminate compounds that target the interface between CDK4 and cyclin D, or that perturb the conformational dynamics of CDK4, respectively, from ATP-pocket binding compounds. Fluorescence-based screening assays performed with these biosensors lead to identification of hits, which were validated and characterized in vitro and in cell proliferation assays, and which constitute promising candidates for selective chemotherapy in melanoma
Saccucci, Laurent. "INTERET THERAPEUTIQUE DE LA PROTEINE A20 DES ORTHOPOXVIRUS COMME CIBLE PERTINENTE D'APTAMERES PEPTIDIQUES ET DE COMPOSES CHIMIQUES BLOQUANT SES INTERACTIONS ESSENTIELLES A L'INTERIEUR DU COMPLEXE DE REPLICATION VIRALE." Phd thesis, 2009. http://tel.archives-ouvertes.fr/tel-00440047.
Timchenko, Natalia. "La dihydrofolate réductase R67, comme une cible d’antibiotiques et biocatalyseur potentiel." Thèse, 2010. http://hdl.handle.net/1866/5196.
Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.