We assessed influence of three acclimation regimes and influence of recovery after cold shock (exposure to 0°C for a period of time corresponding to Lt25) on the relative mRNA levels of selected stress proteins using qRT-PCR method. Larvae acclimated at 25°C showed relatively weak upregulation responses to cold shock. Much stronger responses were observed in the larvae that were cold-acclimated at 15°C or 15°C ? 6°C prior to cold shock. Two different general trends were distinguished in the response to cold acclimation and cold shock: (a) proteins from families SP70 and SP90 and splice variants c and d of the transcription factor HSF were upregulated in response to cold acclimation and the levels of their mRNA transcripts further increased after cold shock (for instance, the abundance of hsp70Aa mRNA increased up to 300-fold after cold shock (acclimation variant 15°C ? 6°C)); (b) four members of the small Hsp family (22, 23, 26 and 27 kDa) and splice variants a and b of the transcription factor HSF were down-regulated during cold acclimation (for instance, 10-fold in the case of hsp22) and the levels of their mRNA transcripts were either unchanged or increased only moderately after the cold shock. A third group of proteins, namely Hsc70, Hsp40 showed no or relatively small changes.

No description available.

No / The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

Lors de ce travail, une étude comparative entre trois souches de C. glutamicum a été réalisée. Celles-ci sont C. glutamicum 2262, une souche surproductrice de glutamate suite à l’élévation de température du milieu de culture de 33 à 39°C, C. glutamicum 2262 NP un variant incapable d’excréter du glutamate dans ces mêmes conditions et C. glutamicum 2262 ∆pks13 un mutant dépourvu de bicouche mycolique externe. Un modèle métabolique original reprenant les différentes modifications physiologiques aboutissant à l’excrétion du glutamate au cours du procédé thermo-induit a été établi. La bicouche mycolique joue un rôle primordial puisque son absence affecte sévèrement la production du glutamate. Dans un premier temps, l’élévation de température serait ressentie au niveau de cette bicouche. Ce ressenti, visualisé par l’accumulation de protéines caractéristiques d’un stress thermique, est nécessaire pour que la bactérie soit en capacité de surproduire le glutamate. Par la suite, la production de glutamate est régulée au niveau de l’α-cétoglutarate déshydrogénase (ODH) grâce à la phosphoprotéine OdhI. Suite au changement de température, celle-ci est déphosphorylée ce qui lui permet d’interagir avec ODH et de provoquer l’inhibition de cette dernière. Ceci se traduit par la redirection sur flux carboné vers la synthèse du glutamate. Aucun de ces évènements n’est observé chez C. glutamicum 2262 ∆pks13. Par ailleurs, l’élévation de température induit une modification de la composition de l’enveloppe cellulaire qui semble intervenir dans le processus physiologique aboutissant à l’excrétion du glutamate puisque très peu de changements sont observés chez C. glutamicum 2262 NP / During this work, a comparative study between three strains of Corynebacterium glutamicum was carried out. These strains were C. glutamicum 2262 which overproduces glutamate after an increase in the culture temperature from 33 to 39°C, C. glutamicum 2262 NP which is unable to produce glutamate in the same culture conditions and C. glutamicum 2262 ∆pks13 devoid of outer corynomycolic acid bilayer. An original metabolic model describing the successive physiological modifications responsible for the glutamate excretion during the temperature-triggered process was established. The presence of the corynomycolic acid bilayer appeared to be necessary since its lack affected dramatically the glutamate production. The temperature increase would be first sensed at the level of the external corynomycolic acid layer. This sensing was visualised through the accumulation of thermal stress proteins. In C. glutamicum 2262 ∆pks13, the synthesis of these proteins was not induced. The glutamate production is regulated at the oxoglutarate dehydrogenase (ODH) level by the phosphoprotein OdhI. A consequence of the temperature increase was the dephosphorylation of this regulatory protein and its interaction with ODH, provoking its inhibition. The carbon flux was then reoriented toward the glutamate synthesis. In C. glutamicum 2262 ∆pks13, no dephosphorylation of OdhI and no change in the ODH activity were not determined. The thermal stress also induced a change in the composition of the corynomycolic acid layer which was correlated with the ability of C. glutamicum 2262 to overproduce glutamate. In C. glutamicum 2262 NP, the composition of the corynomycolic acid layer remained unchanged

Malaria is caused by a protozoan parasite belonging to the genus Plasmodia. Plasmodium falciparum is responsible for the fatal form of human malaria. Spread of drug resistant parasites warrants for sound biological understanding of the parasite at both cellular and biochemical level. Heat shock proteins are highly conserved group of proteins required for correct folding, transport, and degradation of substrate proteins in vivo. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where in cooperation with Hsp10, it participates in protein folding. Keeping in mind the central importance of chaperones in biological processes, our lab has been interested in examining roles of heat shock proteins in malarial parasite during its asexual growth in human erythrocytes. During its life cycle, the parasite continually shuttles between a cold-blooded insect vector with the body temperature of 27°C and a warm-blooded human host with the body temperature of 37°C and parasite experiences episodes of heat shock periodically. Therefore malaria parasite serves as good model to study heat shock protein functions. Like all biological systems, the malaria parasite expresses several chaperones including proteins of the Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families. Towards this we have systematically characterized different families of stress proteins Hsp40, Hsp60, Hsp70, Hsp90 as well as Hsp100. In addition to cloning their genes we have studied their expression, localization, abundance, complexes and their biological roles. Earlier studies from our lab showed PfHsp90 is essential for parasite growth and survival in human erythrocytes. Our present study attempts to study heat shock protein 60 of the malarial parasite (PfHsp60). In this connection we have been successful to clone and express PfHsp60 gene from Plasmodium falciparum in E. coli and to raise antibodies specific to PfHsp60. We have examined its expression and import in the mitochondrion of malarial parasite during its asexual growth in human erythrocytes. Analysis of the total parasite lysates resolved by two-dimensional gel electrophoresis followed by western blotting using specific antibodies showed PfHsp60 exhibits an isoelectric point corresponding to its signal uncleaved precursor (pI - 6.2). Mass spectrometric analysis of the spot corresponding to precursor PfHsp60 confirmed the presence of signal peptide region. Co-immunoprecipitation analysis of total parasite lysates with antibodies specific to PfHsp60 showed precursor PfHsp60 to be associated with PfHsp70 and PfHsp90. Co-immunoprecipitation from the mitochondrial and cytoplasmic fraction confirmed the position of mature PfHsp60. Indirect immunofluorescence analysis also showed presence of a pool of PfHsp60 in the cytoplasm of the parasite, in addition to its expected localization in the mitochondrion. Treatment of parasite infected erythrocytes with an inhibitor of Hsp90 disrupted its association with cytoplasmic chaperones and targeted precursor Pfhsp60 for intracellular degradation. On the other hand treatment with the mitochondrial import inhibitor further inhibited the import of precursor PfHsp60 into the mitochondrion and stabilized its interaction with cytosolic chaperones. Previous reports have shown that there are four fold accumulations of PfHsp60 transcripts in heat shocked parasites. However, the expression of PfHsp60 was not induced upon heat shock in the blood stages of P.falciparum. Biochemical data indicate that the mitochondrion is not the source of ATP in the parasite. Furthermore the genome does not seem to encode the critical subunits of Fo-F1 ATP synthase. Yet, the active mitochondrial electron transport chain serves for regeneration of ubiquinone required for pyrimidine biosynthesis. The active electron transport chain is critical for parasite survival. Recent study with the lab-grown 3D7 strain of malaria parasite concluded that mitochondria are not required for energy conversion. Transcriptome analysis of the parasite derived directly from blood samples of infected patients showed that genes encoding the proteins of mitochondrial biogenesis, oxidative phosphorylation, respiration and highlighted the mean expression level for PfHsp60 is dramatically up regulated in parasites. Gene up regulation doesn’t always translate to increase in protein function or metabolic up regulation. When we analyzed the total parasite lysates of field isolates resolved by two-dimensional gel electrophoresis also showed presence of the precursor form of Pfhsp60 in the cytoplasm of the parasite. Overall, our observations indicated accumulation of precursor PfHsp60 in the parasite cytoplasm suggesting an inefficient mitochondrial protein import in the malarial parasite. The defect in mitochondrial protein import is possibly reflective of the compromised energy state of the parasite mitochondrion. This fits with the model that has been reported in mutant strains of yeast, Saccharomyces cerevisiae lacking functional F o-F1-ATPase. These strains were found to grow very poorly under anaerobic conditions and are known to accumulate Hsp60 protein in the cytoplasm mainly its precursor form. Under optimal growth conditions most eukaryotes maintain close co-ordination between gene expression, translation and translocation efficiently. As a result, mitochondrial precursor proteins are usually not found to accumulate in the cytoplasm. To our knowledge this the first report suggesting an inefficient co-ordination in the synthesis and translocation of precursor PfHsp60 and possibly other proteins during asexual growth of malarial parasite in human erythrocytes under optimal growth conditions. Finally, expression of the PfHsp60 gene in E.coli resulted in its association with bacterial GroEL subunits co-fractionating with a size of 920 kDa, corresponding to the tetra decameric form. The observation indicated possible existence of a hybrid chaperonin complex consisting of subunits from ectopically expressed PfHsp60 and endogenous GroEL.

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

The genus Salmonella consists of pathogenic gram negative organisms which infect intestines of birds, animals and humans. They are the causative agents of salmonellosis which is characterised by diarrhoea, nausea, fever and abdominal cramps. If not treated in time, salmonellosis can also be fatal. Salmonella genus is divided into two species Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies out of which the subspecies enterica has many of the pathogenic serovars of this species. Salmonella typhimurium is a server in the subspecies enterica of Salmonella enterica species. Transmission of salmonellosis takes place through contaminated food and water. When the organism enters a host, it encounters a range of hostile environments such as acidic pH, lack of oxygen as well as immune response of the host. In order to establish infection, the bacterium needs to survive under stressful conditions and propagate itself. Various proteins are induced in cells under unfavourable conditions that protect them in such situations. One such group of proteins belongs to the Universal Stress Protein (USP) family. Universal Stress Proteins are a set of proteins induced in organisms when it is exposed to a variety of environmental insults including heat shock, nutrient starvation, presence of toxic compounds, etc. Although survival in adverse conditions is mediated by induction of this group of proteins, the precise mechanism of cellular protection has not been elucidated yet. The functional role of a protein is directly related to its three-dimensional structure and hence important insights can be gained regarding the role of these proteins by determining their structures. The structures of two Universal Stress Proteins from S. typhimurium; a single domain protein, YnaF and another tandem USP domain protein, YdaA were determined by X-ray crystallography and biochemical analysis was carried out on them. Guided by structure, plausible roles for both the proteins in stress tolerance of S. typhimurium have been proposed. Additionally, work was also carried out on phosphomannose isomerise from S. typhimurium. Phosphomannose isomerase is a housekeeping enzyme which catalyses the interconversion of mannose-6-phosphate and fructose-6-phosphate. Mannose is important for mannosylation of various lipids and proteins which form an important component of bacterial and fungal cell walls. Presence of a functional phosphomannose isomerise enzyme is important as it helps the organism survive adverse conditions by forming a strong cell wall which shields it from harmful environments. Moreover, phosphomannose isomerase was also found to be essential for virulence of Leishmania mexicana and Cryptococcus neoformans. The structure of phosphomannose isomerase from S. typhimurium was determined in our laboratory in the year 2009. However, in the earlier studies, the catalytically important residues had not been identified and mechanism of isomerisation was not established. Structural analysis, site directed mutagenesis and biochemical assays were used to identify key residues in the active site of StPMI. Identification of these residues might help in deciphering the catalytic mechanism which will eventually be useful to develop inhibitors that arrest the growth of Salmonella as well as other microorganisms. The work reported in this thesis describes the efforts made to enhance our understanding of functional aspects of the two Universal Stress Proteins, YnaF and YdaA and phosphomannose isomerase from S. typhimurium. Chapter 1 begins with a brief introduction to the kinds of unfavourable environments encountered by microorganisms and their strategies of adaptation. This is followed by a review of the literature on Universal Stress Proteins, which are induced in many organisms in response to arrest of or perturbations in the growth rate. Structural, biochemical and evolutionary aspects of members of the family have also been discussed. Subsequently, a brief description of the earlier work carried out on another enzyme important in stress tolerance, phosphomannose isomerase, has been documented. A detailed account of mechanisms of isomerisation carried out by aldose ketose isomerases and identification of important strategies for determination of mechanism of phosphomannose isomerase catalysed reaction have then been provided. The chapter ends with a summary of aims and objectives of the present work. Chapter 2 describes the various experimental techniques and computational methods used during the course of this thesis work. Isolation of plasmids, overexpression and purification of protein, site directed mutagenesis, biochemical assays, crystallisation of proteins, X ray diffraction data collection form a part of the experimental aspect and have been described in detail. Brief descriptions of the programs used and principles behind computational methods used for structure determination (including data processing, phasing, model building and refinement), validation and analysis have also been provided. Chapter 3 includes the structural and functional studies carried out on YdaA, a tandem USP domain protein from S. typhimurium. Expression, purification, crystallisation and structure determination of YdaA in its native and ADP bound forms are described in the chapter. Biochemical assays with radiolabelled ATP showed that YdaA was an ATPase. The crystal structure of YdaA complexed with ATP revealed the presence of ADP (hydrolysis product of ATP) only in the C-terminal domain of the protein. Based on structural analysis and presence of ATP binding motif in the C-terminal domain, it could be hypothesized that ATP hydrolysis activity of the protein is confined to the C-terminal domain of the protein. The N-terminal domain of the protein was found to play another interesting role. A zinc binding site could be identified in the N terminal domain based on structural analysis and elemental X-ray absorption studies done at the synchrotron. Site directed mutagenesis and biochemical experiments suggested that zinc binding in the N-terminal domain was not related to ATPase activity of the C-terminal domain. Additionally, an intermediate of lipid A biosynthesis pathway UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetyl glucosamine was found bound to the N-terminal domain of YdaA. Lipid A is the membrane anchor of polysaccharides in the outer membrane of gram negative organisms and the intermediate occurs at the committed step of the pathway. However, no similarities could be identified between YdaA and members of the relevant biosynthetic pathway. Therefore, YdaA is unlikely to play a catalytic role in the same pathway but can function as a carrier molecule. A plausible link between the N- and C-terminal domains of YdaA could be identified by structural analysis. Many catalytically suitable residues from the N-terminal domain were found to be close to the β-phosphate of ADP bound to the C-terminal domain. Hence YdaA was identified to be a zinc binding ATPase which might play some yet unidentified role in lipid A biosynthesis pathway. Chapter 4 describes the attempts made towards understanding the functional role of YnaF, a single domain USP from S. typhimurium. A description of the expression, purification, crystallisation and X ray diffraction techniques used for structure determination of YnaF and its single site mutant have been provided in detail. Gel filtration, dynamic light scattering studies and the crystal structure determination of YnaF showed a tetrameric organisation of four USP protomers stabilised in the centre by chloride ions. Additionally, YnaF crystallised with a bound ATP even though ATP was not included in the crystallisation cocktail. Biochemical assays on YnaF with radiolabelled ATP showed that it was inactive with respect to ATP hydrolysis. When selected mutations that disrupt chloride binding were made, YnaF was converted to an active ATPase. The crystal structure of the mutant complexed with an ATP analogue revealed key differences at the active site in comparison with that of the wild type and allowed identification of residues that might be important for ATP hydrolysis in this group of proteins. Hence YnaF might play the role of a sensor protein in some signal transduction pathway involving chloride ions in bacteria. A structure based analysis and comparison of USPs from the Protein Data Bank with the structures of YnaF and YdaA is summarised at the end of this chapter. Chapter 5 describes the efforts carried out towards determination of mechanism of isomerisation catalysed by phosphomannose isomerise (PMI). Earlier reports suggest that the enzyme catalyses the reversible isomerisation of mannose-6-phosphate and fructose-6-phosphate via formation of a cis-enediol intermediate. The structure of phosphomannose isomerase from S. typhimurium has been reported by our laboratory. The enzyme is a monomer with three domains; a catalytic domain, a carboxy terminal domain and an α-helical domain. Residues from the catalytic domain were found to coordinate a zinc ion. Overexpression, purification, co crystallisation experiments and soaking studies carried out on crystals of PMI and its single site mutants are outlined in this chapter. The structure of a complex of PMI with mannose-6-phosphate at pH 7.0 revealed the presence of a blob of density close to the zinc binding site which was confirmed to be the active site by analysis of conservation of residues in the site. Based on site directed mutagenesis, activity studies and analysis of structure of PMI, zinc was identified to play an important role in maintaining the structural integrity of the active site. Electrostatic surface analysis of the structure of PMI revealed that the zinc ion might also play the role of anchoring phosphate moiety of the substrate in a highly negatively charged active site pocket. Activity assays following site directed mutagenesis studies eliminated the role of Glu264 in catalysis and implicated two lysines, Lys86 and Lys132 as the possible base in the reaction. The plausible role of a highly conserved residue Arg274 was also proposed based on comparison of structures of wild type and mutant PMIs. The future prospects of the work are briefly discussed towards the end of the thesis. Further experiments and analysis required to obtain better understanding of the functions of these proteins have been discussed. The Appendix section describes extensive crystallisation attempts that were carried out on the enzyme sorbitol-6-phosphate-dehydrogenase from S. typhimurium which catalyses the isomerisation reaction between sorbitol-6-phosphate and glucose-6-phosphate using NADPH as the cofactor. Needle shaped crystals were obtained which diffracted to a poor resolution of 7-8 Å at our in house X ray facility. Attempts to improve the quality of the crystals like co crystallisation with substrate and its analogues, soaking in various compounds and seeding are briefly described. The following manuscripts based on work described in this thesis have been published or will be communicated for publication. 1. Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance. Bangera M., Panigrahi R., Sagurthi S.R., Savithri H.S., Murthy M.R.N. Journal of Structural Biology, 2015 Mar; 189 (3): 238-50. 2. Structural and functional insights into phosphomannose isomerise: role of zinc and catalytic residues. Bangera M., Savithri H.S., Murthy M.R.N. Manuscript under preparation

Notre équipe vient de découvrir une méthode originale d’obtention d’extraits de Bacteroides thetaiotaomicron (E) qui préserve sa viabilité. Après culture anaérobie de ce commensal intestinal en milieu gélosé pauvre en facteurs de croissance, puis exposition à l’air, la bactérie semble posséder et générer dans E tout l’équipement de détoxication des espèces réactives de l’oxygène in vitro. Il laisse alors augurer d’un pouvoir thérapeutique à visée anti-inflammatoire.Objectifs et méthodes : Le but est d’abord de caractériser E, aux plans glucidique, lipidique et protéique. Dans ce dernier cas, il s’agit de séparer les protéines produites par la bactérie vivante et contenues dans E par électrophorèse bidimensionnelle et de les identifier par la technique des cartes peptidiques massiques. Les gels (n≥6) sont traités statistiquement (PDQuest®, Bio-Rad). Pour mieux localiser ces protéines dans la bactérie, elles sont comparées avec celles obtenues par destruction de B. thetaiotaomicron et identifiées dans la fraction cellulaire relative à la membrane bactérienne externe. Un travail de microscopie électronique est aussi entrepris pour visualiser les éventuels évènements intervenant pendant l’extraction.Le but est alors de vérifier, in vitro, l’effet antioxydant de l’extrait bactérien standardisé et d’en contrôler l’innocuité en modèles cellulaires utilisant le granulocyte neutrophile. L’effet thérapeutique anti-inflammatoire est ensuite recherché chez l’animal. L’action de E est d’abord évaluée en modèle murin d’inflammation cutanée auriculaire induite par dépôt de chlorure de benzalkonium, sous anesthésie générale. Des témoins positifs et négatifs de traitement et d’autres ne subissant pas d’irritation sont testés en parallèle. L’épaisseur des oreilles est mesurée toutes les heures pendant 5 h et des coupes histologiques d’oreilles, effectuées au bout de 2 h chez certains animaux. Deux colorations différentes permettent alors d’évaluer la quantité de mastocytes dégranulant localement.L’action de E, administré par voie intra-rectale (IR), est ensuite testée chez des souris subissant les premières phases d’un processus inflammatoire, en modèle de colite aiguë. Celle-ci est induite per os par du dextran sulfate sodium (DSS) ; elle évolue sur 8 jours. Sont considérés en parallèle des témoins positifs et négatifs de traitement et d’autres ne subissant pas de colite. Des scores cliniques et des scores histologiques de sévérité sont établis tous les jours de l’expérience. Des marqueurs de l’inflammation sont suivis dans les tissus murins après autopsie des animaux. [...] / Our team had discovered a new method to obtain extracts of Bacteroides thetaiotaomicron (E) which preserved its viability. This intestinal symbiont was anaerobically grown on an agar medium poorly supplemented in growth factors. After exposure to air, the bacterium seemed to possess and generate in E all the equipment able in vitro to detoxify reactive oxygen species. It let us expect a therapeutic power referred to anti-inflammatory properties.Objectives and methods: The aim was first to characterize E, in terms of carbohydrates, lipids and proteins. To achieve this last-mentioned goal, proteins contained in E coming from living bacteria were separated by two-dimensional electrophoresis and identified by the peptide mass fingerprinting technique. The gels (n ≥ 6) were statistically analyzed (PDQuest®, Bio-Rad). To find the origin of these proteins in bacteria, they were compared with those obtained by destruction of B. thetaiotaomicron (BT) and identified in the cell fraction containing the bacterial outer membrane proteins. Electron microscopy work was also undertaken to visualize any event occurring during extraction.The antioxidative effect of standardized E extracts was checked in vitro. E safety was also controlled in cell models using polymorphonuclear neutrophils. An E anti-inflammatory effect was then searched in animal models. E was first evaluated using a skin irritation mouse model. Inflammation was induced by benzalkonium chloride on ears of anesthetized mice. Positive and negative controls were treated in parallel. The ear thickness was measured every hour for 5 h and histological ear sections were performed after 2h for some animals. Two different staining methods enabled the enumeration of degranulating mast cells in ear sections.The effect of the bacterial extract was next tested locally by intrarectal (IR) instillations in mice undergoing the early stages of inflammation in a dextran sodium sulfate (DSS)-induced colitis. This acute model evolved over 8 days. In parallel, positive and negative animal controls underwent or not the colitis and were treated or not. Clinical and colonic histological severity scores were daily determined. Inflammation markers were measured in mouse colonic tissues after animal autopsy. [...]

No description available.