Search

Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort.

International audience; Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Drosophila melanogaster is a compelling genetic model to better understanding human neurological diseases owing to its high conservation in signaling pathways and cellular content of the brain. Here, glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by 1H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state 1H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism.

Drosophila flies are versatile animal models for the study of gene mutations in neuronal pathologies. Their small size allows performing in vivo Magic Angle Spinning (MAS) experiments to obtain high-resolution 1H nuclear magnetic resonance (NMR) spectra. Here, we use spatially-resolved 1H high-resolution MAS NMR to investigate in vivo metabolite contents in different segments of the fly body. A comparative study of metabolic changes was performed for three neurodegenerative disorders: two cell-specific neuronal and glial models of Huntington disease (HD) and a model of glutamate excitotoxicity. It is shown that these pathologies are characterized by specific and sometimes anatomically localized variations in metabolite concentrations. In two cases, the modifications of 1H MAS NMR spectra localized in fly heads were significant enough to allow the creation of a predictive model.

International audience; Huntington’s disease (HD) is an inherited neurodegenerative disorder, for which diagnostic development and discovery of new therapeutic targets are urgently required. In this study, a model of HD in Drosophila melanogaster has been used to identify metabolic biomarkers at presymptomatic and symptomatic stages of the disease. The pan-neuronal expression of a pathogenic fragment of the human Huntingtin (HTT) protein containing a 93-repeat polyglutamine expansion (Httex1p Q93) in transgenic flies induces a neuropathology with several characteristics of the human disease. The discriminant metabolites between the diseased flies and their controls were identified by 1H nuclear magnetic resonance and orthogonal partial least squares discriminant multivariate analysis. The experiments carried out with 10-day-old flies allowed us to identify a set of 10 biomarkers of the presymptomatic stage: NAD+, AMP, fumarate, asparagine, dimethylamine, β-alanine, glutamine, succinate, glutamate, and ethanol. Remarkably, the experiments conducted with 16-day-old flies, when the symptoms of the disease were present, highlighted a different set of 6 biomarkers: phosphocholine, ethanolamine, 2-oxoglutarate, succinate, pyruvate, and acetate. Our results provide a better understanding of the metabolic impairments in a widely used HD model and demonstrate that metabolism perturbations change dramatically during the development of the disease.

International audience; Leucine‐rich repeat and immunoglobin‐domain containing (LRRIG) proteins that are commonly involved in protein‐protein interactions play important roles in nervous system development and maintenance. LINGO‐1, one of this family members, is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. Three LINGO‐1 homologs named LINGO‐2, LINGO‐3, and LINGO‐4 have been described. However, their relative expression and functions remain unexplored. Here, we show by in situ hybridization and quantitative polymerase chain reaction that the transcripts of LINGO homologs are differentially expressed in the central nervous system. The immunostaining of brain slices confirmed this observation and showed the co‐expression of LINGO‐1 with its homologs. Using BRET (bioluminescence resonance energy transfer) analysis, we demonstrate that LINGO proteins can physically interact with each of the other ones with comparable affinities and thus form the oligomeric states. Furthermore, co‐immunoprecipitation experiments indicate that LINGO proteins form heterocomplexes in both heterologous systems and cortical neurons. Since LINGO‐1 is a promising target for the treatment of demyelinating diseases, its ability to form heteromeric complexes reveals a new level of complexity in its functioning and opens the way for new strategies to achieve diverse and nuanced LINGO‐1 regulation.

The phosphatidylethanolamine binding proteins (PEBPs) family is evolutionarily conserved and involved in different physiological phenomena. PEBPs were found in many species from bacteria to mammals. Despite numerous studies, PEBPs' biological function and mode of action remain elusive. Based on sequence homology, seven PEBP genes were detected in the Drosophila genome. Only one of them, the odorant binding protein (OBP), has been characterized. To date nothing is known concerning the expression pattern and biological roles of the six other PEBP genes. By RT-PCR and Western blot analysis, we examined expression of the PEBPs in different tissues and embryos. The 6 PEBPs were differentially expressed. Only one, CG10298, is specific of only one tissue: the testis. Additionally, by comparing in wild type and male-sterile mutants we show that CG10298 is present only during spermatid differentiation. Furthermore, by comparing structural parameters of the six PEBP proteins with those of human PEBP-1, we have established that PEBP CG10298 is most closely related to human PEBP.

Summary: DSP1 is an HMG-box protein which has been implicated in the regulation of homeotic genes in Drosophila melanogaster. Here we report that DSP1 is also involved in the regulation of the kni gap gene. Analysis of the phenotype of a null mutation of dsp1 (dsp11) reveals that the absence of maternal DSP1 results in A4 segmentation defects that are correlated with a diminution of the kni expression domain. Genetic interaction studies demonstrate that a bcd mutation enhances the A4 defect of dsp11. We present in vitro and in vivo evidences for a direct interaction between DSP1 and Bicoid, mediated by the BCD homeodomain and the HMG box of DSP1. Finally, we show by immunoprecipitation of cross-linked chromatin the association of DSP1 with the kni-regulating region and discuss the potential mechanism of DSP1-mediated activation of kni. genesis 36:104–113, 2003. © 2003 Wiley-Liss, Inc.

Lakhal W., Boizot N., Lesage-Descauses M-C., Rogier O., Lainé-Prade V., Laurans F., Ader K., Charpentier J-P., Decoville M., Bénédetti H., Leplé J-C., Pilate G., Déjardin A, 2015. Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG Meeting, 6-8 October 2015, Nancy, France, communication orale.; Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG-Meeting 2015

International audience; Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 muM), 52 for larvae (157 muM/3 muM) and more than 172 for females (>3.1 mM/18 muM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration: 0.391 nM) was 46 000 times lower than the chronic LC50 for males; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.

International audience; In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10 10 80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.

International audience; Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB-like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD-box RNA helicase, Rm62, as partner of DSP1 in a 250-kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3- to 12-h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes.

International audience; CG7054 is a 179-residue protein from Drosophila melanogaster that has been shown from sequence analysis to be a member of the ‘Phosphatidyl-Ethanolamine-Binding Protein' (PEBP) family1 [Pfam PF01161]. This family containing more than 500 proteins is widely distributed from bacteria to mammals. In mammals, bovine PEBP (bPEBP) was initially characterized as a phospholipid binding protein2, but has also been shown to bind nucleotides and opioids3. The most abundant isoform in human, PEBP-1 (hPEBP-1/RKIP), has been described to modulate important cell mechanisms such as the control of heterotrimeric G-proteins4, the inhibition of mitogen-actived protein-kinase5, nuclear factor-kappa B signaling pathways6 and of serine proteases thrombin, neuropsin and chymotrypsin7. Human PEBP-1 is implicated in metastasis formation8 and Alzheimer's disease

International audience

Background The nuclear factor kappa B (NFκB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NFκB DNA binding sequence that promotes an efficient plasmid nuclear import. Methods On the basis of structural studies, the 5′-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3′ segment (termed 3NF) comprising three 10-bp κB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NFκB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical κB motifs. Results A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NFκB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NFκB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. Conclusions The results obtained in the present study demonstrate the NFκB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NFκB, the 3NF sequence is a very promising helper for a nonviral gene delivery system. Copyright © 2009 John Wiley & Sons, Ltd.

Background Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1. Results We show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp1 1 sex comb phenotypes and enhanced Antp Scx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells. Conclusion Our results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFkappaB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly beta-sheets at pH 7. The good dispersion of the crosspeaks on the 1H-15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family.

Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development1. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development2. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.

Background information. The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. Results. In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1. We report here, using a transgenic line for the Scr10.0XbaI-regulatory element, that lack of DSP1 affects the function of a reporter gene in legs' imaginal discs but not in embryos. We show by immunolocalization that DSP1 is recruited on polytene chromosomes to the insertion site of the transgene. Moreover, using chromatin immunoprecipitation experiments, we identify two regions of 1 kb in Scr10.0XbaI as the main DSP1 targets. Conclusion. These results provide strong evidence that the Scr gene expression is influenced by direct interaction between DSP1 and two Scr regulation elements. In addition, our results show that this interaction undergoes dynamic changes during development.

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ( 2 5 3 KKRK 2 5 6 ) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.

International audience; The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.

International audience; Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses zen amongst others. Recently, several proteins have been shown to assist Dorsal in the repression of zen, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of zen but not in the activation of twist. We show that Dorsal has the ability to interact with DSP1 in vitro as well as with rat HMG1. Using truncated versions of the proteins we located the domains of interaction as being the HMG boxes for DSP1 and HMG1 and the Rel domain for Dorsal. Finally, studies of the zen DNA binding properties of Dorsal and another related Rel protein (Gambif1 from Anopheles gambiae) revealed that their DNA binding affinities were increased in the presence of DSP1 and HMG1.

Background Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Results Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Conclusions Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.

International audience; Two DNA restriction fragments containing either a d(GC)5 or a d(TTGCTTGATTAGTTGTGTT) insert were subjected to reaction with cis-diamminedichloroplatinum(II) and were then used as templates for RNA synthesis by T7 RNA polymerase. Within the d(GC)5 insert, interstrand cross-links are preferentially formed. Within the second insert, the reactivity order of the potential binding sites is d(ApG) > d(GpC/GpC) = d(GpA) > d(GpTpG). In the presence of cyanide ions, the adducts are much less stable at the d(GpA) sites than at the d(GpCpG) sites, in double-stranded DNA.

International audience; A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5' part, showing similarity with the 5' end of L1Md-A2, and seven long A + T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.

International audience; A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.

The antibody response against the H-2.2 specificity has been studied in three H-2 d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F1 and (B10.D2 × BALB/c)F1. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 d /H-2 k hybrids (B10.BR × B10.D2)F1 or (B10.BR × DBA/2)F1 are responders when immunized with C57BL/10 (H-2 b ) but not with B10.A(2R) (H-2 h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.

Pesticides have been used extensively and induce harmful effects on organisms and their environment. The aim of my PhD work was to investigate the effects of two insecticides by using Drosophila as a model. The first study concerns the effect of a neonicotinoid, imidacloprid. The results obtained have shown that a chronic exposure at sublethal doses of this insecticide affects reproduction in flies. Moreover, acute exposure at high doses has pointed out a resistance phenomenon in females of a field strain. We have shown that two mechanisms are implicated: (i) low expression of a subunit of a nicotinic acetylcholine receptor that is the target of imidaclopride and (ii) role of glutathione S-transférases, detoxication enzymes, in imidaclopride metabolism. These results highlight impact of insecticides, in particular imidaclopride, on non target organisms. The aim of the second study was to characterize in Drosophila, effects of an organochlorine, dieldrin, potentially implicated in the etiology of Parkinson’s disease in humans. Dieldrin exposure at low doses promotes neurodegeneration of dopaminergic neurons and alters structures of particular regions of the brain. Disruption of metabolism is also induced after exposition to dieldrin. In addition, we have shown that epigenetic processes are implicated in neurodegenration induced by dieldrin. This work show advantages of new techniques such as Magnetic Resonance Imaging (MRI) and High Reslution Magic Angle Spinning in this kind of study.; L’utilisation intensive des produits phytosanitaires, en particulier les insecticides, provoque des effets indésirables sur les organismes vivants et leur environnement. Mon travail de thèse a consisté à évaluer l’effet de deux insecticides chez un insecte modèle la drosophile. Une première étude concernait l’effet d’un néonicotinoïde, l’imidaclopride. Nous avons pu montrer que l’exposition chronique à des doses sublétales de cet insecticide perturbe la fonction de reproduction chez la drosophile. D’autre part, une exposition aiguë à l’imidaclopride a mis en évidence une résistance chez les femelles d’une souche de drosophile dite ``des champs´´. Deux mécanismes différents ont été mis en évidence dans la résistance à l’imidaclopride de cette souche. Le premier concerne la sous expression d’une sous-unité (D1) du récepteur nicotinique à l’acétylcholine, cible de l’imidaclopride. Le deuxième concerne l’implication des glutathion S-transférases, enzymes de détoxification, dans le métabolisme de l’imidaclopride. Ces études montrent que les insecticides peuvent avoir en plus des effets sur les insectes ravageurs, des effets néfastes sur des organismes non cibles. La deuxième étude avait pour but de modéliser chez la drosophile, l’impact d’un organochloré, la dieldrine, potentiellement impliquée dans la maladie de Parkinson chez l’homme. L’exposition à cet insecticide conduit à une dégénérescence des neurones dopaminergiques ainsi qu’une perturbation de la structure de régions particulières du cerveau. Nous avons également montré des altérations du métabolisme et l’implication de processus épigénétiques dans la neurodégénérescence induite par la dieldrine. Au cours de ce travail, nous avons pu montrer l’intérêt de nouvelles méthodes comme l’Imagerie par Résonance Magnétique (IRM) ou le High Resolution Magic Angle Spinning (HRMAS) dans ce type d’étude.

Pesticides have been used extensively and induce harmful effects on organisms and their environment. The aim of my PhD work was to investigate the effects of two insecticides by using Drosophila as a model. The first study concerns the effect of a neonicotinoid, imidacloprid. The results obtained have shown that a chronic exposure at sublethal doses of this insecticide affects reproduction in flies. Moreover, acute exposure at high doses has pointed out a resistance phenomenon in females of a field strain. We have shown that two mechanisms are implicated: (i) low expression of a subunit of a nicotinic acetylcholine receptor that is the target of imidaclopride and (ii) role of glutathione S-transférases, detoxication enzymes, in imidaclopride metabolism. These results highlight impact of insecticides, in particular imidaclopride, on non target organisms. The aim of the second study was to characterize in Drosophila, effects of an organochlorine, dieldrin, potentially implicated in the etiology of Parkinson’s disease in humans. Dieldrin exposure at low doses promotes neurodegeneration of dopaminergic neurons and alters structures of particular regions of the brain. Disruption of metabolism is also induced after exposition to dieldrin. In addition, we have shown that epigenetic processes are implicated in neurodegenration induced by dieldrin. This work show advantages of new techniques such as Magnetic Resonance Imaging (MRI) and High Reslution Magic Angle Spinning in this kind of study., L’utilisation intensive des produits phytosanitaires, en particulier les insecticides, provoque des effets indésirables sur les organismes vivants et leur environnement. Mon travail de thèse a consisté à évaluer l’effet de deux insecticides chez un insecte modèle la drosophile. Une première étude concernait l’effet d’un néonicotinoïde, l’imidaclopride. Nous avons pu montrer que l’exposition chronique à des doses sublétales de cet insecticide perturbe la fonction de reproduction chez la drosophile. D’autre part, une exposition aiguë à l’imidaclopride a mis en évidence une résistance chez les femelles d’une souche de drosophile dite ``des champs´´. Deux mécanismes différents ont été mis en évidence dans la résistance à l’imidaclopride de cette souche. Le premier concerne la sous expression d’une sous-unité (D1) du récepteur nicotinique à l’acétylcholine, cible de l’imidaclopride. Le deuxième concerne l’implication des glutathion S-transférases, enzymes de détoxification, dans le métabolisme de l’imidaclopride. Ces études montrent que les insecticides peuvent avoir en plus des effets sur les insectes ravageurs, des effets néfastes sur des organismes non cibles. La deuxième étude avait pour but de modéliser chez la drosophile, l’impact d’un organochloré, la dieldrine, potentiellement impliquée dans la maladie de Parkinson chez l’homme. L’exposition à cet insecticide conduit à une dégénérescence des neurones dopaminergiques ainsi qu’une perturbation de la structure de régions particulières du cerveau. Nous avons également montré des altérations du métabolisme et l’implication de processus épigénétiques dans la neurodégénérescence induite par la dieldrine. Au cours de ce travail, nous avons pu montrer l’intérêt de nouvelles méthodes comme l’Imagerie par Résonance Magnétique (IRM) ou le High Resolution Magic Angle Spinning (HRMAS) dans ce type d’étude.

In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.; Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l’activation oula répression des gènes. Les profils d’expression génique mis en place vont perdurer d’une générationcellulaire à l’autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d’expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l’activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d’identifier les partenaires de la protéineDSP1 dans l’embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j’aimis en place puis effectué l’immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d’identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j’aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1.