Post-translational modifications regulate distinct functions of CARMA1 and BCL10.

Activation of the transcription factor nuclear factor (NF)-kappaB is essential for the normal functioning of the immune system. Deregulated NF-kappaB signalling in lymphocytes can lead to immunodeficiency, but also to autoimmunity or lymphomas. Many of the signalling components controlling NF-kappaB activation in lymphocytes are now known, but it is less clear how distinct molecular components of this pathway are regulated. Here, we summarize recent findings on post-translational modifications of intracellular components of this pathway. Phosphorylation of the CARMA1 and BCL10 proteins and ubiquitylation of BCL10 affect the formation and stability of the CARMA1-BCL10-MALT1 (CBM) complex, and also control negative feedback regulation of the NF-kappaB signalling pathway. Moreover, the study of BCL10 phosphorylation isoforms has revealed a new mechanism controlling BCL10 nuclear translocation and an unexpected role for BCL10 in the regulation of the actin cytoskeleton.

Caffeine consumption attenuates neurochemical modifications in the hippocampus of streptozotocin-induced diabetic rats.

Type 1 diabetes can affect hippocampal function triggering cognitive impairment through unknown mechanisms. Caffeine consumption prevents hippocampal degeneration and memory dysfunction upon different insults and is also known to affect peripheral glucose metabolism. Thus we now characterized glucose transport and the neurochemical profile in the hippocampus of streptozotocin-induced diabetic rats using in vivo(1)H NMR spectroscopy and tested the effect of caffeine consumption thereupon. We found that hippocampal glucose content and transport were unaltered in diabetic rats, irrespective of caffeine consumption. However diabetic rats displayed alterations in their hippocampal neurochemical profile, which were normalized upon restoration of normoglycaemia, with the exception of myo-inositol that remained increased (36 +/- 5%, p < 0.01 compared to controls) likely reflecting osmolarity deregulation. Compared to controls, caffeine-consuming diabetic rats displayed increased hippocampal levels of myo-inositol (15 +/- 5%, p < 0.05) and taurine (23 +/- 4%, p < 0.01), supporting the ability of caffeine to control osmoregulation. Compared to controls, the hippocampus of diabetic rats displayed a reduced density of synaptic proteins syntaxin, synaptophysin and synaptosome-associated protein of 25 kDa (in average 18 +/- 1%, p < 0.05) as well increased glial fibrillary acidic protein (20 +/- 5%, p < 0.05), suggesting synaptic degeneration and astrogliosis, which were prevented by caffeine consumption. In conclusion, neurochemical alterations in the hippocampus of diabetic rats are not related to defects of glucose transport but likely reflect osmoregulatory adaptations caused by hyperglycemia. Furthermore, caffeine consumption affected this neurochemical adaptation to high glucose levels, which may contribute to its potential neuroprotective effects, namely preventing synaptic degeneration and astrogliosis.

L'apport de la trace matérielle dans l'enquête criminelle: évaluation de la contribution des liens chimiques issus du profilage de produits stupéfiants par l'analyse des réseaux sociaux

La trace matérielle dispose d'un potentiel incomparable pour mettre en relation des indices et occupe souvent une place centrale lors du procès pénal (Ribaux et Margot, 2008). Au delà de son rôle élémentaire en tant qu'élément de preuve, la trace matérielle véhicule de nombreuses autres informations encore trop peu exploitées en matière d'investigation ou pour servir de base au renseignement criminel. C'est particulièrement le cas des liens chimiques découverts grâce au profilage physico-chimique de produits stupéfiants saisis par les autorités. Le but de ce travail est d'estimer l'apport de ces liens chimiques à la perception de la structure des réseaux criminels. Pour ce faire, nous nous appuyons sur des modèles d'analyse des réseaux sociaux (SNA, Social Network Analysis). Ces modèles sont toujours plus utilisés pour traiter des affaires qui relèvent de la criminalité organisée, notamment pour comprendre la structure et la dynamique de ces réseaux d'acteurs. L'analyse des réseaux sociaux (SNA) procède par le calcul de différents indicateurs pour mettre en évidence des éléments pertinents de leur structure et leurs acteurs clés. Par exemple, l'identification des vulnérabilités de l'organisation et des mécanismes mis en place par les acteurs pourra orienter les actions policières. Cette recherche se base sur une enquête concernant un réseau international de trafic de produits stupéfiants. En plus des autres informations d'enquête traditionnellement disponibles, des liens chimiques ont été mis en évidence par le profilage de l'héroïne saisie à différents endroits et périodes, puis utilisés par les enquêteurs. Cette recherche consiste à utiliser les indicateurs SNA pour évaluer l'influence de ces liens chimiques sur la structure de ce réseau construit à partir des autres indices. Les premiers résultats montrent que les liens chimiques sont cohérents avec les autres types de relations, et qu'ils tendent, à eux seuls, à mettre en évidence les positions centrales de certains acteurs clés. De plus, l'intégration de ces liens renforce les centralités de certains acteurs et permet ainsi de les considérer de manière plus centrale qu'ils ne l'étaient auparavant. Enfin, ces premiers résultats laissent entrevoir des perspectives intéressantes notamment au travers de la pondération de ces relations afin d'évaluer de manière plus globale l'apport du potentiel informatif de ce type de renseignement.

Médicaments et grossesse : modifications pharmacocinétiques et place du suivi thérapeutique pharmacologique [Pharmacokinetic Alterations in Pregnancy and Use of Therapeutic Drug Monitoring].

Following the thalidomide tragedy, pharmacological research in pregnant women focused primarily on drug safety for the unborn child and remains only limited regarding the efficacy and safety of treatment for the mother. Significant physiological changes during pregnancy may yet affect the pharmacokinetics of drugs and thus compromise its efficacy and/or safety. Therapeutic drug monitoring (TDM) would maximize the potential effectiveness of treatments, while minimizing the potential risk of toxicity for the mother and the fetus. At present, because of the lack of concentration-response relationship studies in pregnant women, TDM can rely only on individual assessment (based on an effective concentration before pregnancy) and remains reserved only to unexpected situations such as signs of toxicity or unexplained inefficiency.

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Optimisation de la cryo microscopie électronique pour les études des minicercles d'ADN

RESUME : Bien que les propriétés physiques de la structure de l'ADN aient été intensivement étudiées pendant plus de 50 ans il y a encore beaucoup de questions importantes qui attendent des réponses. Par exemple, qu'arrive-t-il à la structure de la double hélice d'ADN nue (sans protéines liées) lorsqu'elle est fortement courbée, de la même manière que dans les nucléosomes? Cet ADN nu est-il facilement plié (il reste dans le régime élastique) ou réduit-il la contrainte de flexion en formant des sites hyperflexibles «kinks» (il sort du régime élastique en cassant l'empilement des paires de bases à certains endroits) ? La microscopie électronique peut fournir une réponse à cette question par visualisation directe des minicercles d'ADN de la longueur d'un tour de nucléosome (environ 90 paires de bases). Pour que la réponse soit scientifiquement valide, on doit observer les molécules d'ADN lorsqu'elles sont en suspension dans la solution d'intérêt et sans que des colorations, produits chimiques ou fixatifs n'aient été ajoutés, étant donné que ceux-ci peuvent changer les propriétés de l'ADN. La technique de la cryo-microscopie électronique (cryo-EM) développée par le groupe de Jacques Dubochet au début des années 80, permet la visualisation directe des molécules d'ADN suspendues dans des couche minces vitrifiées de solutions aqueuses. Toutefois, le faible contraste qui caractérise la cryo-EM combinée avec la très petite taille des minicercles d'ADN rendent nécessaire l'optimisation de plusieurs étapes, aussi bien dans la préparation des échantillons que dans le processus d'acquisition d'images afin d'obtenir deux clichés stéréo qui permettent la reconstruction 3-D des minicercles d'ADN. Dans la première partie de ma thèse, je décris l'optimisation de certains paramètres pour la cryoEM et des processus d'acquisition d'image utilisant comme objets de test des plasmides et d'autres molécules d'ADN. Dans la deuxième partie, je .décris comment j'ai construit les minicercles d'ADN de 94 bp et comment j'ai introduit des modifications structurelles comme des coupures ou des lacunes. Dans la troisième partie, je décris l'analyse des reconstructions des rninicercles d'ADN. Cette analyse, appuyée par des tests biochimiques, indique fortement que des molécules d'ADN sont capables de former de petites molécules circulaires de 94 bp sans dépasser les limites d'élasticité, indiquant que les minicercles adoptent une forme circulaire régulière où la flexion est redistribuée le long la molécule. ABSTRACT : Although physical properties of DNA structure have been intensively studied for over 50 years there are still many important questions that need to be answered. For example, what happens to protein-free double-stranded DNA when it is strongly bent, as in DNA forming nucleosomes? Is such protein-free DNA smoothly bent (i.e. it remains within elastic limits of DNA rigidity) or does it release its bending stress by forming sharp kinks (i.e. it exits the elastic regime and breaks the stacking between neighbouring base-pairs in localized regions)? Electron microscopy can provide an answer to this question by directly visualizing DNA minicircles that have the size of nucleosome gyres (ca 90 bp). For the answer to be scientifically valid, one needs to observe DNA molecules while they are still suspended in the solution of interest and no staining chemicals or fixatives have been added since these can change the properties of the DNA. CryoEM techniques developed by Jacques Dubochet's group beginning in the 1980's permit direct visualization of DNA molecules suspended in cryo-vitrified layers of aqueous solutions. However, a relatively weak contrast of cryo-EM preparations combined with the very small size of the DNA minicircles made it necessary to optimize many of the steps and parameters of the cryo-EM specimen preparation and image acquisition processes in order to obtain stereo-pairs of images that permit the 3-D reconstruction of the observed DNA minicircles. In the first part of my thesis I describe the optimization of the cryo-EM preparation and the image acquisition processes using plasmid size DNA molecules as a test object. In the second part, I describe how I formed the 94 by DNA minicircles and how I introduced structural modifications like nicks or gaps. In the third part, I describe the cryo-EM analysis of the constructed DNA minicircles. That analysis, supported by biochemical tests, strongly indicates that DNA minicircles as small as 94 by remain within the elastic limits of DNA structure, i.e. the minicircles adopt a regular circular shape where bending is redistributed along the molecules.

Evaluation des modifications anatomiques et fonctionnelles de la macula après chirurgie des membranes néovasculaires rétrofovéolaires dans la dégénérescence maculaire liée à l'âge [Evaluation of functional and anatomical changes in the macula after surgery for retro-foveal neovascularization of membranes in age-related macular degeneration].

PURPOSE: The aim of this study was to evaluate the direct effect of surgical treatment of subfoveolar neovascular membranes in age related macular degeneration to macular functions. PATIENTS AND METHODS: Thirteen eyes of 13 patients were included in this study. Macular function was assayed by visual acuity and central visual field using the Octopus perimeter before surgery and in the first three post operative months. Pre and post operative fluorescein angiography frames were digitalized and the size of each lesions were compared. RESULTS: After a 3 months follow up, visual acuity remained stable or improved in 66% of the patients. However, visual acuity was better than 0.1 in 15% of the patients. Central visual field comparison disclosed a significant worsening of the retinal sensitivity in the 3 degree field surrounding the central point. On fluorescein frames, submacular scar was 141% of the size of the neovascular membrane. After a mean follow up of 6.9 months (range 3-14), one case of recurrence occurred. A cataract was observed in 85% of the phakic patients followed for more than six months. CONCLUSION: After a short term follow up, surgery can stabilise visual acuity, even though it remains poor. A worsening of the scotoma in the 3 degrees surrounding the central point is observed. However, patients noticed a subjective visual improvement in 62% of the case.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging.

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Sleep deprivation induces transcriptional modifications of genes related to astrocyte-neuron lactate shuttle in astrocytes

Astrocytes play a key role in the neurometabolic coupling through the glycogen metabolism and the ''Astrocyte-Neuron Lactate Shuttle'' (ANLS). We previously reported that brain glycogen metabolism was affected by sleep deprivation (SD). Therefore, it is of prime interest to determine if a similar sleep loss also affects the ANLS functioning in astrocytes. To address this issue, we sleep deprived transgenic mice expressing the GFP under the control of the GFAP promoter and in which astrocytes can be isolated by FACS. The levels of expression of genes related to ANLS were assessed by qRT-PCR in the GFP-positive cells (GFPþ). The FVB/NTg( GFAP-GFP)Mes14/j mice were weaned at P20-P21 and underwent an instrumental 6 h SD at P23-P27. The SD was realized using the ''CaResS'' device which has been designed to minimize stress during SD. Control group corresponds to undisturbed mice. At the end of SD, mice were sacrificed and their cerebral cortex was rapidly dissected, cut in small pieces and enzymatically digested. After cell dissociation, GFPþ and GFP- cells were sorted by FACS and treated for RNA extraction. A quantitative RTPCR was realized using specific probes against different genes involved in ANLS. Results indicate that genes encoding the LDHb, the GLT1, the alpha2 subunit of the Na/KATPase pump as well as the GLUT1, were significantly increased in the GFPþ cells from SD mice. No significant change was observed in the GFP- cells from the same group. These results indicate that this approach is suitable to determine the transcriptional signature of SD in glial cells from juvenile animals. They also indicate that sleep loss induces transcriptional changes of genes involved in ANLS specifically in astrocytes. This could suggest that an adaptation of the ANLS at the transcriptional levels exists in pathophysiological conditions where neuronal activity is enhanced.

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation - A Review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.

Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.