The UniProtKB/Swiss-Prot Tox-Prot program: A central hub of integrated venom protein data.

Animal toxins are of interest to a wide range of scientists, due to their numerous applications in pharmacology, neurology, hematology, medicine, and drug research. This, and to a lesser extent the development of new performing tools in transcriptomics and proteomics, has led to an increase in toxin discovery. In this context, providing publicly available data on animal toxins has become essential. The UniProtKB/Swiss-Prot Tox-Prot program (http://www.uniprot.org/program/Toxins) plays a crucial role by providing such an access to venom protein sequences and functions from all venomous species. This program has up to now curated more than 5000 venom proteins to the high-quality standards of UniProtKB/Swiss-Prot (release 2012_02). Proteins targeted by these toxins are also available in the knowledgebase. This paper describes in details the type of information provided by UniProtKB/Swiss-Prot for toxins, as well as the structured format of the knowledgebase.

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hibridization whit the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O?H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products - for example, adhesins and toxins - and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

Enterotoxigenic and nontoxigenic Bacteroides fragilis strains isolated in Brazil

The presence of enterotoxigenic Bacteroides fragilis and nontoxigenic B. fragilis (NTBF) among 109 strains isolated from 1980-2008 in Brazil were investigated by PCR. One strain, representing 0.9% of the total analyzed strains, harbored the bft gene which was identified as bft-1 isoform based on PCR-RFLP and sequencing. Forty-nine strains (44.9%) exhibited the NTBF pattern III which possesses the flanking region required for pathogenicity island acquisition in which the bftgene is codified. These data reinforce the potential of B. fragilis as an emerging enteropathogen in our country.

Necrotising pneumonia due to influenza A (H1N1) and community-acquired methicillin-resistant Staphylococcus aureus clone USA300: successful management of the first documented paediatric case.

Necrotising pneumonia in young, previously healthy patients due to Panton–Valentine leucocidin (PVL) producing Staphylococcus aureus has been increasingly recognised. PVL pneumonia is often associated with influenza co-infection and high mortality. This case report describes the successful management of the first documented paediatric case of a previous healthy adolescent who developed necrotising pneumonia due to community-acquired methicillin-resistant (CA-MRSA) clone USA300 with pandemic influenza A (H1N1) co-infection, and highlights the importance of early recognition and initiation of appropriate therapy for this potentially fatal co-infection. PCR remains the gold standard to diagnose pandemic H1N1 since it may not be detected by rapid antigen tests. Bacterial necrotising pneumonia should be suspected in those presenting with worsening flu-like symptoms and clinical and/or radiological evidence of PVL infection (multifocal infiltrates, effusion and cavitation). These patients may benefit from the administration of toxin neutralising agents. In light of the current H1N1 pandemic, healthcare professionals will be increasingly confronted with this clinical scenario.

Rac2 GTPase activation by angiotensin II is modulated by Ca2C/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.

Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

Analysis of GPCR properties of Frizzled receptors and establishment of the "in vitro" platform for screening of Frizzled agonists/antagonists as potential therapeutic agents

Morphogens of the Wnt protein family are the secreted lipoglycoprotein ligands which initiate several pathways heavily involved in the coordination of various developmental stages of organisms in the majority of animal species. Deregulation of these pathways in the adult leads to formation and sustaining of multiple types of cancer. The latter notion is reinforced by the fact that the very discovery of the first Wnt ligand was due to its role as the causative factor of carcinogenic transformation (Nusse and Varmus, 1982). Nowadays our knowledge on Wnt signaling has "moved with the times" and these pathways were identified to be often crucial for tumor formation, its interactions with the microenvironment, and promotion of the metastases (Huang and Du, 2008; Zerlin et al., 2008; Jessen, 2009). Thus the relevance of the pathway as the target for drug development has further increased in the light of modern paradigms of the complex cancer treatments which target also spreading and growth- promoting factors of tumors by specific and highly efficient substances (Pavet et al., 2010). Presently the field of the Wnt-targeting drug research is almost solely dominated by assays based on transcriptional activation induced by the signaling. This approach resulted in development of a number of promising substances (Lee et al., 2011). Despite its effectiveness, the method nevertheless suffers from several drawbacks. Among the major ones is the fact that this approach is prone to identify compounds targeting rather downstream effectors of the pathway, which are indiscriminately used by all the subtypes of the Wnt signaling. Additionally, proteins which are involved in several signaling cascades and not just the Wnt pathway turn out as targets of the new compounds. These issues increase risks of side effects due to off-target interactions and blockade of the pathway in healthy cells. In the present work we put forward a novel biochemical approach for drug development on the Wnt pathway. It targets Frizzleds (Fzs) - a family of 7-transmbembrane proteins which serve as receptors for Wnt ligands. They offer unique properties for the development of highly specific and effective drugs as they control all branches of the Wnt signaling. Recent advances in the understanding of the roles of heterotrimeric G proteins downstream from Fzs (Katanaev et al., 2005; Liu et al., 2005; Jernigan et al., 2010) suggest application of enzymatic properties of these effectors to monitor the receptor-mediated events. We have applied this knowledge in practice and established a specific and efficient method based on utilization of a novel high-throughput format of the GTP-binding assay to follow the activation of Fzs. This type of assay is a robust and well-established technology for the research and screenings on the GPCRs (Harrison and Traynor, 2003). The conventional method of detection involves the radioactively labeled non-hydrolysable GTP analog [35S]GTPyS. Its application in the large-scale screenings is however problematic which promoted development of the novel non-radioactive GTP analog GTP-Eu. The new molecule employs phenomenon of the time-resolved fluorescence to provide sensitivity comparable to the conventional radioactive substance. Initially GTP-Eu was tested only in one of many possible types of GTP-binding assays (Frang et al., 2003). In the present work we expand these limits by demonstrating the general comparability of the novel label with the radioactive method in various types of assays. We provide a biochemical characterization of GTP-Eu interactions with heterotrimeric and small GTPases and a comparative analysis of the behavior of the new label in the assays involving heterotrimeric G protein effectors. These developments in the GTP-binding assay were then applied to monitor G protein activation by the Fz receptors. The data obtained in mammalian cultured cell lines provides for the first time an unambiguous biochemical proof for direct coupling of Fzs with G proteins. The specificity of this interaction has been confirmed by the experiments with the antagonists of Fz and by the pertussis toxin-mediated deactivation. Additionally we have identified the specificity of Wnt3a towards several members of the Fz family and analyzed the properties of human Fz-1 which was found to be the receptor coupled to the Gi/o family of G proteins. Another process playing significant role in the functioning of every GPCR is endocytosis. This phenomenon can also be employed for drug screenings on GPCRs (Bickle, 2010). In the present work we have demonstrated that Drosophila Fz receptors are involved in an unusual for many GPCRs manifestation of the receptor-mediated internalization. Through combination of biochemical approaches and studies on Drosophila as the model organism we have shown that direct interactions of the Fzs and the α-subunit of the heterotrimeric G protein Go with the small GTPase Rab5 regulate internalization of the receptor in early endosomes. We provide data uncovering the decisive role of this self-promoted endocytosis in formation of a proper signaling output in the canonical as well as planar cell polarity (PCP) pathways regulated by Fz. The results of this work thus establish a platform for the high-throughput screening to identify substances active in the cancer-related Wnt pathways. This methodology has been adjusted and applied to provide the important insights in Fz functioning and will be instrumental for further investigations on the Wnt-mediated pathways.

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.

Acid-sensing ion channels : modulation by serine-proteases and involvement in acid-induced action potential generation in hippocampal neurons

SUMMARY Acid-sensing ion channels (ASICs) are non-voltage gated sodium channels. They are activated by rapid extracellular acidification and generate an inactivating inward current. Four ASIC genes have been cloned: ASIC1, 2, 3 and 4, with variants a and b for ASIC1and AS1C2. ASICs are expressed in neurons of the central (CNS) and peripheral nervous system (PNS). In the CNS, ASICs have a role in learning, memory, as well as in neuronal death in ischemia. In the PNS, ASICs are involved in the perception of acid-induced pain, as well as in mechanoperception. In one part of my thesis project, we addressed the question of the mechanism of regulation of ASIC1 a by the serine protease trypsin at the molecular level. Trypsin modifies the function of ASIC1 a but not of ASIC1b. In order to identify the channel region responsible for this effect, we created chimeras between ASIC1 a and 1b. Subsequently, to identify the exact trypsin target(s), we mutated predicted trypsin sites in the region identified by the chimera. In the second part of a project, we investigated the role of ASICs at the cellular level, in neuronal signaling. Using the whole-cell patch clamp in hippocampal neuronal culture, we studied the potential involvement of ASICs in action potential (AP) generation. In the first part of the thesis work, we showed that trypsin modifies ASIC1a function: it shifts the pH activation and the steady-state inactivation curve towards more acidic values and accelerates the time course of the channel recovery from inactivation. We also showed that trypsin cleaves ASIC1a and that the functional effect and a channel cleavage correlate. In the inactivated state, channels cannot be modified by trypsin. Cleavage occurs in a channel region that is also important for inactivation of all ASICs; a part of this region is critical for the inhibition of ASIC1 a by the spider toxin Psalmotoxin1. In the second part of the thesis work, we showed that ASIC activity can modulate AP generation. ASIC activity by itself can induce trains of APs. In situations in which this activity by itself is not sufficient to induce APs, it can contribute to AP generation. During high neuronal activity, ASIC activity can block already existing trains of APs. In conclusion, depending on the activity of neuron in a particular moment, ASICs can differently modulate AP generation; they can induce, facilitate or inhibit APs. We also showed that trypsin changes the capability of ASICs to modulate AP generation by shifting the pH dependence to more acidic values, which adapts channel gating to pH conditions which may occur in pathological conditions such as ischemia. Our finding that trypsin modifies ASIC1 a function identifies a novel pharmacological tool, and proposes a mechanism of ASIC1a regulation that may have a physiological importance. The identification of the exact site of trypsin action gives insight to the molecular mechanisms of ASIC regulation. This work proposes a role in modulation of AP generation for ASICs in the CNS. RESUME Les canaux ASIC sont les canaux ioniques activés par l'acidification rapide extracellulaire. Activés, ils génèrent un courant entrant qui inactive en présence de stimulus acide. Quatre gènes ASIC ont été clonés, ASIC1, 2, 3 et 4, avec les variants a et b pour ASIC1 et 2. Les ASICs sont exprimés dans les neurones du système nerveux central (SNC) et périphérique (SNP). Dans le SNC, les ASIC ont un rôle dans le mémoire, apprentissage et la mort neuronale dans t'ischémie. Dans le SNP, ils ont un rôle dans la perception de la douleur et méchanosensation. Dans une partie de mon projet de thèse, nous avons étudié les mécanismes de la régulation d'ASIC1a par la sérine-protéase trypsine au niveau moléculaire. La trypsine modifie la fonction d'ASIC1a et pas ASIC1b. Nous avons créé les chimères entre ASIC1 a et 1 b, afin d'identifier la région du canal responsable pour l'effet. Pour identifier le(s) site(s) exactes de l'action de la trypsine, nous avons muté les sites potentiels de la trypsine dans la région identifiée par les chimères. Dans la deuxième partie du projet, nous avons étudié le rôle des ASICs au niveau cellulaire. En utilisant la technique du patch clamp dans les cultures des neurones de l'hippocampe, nous avons étudié l'implication des ASICs dans la génération des potentiels d'action (PA). Nous avons montré que la trypsine agit sur le canal ASIC1a ; elle décale l'activation et « steady-state » inactivation vers les valeurs plus acides, et elle raccourcit le temps du « recovery » du canal. La trypsine coupe ASIC1a sur le résidu K145 et l'effet fonctionnel et la coupure corrèlent. Nous avons identifié la région du canal responsable pour l'inactivation de tous les ASICs ; une partie de cette région est responsable pour ['inhibition d'ASIC1 a par la Psalmotoxinel . Nous avons montré que les ASICs peuvent moduler la génération des PAs. L'activité des ASICs peut induire les trains des PAs. Quand l'activité des ASICs n'est pas suffisante pour induire le PA, elle peut contribuer à sa génération. Pendant l'activité neuronale forte, l'activité des ASICs peut bloquer les trains des PAs qui existent déjà. En conclusion, dépendant de l'activité neuronale, les ASICs peuvent moduler la génération des PAs différemment ; ils peuvent induire, faciliter ou inhiber les PAs. La trypsine change la capacité des ASICs de moduler les PAs. Après l'action de la trypsine, les ASICs peuvent moduler la génération des PAs dans les conditions légèrement acides, suivies par les fluctuations du pH acide, qui peuvent exister dans l'ischémie. Le fait que la trypsine agit sur ASIC1a définit l'outil pharmacologique et propose le mécanisme de la régulation d'ASICI a qui pourrait avoir l'importance physiologique. L'identification du site de l'action de la trypsine éclaircit les mécanismes moléculaires de la régulation des ASICs. Cette étude propose un rôle des ASICs dans la modulation de la génération des PAs. Résumé pour le public large Les neurones sont les cellules de système nerveux dont la fonction est la signalisation. Comme toutes les autres cellules, les neurones ont une membrane qui sépare l'intérieur du milieu extérieur. Cette membrane est imperméable pour des particules chargées (ions). Dans cette membrane existent les protéines spécifiques, « canaux », qui permettent le transport des ions d'un côté de la membrane à l'autre, comme réponse aux stimuli différents. Ce transport des ions à travers la membrane génère un courant, qu'on peut mesurer. Ce courant est la base de la communication entre les neurones, ou, ce qu'on appelle la signalisation neuronale. Quand ce courant est suffisamment grand, il permet la génération du potentiel d'action, qui est le message principal de communication neuronale. Les canaux ASIC (acid-sensing ion channel), que nous étudions dans le laboratoire, sont activés par les acides. Les acides sont relâchés dans beaucoup de situations dans le système nerveux. Les ASIC ont été découverts récemment (en 1996), et nous ne connaissons pas encore très bien toutes les fonctions de ces canaux. Nous savons qu'ils ont un rôle dans le mémoire, apprentissage, la sensation de la douleur et l'infarctus cérébral. Dans la première partie de ce projet de thèse, nous avons voulu mieux comprendre comment fonctionnent ces canaux. Pour faire ça, nous avons étudié la régulation des ASICs par une protéine, trypsine, qui coupe le canal ASIC. Nous avons étudié ou exactement la trypsine coupe le canal et quels effets ça produit sur la fonction du canal. Dans la deuxième partie du projet de thèse, nous avons voulu mieux connaître comment le canal fonctionne au niveau de la cellule, comment il interagit avec les autres canaux et si il a un rôle dans la génération des potentiels d'action. Nous avons pu montrer que la trypsine change la fonction du canal, ce qui lui permet de fonctionner différemment. Nous avons aussi déterminé ou exactement ta trypsine coupe le canal. Au niveau de la cellule, nous avons montré que les ASIC peuvent moduler la génération des potentiels d'action, étant, dépendant de l'activité du neurone, soit activateurs, soit inhibiteurs. La trypsine est une molécule qui peut être libérée dans le système nerveux pendant certaines conditions, comme l'infarctus cérébral. A cause de ça, les connaissances que la trypsine agit sur le anal ASIC pourraient être important physiologiquement. La connaissance de l'endroit exacte ou la trypsine coupe le canal nous aide à mieux comprendre la relation structure-fonction du canal. La modulation de la génération des potentiels d'actions par les ASIC indique que ces canaux peuvent avoir un rôle important dans la signalisation neuronale.

OnabotulinumtoxinA and multiple sclerosis.

Lower urinary tract dysfunction is present in two of three patients with multiple sclerosis five years after the diagnosis. Most frequent symptoms are related to neurogenic detrusor overactivity, often associated with detrusor-sphincter dyssynergia. From the end of the 1990s, there is growing evidence that neurogenic detrusor overactivity can be effectively managed by intradetrusorial injections of botulinum toxin type A. This treatment has shown, in different randomised placebo-controlled trials, to be safe and effective on clinical and urodynamic parameters with significant improvement in quality of life. The median duration of effect is in mean nine months. The vast majority of studies have been conducted with onabotulinumtoxinA. The dose of onabotulinumtoxinA commonly used to treat neurogenic detrusor overactivity in patients with multiple sclerosis is 200 UI, even if in selected patients lower doses can be preferred. To be considered eligible for treatment, all patients should accept and be instructed to perform clean intermittent self-catheterisation, since the risk of increased post-void residual volume and/or urinary retention after injection is high, especially with 200 UI of onabotulinumtoxinA. However, quality of life and patient satisfaction seem not to be affected by the need of intermittent catheterisation. The risk of urinary infection after the procedure is to be kept in mind, mainly in patients with multiple sclerosis, so that adequate antibiotic prophylaxis is highly recommended.