96 research outputs found

    Pharmacological characterisation of neuronal nicotinic acetylcholine receptors

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    Nicotinic acetylcholine receptors (nAChRs) are the targets for the endogenous neurotransmitter acetylcholine and represent a diverse family of ligand-gated ion channels. They are expressed in the neuromuscular junction, the peripheral nervous system and the central nervous system. In the brain, the most prevalent subtypes are the heteromeric α4β2 and homomeric α7 nAChRs. Neuronal nAChRs are implicated in numerous physiological and pathophysiological functions and are therefore important targets for therapeutic drug discovery for conditions such as Alzheimer’s disease, schizophrenia and tobacco addiction. This thesis aims to further our understanding of the pharmacological and molecular characteristics of neuronal nAChRs. Acetylcholine activates nAChRs by binding at an extracellular orthosteric site. Previous studies have described several ligands that potentiate agonist-evoked responses by binding to an allosteric site of the α7 nAChRs that is distinct from the acetylcholine binding site. These ligands, termed positive allosteric modulators (PAMs) can be described as type I, when they have little or no effect on desensitisation, or type II, when they dramatically slow down the fast desensitisation kinetics of the α7 nAChR subtype. Here, a novel series of α7-selective PAMs with a range of effects on receptor desensitisation is described, using recombinant human receptors. This series consists of PAMs with type I and type II profiles, in addition to PAMs with intermediate properties on desensitisation, therefore increasing the nAChR pharmacological toolbox. Furthermore, the effect of a number of mutations on the pharmacological properties of the receptor is investigated. Three point mutations, two in the transmembrane domain (L247T and M260L) and one in the N-terminal domain (W54A), are shown to have the ability to convert PAMs into agonists. Moreover, the M260L mutation displays this property only with PAMs that have a significant effect on receptor desensitisation. These observations can provide insights into the role of these residues on receptor gating and desensitisation. In addition to the studies on recombinant receptors, the expression and functional properties of nAChRs in neurons derived from human induced pluripotent stem cells (iPSC) is examined. The iPSC-derived neurons represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels in a native environment

    Conclusion

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    De Pascal à Heidegger, des Pensées à Sein und Zeit, nous avons franchi un intervalle de trois siècles pour rencontrer, au cœur de la modernité comme à l’ère de la technique, le même constat angoissé de désontologisation de notre univers. Que les Pensées ne puissent être lues qu’eu égard à un projet moderne aux implications acosmiques, voilà qui, du moins nous l’espérons, relève désormais d’une évidence. L’attention pascalienne à l’hétérogénéité des ordres, le rôle reconnu à l’imagination et l..

    Contrasting Properties of α7-Selective Orthosteric and Allosteric Agonists Examined on Native Nicotinic Acetylcholine Receptors.

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    Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays

    The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

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    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties

    Pathological Features of Breast Cancer seen in Northwestern Tanzania: A Nine Years Retrospective Study.

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    Breast cancer is more common in Western Countries compared to African populations. However in African population, it appears that the disease tends to be more aggressive and occurring at a relatively young age at the time of presentation. The aim of this study was to describe the trend of Breast Cancer in Northwestern Tanzania. This was a retrospective study which involved all cases of breast cancer diagnosed histologically at Bugando Medical Center from 2002 to 2010. Histological results and slides were retrieved from the records in the Pathology department, clinical information and demographic data for patients were retrieved from surgical wards and department of medical records. Histology slides were re-evaluated for the histological type, grade (By modified Bloom-Richardson score), and presence of necrosis and skin involvement. Data was entered and analyzed by SPSS computer software version 15. There were 328 patients histologically confirmed to have breast cancer, the mean age at diagnosis was 48.7 years (+/- 13.1). About half of the patients (52.4%) were below 46 years of age, and this group of patients had significantly higher tendency for lymph node metastasis (p = 0.012). The tumor size ranged from 1 cm to 18 cm in diameter with average (mean) of 5.5 cm (+/- 2.5), and median size of 6 cm. Size of the tumor (above 6 cm in diameter) and presence of necrosis within the tumor was significantly associated with high rate of lymph node metastasis (p = 0.000). Of all patients, 64% were at clinical stage III (specifically IIIB) and 70.4% had lymph node metastasis at the time of diagnosis. Only 4.3% of the patients were in clinical stage I at the time of diagnosis. Majority of the patients had invasive ductal carcinoma (91.5%) followed by mucinous carcinoma (5.2%), Invasive lobular carcinoma (3%) and in situ ductal carcinoma (0.3%). In all patients, 185 (56.4%) had tumor with histological grade 3. Breast cancer in this region show a trend towards relative young age at diagnosis with advanced stage at diagnosis and high rate of lymph node metastasis. Poor Referral system, lack of screening programs and natural aggressive biological behavior of tumor may contribute to advanced disease at the time of diagnosis

    Where two worlds meet: language policing in mainstream and complementary schools in England

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    We compare language policing in two educational contexts in England: mainstream schools and complementary schools. We draw on a varied dataset (policy documents, in-class observations, interviews) collected from mainstream schools and Greek complementary schools in London. We find similarities in how the two types of schools control, regulate, monitor and suppress the language of school students. Both settings hierarchise standardised and non-standardised varieties in institutional policies that delegitimise the non-standardised varieties. Teachers become vehicles for language ideologies in enacting monovarietal policies drawing on discourses around academic success and the primacy of written language over spoken language, including regional varieties such as Cypriot Greek. Our findings suggest that multilingual and multidialectal students in England who attend both mainstream and complementary schools are exposed to similar kinds of prescriptive discourses across the whole spectrum of their educational experiences, which can have a range of negative effects on their learning and the construction of their self-image. We argue that more links need to be forged between the two educational settings and that these should include the development of integrated pedagogies and policies that legitimise students’ whole linguistic repertoires, encompassing both their standardised and their non-standardised varieties as well as their other linguistic resources

    Polyimide hollow fiber membranes for CO2 separation from wet gas mixtures

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    Matrimid®5218 hollow fiber membranes were prepared using the dry-wet spinning process. The transport properties were measured with pure gases (H2, CO2, N2, CH4 and O2) and with a mixture (30% CO2 and 70% N2) in dry and wet conditions at 25 ºC, 50 ºC, 60 ºC and 75 ºC and up to 600 kPa. Interesting values of single gas selectivity up to 60 ºC (between 31 and 28 for CO2/N2 and between 33 and 30 for CO2/CH4) in dry condition were obtained. The separation factor measured for the mixture was 20% lower compared to the single gas selectivity, in the whole temperature range analyzed. In saturation conditions the data showed that water influences the performance of the membranes, inducing a reduction of the permeance of all gases. Moreover, the presence of water caused a decrease of single gas selectivity and separation factor, although not so significant, highlighting the very high water resistance of hollow fiber membrane modules

    Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China

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    Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis

    The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region

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    BACKGROUND: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. RESULTS: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. CONCLUSION: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations
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