362 research outputs found

    Characterisation of M2 muscarinic acetylcholine receptor signalling in dental pulp stem cells

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    Cholinergic signalling is hypothesised to occur in stem cells, and there is evidence that mesenchymal stem cells (MSCs) express a functional cholinergic system. Expression of functional acetylcholine receptors (AChRs) have been reported in several types of MSC, which suggests that MSCs have non-neuronal cholinoceptive properties that may play a role in their regenerative potential. However, this remains relatively unexplored, particularly, in Dental pulp stem cells (DPSCs). This project commenced by reviewing AChRs in MSCs, highlighting DPSCs characteristics, and then investigated the presence of functional AChRs and their role in modulating DPSCs regenerative potential. This study commenced by identifying gene expression of both classes of AChRs, the muscarinic (mAChRs) and the nicotinic (nAChRs), in DPSCs. Protein expression of detected AChRs was assessed via western blotting and immunofluorescence. Functionality of expressed AChRs was assessed using an array of AChRs agonists and antagonists and DPSCs viable count was measured via MTT assay. Subtype selective agonist was used to study the role of the targeted AChR and its influence on DPSCs regenerative potential. Proliferation of DPSCs in response to that stimulation was assessed via measuring viable cell count using MTT assay, Cell Counting Kit-8 (CCK-8), and cell cycle analysis. Survival of DPSCs was assessed via detecting proliferation recovery, measuring Lactate dehydrogenase (LDH) levels, and detecting Annexin V/Propidium iodide staining. Stemness potential of DPSCs was assessed via detecting gene expression of MSCs stemness markers and pluripotency markers. Migration of DPSCs was investigated using a wound healing assays. Osteogenic differentiation of DPSCs was assessed via phenotypic mineralisation stains. Gene expression of cell cycle markers, stemness markers, osteogenic markers were assessed via Real-time polymerase chain reaction (q-PCR). Whole RNA sequencing (RNA-seq) was undertaking to measure transcriptome changes and enriched signalling pathways. Follow-up analysis was undertaking via measuring the phosphorylation and transcripts levels of ERK1 and ERK2 of the Mitogen-activated protein kinase (MAPK) pathway. The results showed transcripts expression for the M2, M3 and M5 mAChRs, and expression of subunits that support the formation of α7 and α4β2-nAChRs. Subtype selective agonists/antagonists results suggest DPSCs to express functional M2 mAChR, α7 nAChRs, and α4β2-nAChRs. This was based on the ability of the agonists to influence DPSCs viable count and the subtype selective antagonist to cancel that effect. The project then focussed on mAChRs and protein expression of M2, M3 and M5 mAChRs were detected. The subsequent work focused on investigating the role of the M2 mAChRs in modulating the function of DPSCs via activating this receptor through its selective agonist Arecaidine propargyl ester (APE). Activation of the M2 mAChR inhibited DPSCs proliferation, in a reversable manner, without affecting DPSCs viability or survival. Further evidence showed that the M2 mAChR inhibits DPSCs proliferation by arresting cell cycle progression. This was further corroborated via expression analysis of key genes involved in the regulating cell cycle. The results also showed that M2 mAChR activation inhibited DPSCs migration and differentiation potential but did not interfere with DPSCs stemness. This was further corroborated via expression analysis of key genes involved in stemness and osteogenesis. The data obtained suggests that M2 mAChR activation induce DPSCs to go into a quiescent state. The RNA-seq results showed that DPSCs responded differently to M2 mAChR activation 4 and 24 hours post activation, with different sets of differentially expressed genes (DEGs). The analysis of the enriched pathways suggested that M2 mAChR activation regulates cellular processes involved in metabolism, growth, adhesion, and response to stimuli. These processes function in proliferation, migration, and cell cycle through several metabolic pathways associated with response to cellular and oxidative stress. Follow up analysis showed upregulation of ERK1 and ERK2 phosphorylation and transcripts, which are downstream effectors of the MAPK pathway. The data obtained suggests that the transcriptomic data support the observed inhibitory effect of the M2 mAChR on DPSCs functions and highlights the many downstream effectors involved in the M2 mAChR downstream signalling. In conclusion, this thesis presents evidence for the expression of a functional M2 mAChR in DPSCs, indicating the involvement of ACh signalling in modulating DPSCs behaviour. It also provides a promising route ultimately to pharmacologically control the regenerative output of DPSCs

    Biological Protein Patterning Systems across the Domains of Life: from Experiments to Modelling

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    Distinct localisation of macromolecular structures relative to cell shape is a common feature across the domains of life. One mechanism for achieving spatiotemporal intracellular organisation is the Turing reaction-diffusion system (e.g. Min system in the bacterium Escherichia coli controlling in cell division). In this thesis, I explore potential Turing systems in archaea and eukaryotes as well as the effects of subdiffusion. Recently, a MinD homologue, MinD4, in the archaeon Haloferax volcanii was found to form a dynamic spatiotemporal pattern that is distinct from E. coli in its localisation and function. I investigate all four archaeal Min paralogue systems in H. volcanii by identifying four putative MinD activator proteins based on their genomic location and show that they alter motility but do not control MinD4 patterning. Additionally, one of these proteins shows remarkably fast dynamic motion with speeds comparable to eukaryotic molecular motors, while its function appears to be to control motility via interaction with the archaellum. In metazoa, neurons are highly specialised cells whose functions rely on the proper segregation of proteins to the axonal and somatodendritic compartments. These compartments are bounded by a structure called the axon initial segment (AIS) which is precisely positioned in the proximal axonal region during early neuronal development. How neurons control these self-organised localisations is poorly understood. Using a top-down analysis of developing neurons in vitro, I show that the AIS lies at the nodal plane of the first non-homogeneous spatial harmonic of the neuron shape while a key axonal protein, Tau, is distributed with a concentration that matches the same harmonic. These results are consistent with an underlying Turing patterning system which remains to be identified. The complex intracellular environment often gives rise to the subdiffusive dynamics of molecules that may affect patterning. To simulate the subdiffusive transport of biopolymers, I develop a stochastic simulation algorithm based on the continuous time random walk framework, which is then applied to a model of a dimeric molecular motor. This provides insight into the effects of subdiffusion on motor dynamics, where subdiffusion reduces motor speed while increasing the stall force. Overall, this thesis makes progress towards understanding intracellular patterning systems in different organisms, across the domains of life

    The temporal pattern of impulses in primary afferents analogously encodes touch and hearing information

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    An open question in neuroscience is the contribution of temporal relations between individual impulses in primary afferents in conveying sensory information. We investigated this question in touch and hearing, while looking for any shared coding scheme. In both systems, we artificially induced temporally diverse afferent impulse trains and probed the evoked perceptions in human subjects using psychophysical techniques. First, we investigated whether the temporal structure of a fixed number of impulses conveys information about the magnitude of tactile intensity. We found that clustering the impulses into periodic bursts elicited graded increases of intensity as a function of burst impulse count, even though fewer afferents were recruited throughout the longer bursts. The interval between successive bursts of peripheral neural activity (the burst-gap) has been demonstrated in our lab to be the most prominent temporal feature for coding skin vibration frequency, as opposed to either spike rate or periodicity. Given the similarities between tactile and auditory systems, second, we explored the auditory system for an equivalent neural coding strategy. By using brief acoustic pulses, we showed that the burst-gap is a shared temporal code for pitch perception between the modalities. Following this evidence of parallels in temporal frequency processing, we next assessed the perceptual frequency equivalence between the two modalities using auditory and tactile pulse stimuli of simple and complex temporal features in cross-sensory frequency discrimination experiments. Identical temporal stimulation patterns in tactile and auditory afferents produced equivalent perceived frequencies, suggesting an analogous temporal frequency computation mechanism. The new insights into encoding tactile intensity through clustering of fixed charge electric pulses into bursts suggest a novel approach to convey varying contact forces to neural interface users, requiring no modulation of either stimulation current or base pulse frequency. Increasing control of the temporal patterning of pulses in cochlear implant users might improve pitch perception and speech comprehension. The perceptual correspondence between touch and hearing not only suggests the possibility of establishing cross-modal comparison standards for robust psychophysical investigations, but also supports the plausibility of cross-sensory substitution devices

    Characterization of a double-hit mouse model of schizophrenia. Focus on inflammatory and epigenetic processes, pharmacological modulation and sex bias

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    333 p.Los déficits cognitivos de la esquizofrenia son un síntoma central, son indicadores del pronóstico de la enfermedad y son resistentes a los tratamientos actuales. Se sospecha que los antipsicóticos atípicos podrían empeorar los déficits cognitivos, y se ha propuesto que este proceso tiene lugar a través de una regulación epigenética de las histonas deacetilasas y que podría ser inducido por un exceso de actividad neuroinflamatoria. En la etiopatogenia de esquizofrenia se ha sugerido la existencia de eventos prenatales que generan vulnerabilidad y que posteriormente se reactivan por procesos estresantes durante la adolescencia, constituyendo un fenómeno de "doble-hit". Se considera que la fisio- y psicopatología de la enfermedad podría vincularse a ese fenómeno a través de un exceso de actividad neuroinflamatoria. Teniendo en cuenta todo lo anterior, la presente Tesis Doctoral se centra en la validación de un modelo animal ¿doble-hit¿ de esquizofrenia en ratones macho y hembra. El modelo doble-hit está basado en la activación inmune materna durante la gestación -mediante la administración de un análogo de ARN viral, denominado Poly (I:C)- seguido de un periodo de aislamiento social durante la pubertad. El modelo doble-hit ha demostrado poseer alteraciones comportamentales asociadas con esquizofrenia, así como alteraciones inflamatorias y epigenéticas relacionadas. con la enfermedad. Asimismo, se ha evaluado el efecto de un tratamiento crónico con antipsicóticos atípicos y fármacos anti-inflamatorios. Los hallazgos obtenidos en este trabajo demuestran que el modelo doble-hit es una herramienta válida para el estudio de la esquizofrenia

    25th Annual Computational Neuroscience Meeting: CNS-2016

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    Abstracts of the 25th Annual Computational Neuroscience Meeting: CNS-2016 Seogwipo City, Jeju-do, South Korea. 2–7 July 201

    A Meeting of Minds: In Recognition of the Contributions of Randall J. Cohrs

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    A Special Issue in memory of Randall J. Cohrs, Ph.D. Topics include original research reports on a variety of viruses as well as reviews and commentaries on Randy’s contributions to many investigations
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