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Characterization of mouse two-pore channels (TPCs) in NAADP-mediated Ca(2+) signallingChuang, Kai-Ting January 2011 (has links)
Recent studies have identified Two-Pore Channels (TPCs) as the channels activated by NAADP. To date, most studies that characterized these channels have employed heterologous expression or overexpression systems. The research reported here has focused principally on endogenous TPC activity by using single and dual gene knockout (KO) in a mouse system and has yielded insights into TPC expression levels, subcellular localisation, NAADP binding, and channel function. Mouse models that had been generated by both the “gene-trapping” and the “genetargeting” techniques were obtained and validated. These included a knock-down strain (“hypomorph”). Surprisingly, all TPC mutant mice showed no gross phenotypes. In addition to the two known isoforms in mouse, TPC1 and TPC2, the expression of a shorter variant of TPC1 was discovered; this has an alternative (truncated) N-terminus, and has been termed (DELTA)N-TPC1. All TPC variants/isoforms were widely expressed in all mouse tissue types tested. Overexpression of mouse TPCs in mouse embryonic fibroblasts showed that (DELTA)N-TPC1 and TPC2 were expressed primarily in late endosomes/lysosomes while TPC1 was expressed in both endosomes and lysosomes. Dileucine sorting motifs target TPCs to late endosomes/lysosomes; it was shown that truncation or mutation of dileucine motifs significantly reduced localization in late endosomes/lysosomes. Furthermore, TPCs were shown not to be the direct binding target of NAADP, as the high affinity NAADP binding was retained in hepatic membranes from TPC double KO (DKO) mice. It is concluded that NAADP binds to an (as yet, unidentified) accessory protein. The functional role of TPCs was studied in depth using mouse pancreatic acinar cells. NAADP is known to release Ca<sup>2+</sup> from the acidic stores in response to the stimulation by the hormone cholecystokinin (CCK). In all TPC mutant mice, CCK was still able to evoke Ca<sup>2+</sup> oscillations, but with slower and attenuated oscillations in the TPC1 hypomorph, and with slower oscillations in TPC DKO. In all TPC KOs, oscillations were disrupted by known inhibitors of the NAADP-signalling pathway (Ned-19, GPN and bafilomycin A1), indistinguishable from the responses with wild-type cells. This suggests that TPCs are not involved in CCK signalling, although it is possible that functional compensation masked the phenotype arising from the impaired signalling.
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Ca2+/Calmodulin signalling during colony initiation in Neurospora crassaChang, Chia-Chen January 2015 (has links)
The primary research aims of this thesis were to analyse the mechanism of Ca2+/calmodulin (CaM) signalling during conidial germination and conidial anastomosis tube (CAT)-mediated fusion in Neurospora crassa. Ca2+ is an ubiquitous signalling molecule that regulates many important processes in filamentous fungi including spore germination, hyphal growth, mechanosensing, stress responses, circadian rhythms, and the virulence of pathogens. Transient increases in cytosolic free calcium ([Ca2+]c) act as intracellular signals. As the primary intracellular Ca2+ receptor, calmodulin (CaM) converts these Ca2+ signals into responses by regulating the activities of numerous target proteins. Ca2+-free medium, antagonists of L-type Ca2+ channels, CaM and calcineurin were found to inhibit CAT fusion. In addition, my results showed that CAT chemotropism is dependent on extracellular Ca2+. 65 genes were identified as likely components of the Ca2+ signalling machinery of N. crassa based on a comparative genomic analysis of S. cerevisiae, A. fumigatus and C. albicans. Deletion mutants of 29 of these genes were characterized in relation to their possible roles during colony initiation and development. Four of these mutants (Δcna-1, Δcnb-1, Δcamk-1, Δplc-2, and Δrgs-1), which were homokaryons, exhibited strong morphological phenotypes associated with CAT fusion. To identify the protein machinery involved in Ca2+/CaM signalling during colony initiation, proteins that directly or indirectly interacted with CaM were isolated from germlings by immunoprecipitation and analyzed by mass spectroscopy. A total of 286 putative Ca2+/CaM-interacting proteins were identified in this way and 30 of these proteins contained CaM-binding motifs. This proteomics analysis provided evidence for Ca2+/CaM signalling playing a role in regulating the activity of a wide range of proteins including MAP kinases in the cell integrity pathway, Ras/Rho signalling pathway, and microtubule and actin cytoskeletal proteins. GFP labelled CaM localized as dynamic spots associated with the plasma membrane and cytoplasm in both germ tubes and CATs. Significant CaM accumulation was observed in the tips of CATs growing towards each other, around fusion pores at sites of CAT fusion, and at developing septa in germ tubes. CaM localization was influenced by the actin and microtubule cytoskeleton during the colony initiation. Inhibition of F-actin polymerization with latrunculin-A suppressed the pronounced accumulation of CaM at growing germ tube and CAT tips. The movement of CaM associated with spindle pole bodies was prevented by treatment with the microtubule polymerization inhibitor benomyl. The absence of myo-5 resulted in reduced CAT fusion and the lack recruitment of CaM at growing tips indicating a role for the motor protein, myosin-5, in these processes. Finally, by expressing the genetically encoded Ca2+ sensor GCaMP6s under the control of tef-1 promoter in N. crassa, I have been able to image [Ca2+]c dynamics in this fungus for the first time. Using this I have been able to detect localized [Ca2+]c spikes and waves in conidia, germ tubes and CATs. However, I obtained no clear evidence for localized [Ca2+]c changes being associated with CAT chemotropism or fusion.
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Investigating temperature signalling pathways in Arabidopsis thaliana using small moleculesSchoepfer, David January 2019 (has links)
Upon exposure to heat or cold, Arabidopsis thaliana seedlings undergo rapid transcriptional reprogramming of several hundreds of genes that promote stress tolerance. Despite extensive characterisation of the transcriptional responses to these stimuli, however, relatively little is known about the mechanisms by which temperature signals are perceived and transduced in plant cells. High or low seasonal temperatures have large impacts on crop productivity and are expected to intensify given current global climatic projections. It is therefore of agricultural importance to better understand temperature signalling pathways in plants in order to find solutions to this problem. In this thesis, a chemical genomics screen for molecules activating or repressing heat-inducible genes in A. thaliana was performed in collaboration with Syngenta and the biological targets of these chemicals were predicted based on structural similarities to compounds with known modes of action. Many molecules that affect the function of chloroplasts or mitochondria either activate or repress heat-responsive genes, thus implicating these organelles in the regulation of plant temperature responses. In addition, the translation inhibitor cycloheximide was identified as a repressor of heat-inducible genes and an activator of early cold-inducible genes. Diverse translation inhibitors trigger a cytosolic influx of calcium ions and several inhibitors of translation elongation were found to strongly activate cold-inducible gene expression in a calcium-dependent manner. Furthermore, it was demonstrated that cold shock causes rapid translation repression in A. thaliana seedlings and that the elongation factor LOS1 is involved in cold- or cycloheximide-induced gene expression, thus implicating translational machinery in the regulation of temperature signalling in plants. Finally, one of the chemicals identified in the screen, S01A463859Y, was found to improve heat resilience in A. thaliana and may therefore be of potential use in enhancing crop productivity during thermal stress.
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Effects of Chronic Oxidative Stress on TRPM2 and TRPC3 Channels: Potential Implications for Bipolar DisorderRoedding, Angela 09 August 2013 (has links)
Intracellular calcium and oxidative stress dyshomeostasis, which can be highly interactive, occur in bipolar disorder (BD), but the pathogenesis of these disturbances is unknown. The transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) calcium-permeable non-selective ion channels, already implicated in BD, are involved in calcium and oxidative stress signalling. Thus, I sought to determine whether the expression and function of these channels are modulated by oxidative stress exposure in rat cortical neurons, astrocytes, and in human B lymphoblast cell lines (BLCLs), a cell model that reports diagnostically relevant abnormalities in BD.
This thesis work demonstrated that TRPC3 expression and function are decreased after chronic, but not acute oxidative stress exposure in both human and rat cell models. TRPM2 expression, on the other hand, was increased after both acute and chronic stressor treatments in rat cortical neurons. In BLCLs, TRPM2-mediated calcium entry was blunted although no difference in TRPM2 mRNA expression was detected. Moreover, BLCLs from BD-I patients exhibited greater susceptibility to cell death and a differential sensitivity of TRPM2-mediated calcium influx to acute oxidative stress compared with healthy subjects, further supporting reduced cellular resilience in the pathophysiology of BD-I. I also demonstrated that TRPC3 protein is expressed in human brain from 8 days to 83 years old supporting an ongoing role in the developing and adult human brain.
These findings support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress, and in transducing oxidative stress signalling to intracellular calcium homeostatic and cellular stress responses, which have been implicated in the pathophysiology of BD. Finally, this work has highlighted an inherent difference in TRPM2 channel functionality in BD type I subjects compared with controls, adding functional evidence to the genetic and differential expression findings implicating TRPM2 dysfunction in BD.
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Effects of Chronic Oxidative Stress on TRPM2 and TRPC3 Channels: Potential Implications for Bipolar DisorderRoedding, Angela 09 August 2013 (has links)
Intracellular calcium and oxidative stress dyshomeostasis, which can be highly interactive, occur in bipolar disorder (BD), but the pathogenesis of these disturbances is unknown. The transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) calcium-permeable non-selective ion channels, already implicated in BD, are involved in calcium and oxidative stress signalling. Thus, I sought to determine whether the expression and function of these channels are modulated by oxidative stress exposure in rat cortical neurons, astrocytes, and in human B lymphoblast cell lines (BLCLs), a cell model that reports diagnostically relevant abnormalities in BD.
This thesis work demonstrated that TRPC3 expression and function are decreased after chronic, but not acute oxidative stress exposure in both human and rat cell models. TRPM2 expression, on the other hand, was increased after both acute and chronic stressor treatments in rat cortical neurons. In BLCLs, TRPM2-mediated calcium entry was blunted although no difference in TRPM2 mRNA expression was detected. Moreover, BLCLs from BD-I patients exhibited greater susceptibility to cell death and a differential sensitivity of TRPM2-mediated calcium influx to acute oxidative stress compared with healthy subjects, further supporting reduced cellular resilience in the pathophysiology of BD-I. I also demonstrated that TRPC3 protein is expressed in human brain from 8 days to 83 years old supporting an ongoing role in the developing and adult human brain.
These findings support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress, and in transducing oxidative stress signalling to intracellular calcium homeostatic and cellular stress responses, which have been implicated in the pathophysiology of BD. Finally, this work has highlighted an inherent difference in TRPM2 channel functionality in BD type I subjects compared with controls, adding functional evidence to the genetic and differential expression findings implicating TRPM2 dysfunction in BD.
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The role of Ca2+ signalling in the mode-of-action of PAF26Alexander, Akira January 2017 (has links)
PAF26 is a synthetic hexapeptide that has been shown to be highly effective at killing filamentous fungi whilst showing low toxicity against human and bacterial cells. Unlike membrane permeabilising antimicrobials, PAF26 at low fungicidal concentrations is endocytically internalised by fungal cells and accumulates in the vacuole, which undergoes expansion. At a certain point, PAF26 is transported out of the vacuole into the cytoplasm and this is followed by cell death. Previously, cytosolic free Ca2+ ([Ca2+]cyt) has been shown to exhibit a dose-dependent increase in response to PAF26 but the significance of this was not understood. The main objective of my PhD research was to analyse what role(s) Ca2+ signalling has in the mode-of-action of PAF26 using Neurospora crassa as an experimental model. In the first part of my research, evidence for Ca2+ signalling having a significant role in the PAF26 mode-of-action was obtained by testing the PAF26 sensitivity of deletion mutants defective in different components of their Ca2+ signalling machinery. The Deltacch-1, Deltayvc-1 and Deltanca-2 strains were found to exhibit the greatest resistance against PAF26. Screening of a range of vacuolar fusion mutants provided evidence for PAF26-induced vacuolar expansion involving the fusion of small vacuoles. Evidence was also obtained for PAF26-induced programmed cell death occurring via the pathway involved in HET-C-mediated heterokaryon incompatibility. In the second part of my research, I used live-cell imaging with fluorescently labelled PAF26 to investigate the pattern and kinetics of peptide interaction, internalization and distribution within the cells of the Deltacch-1, Deltayvc-1 and Deltanca-2 PAF26-resistant strains compared with the parental wild type. From these studies I obtained evidence for: CCH-1 being required for endocytic internalization of PAF26; YVC-1 being required for PAF26-induced vacuolar expansion; NCA-2 being involved in PAF26 binding to the cell envelope and subsequent endocytic cell internalization and transport to vacuoles; and cell death not being simply induced by PAF26 making contact with the cytoplasm. In the third part of my research I used the genetically encoded bioluminescent Ca2+ reporter aequorin to investigate differences in the average [Ca2+]cyt responses to PAF26 in cell populations of the Deltacch-1, Deltayvc-1 and Deltanca-2 mutant strains compared to that of the wild type. The Ca2+ signatures of the dose-dependent [Ca2+]cyt increases of each mutant were significantly different to each other and to the wild type, and external Ca2+ was required to initiate the [Ca2+]cyt responses. Evidence was obtained for an as yet unknown mechanism of Ca2+ entry into the fungal cell that was independent of the Ca2+ channels, CCH-1 and YVC-1.In the fourth part of my research, I used the genetically encoded fluorescent Ca2+ reporter GCaMP6 to visualise the Ca2+ signatures within individual cells in response to a low fungicidal concentration (3.5 M) of PAF26. This peptide treatment resulted in [Ca2+]cyt spiking at irregular intervals within all cells but there was considerable heterogeneity in the Ca2+ signatures of individual cells of a germling population. Furthermore, subcellular regions within which transient [Ca2+]cyt increases occurred were spatially correlated with increased membrane fusion events between vacuolar-like structures. In the fifth part of my research, YVC-1 and CCH-1 were labelled with GFP. Whilst YVC-1 was consistently associated with the vacuolar membrane, CCH-1 was harder to visualise. CCH-1 appeared to localise to intracellular membranes in conidia and germlings and to the plasma membrane in mature hyphae. In the final part of my thesis I investigated the hypothesis, based on previous evidence, that PAF26 disrupts pH homeostasis by using live cell imaging with the fluorescent, ratiometric pH dye, carboxy SNARF-1. PAF26 treatment was found to cause acidification of the cytoplasm.
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Studies relating to the differentiation of human embryonic stem cellsAnyfantis, Georgios January 2015 (has links)
Human embryonic stem cells (hESCs) have been a useful tool in the study of the embryo development and could be used by drug developing companies to create disease models and assist in the production of new medicines. One of the models that has been studied before, is the development of the pancreas. Scientists have obtained mixed results so far in the generation of functional pancreatic cells from hESCs. We studied the differentiation potential of hESCs. As purinergic signalling is involved in may physiological processes, including cell proliferation and differentiation, a study of purinergic signalling in hESCs would help us deeper understand the hESC physiology. In order to study the purinergic profile of hESCs we established a culture system that allowed the transfer and attachment of pluripotent hESC colonies on glass coverslips. We then studied the functional purinergic profile of hESCs and found that they do not express functional P2X1 receptors, but they do express functional P2Y6 receptors, which might be implicated in the hESC differentiation. In parallel to these studies, we developed a reporter gene lentivirus, where the mouse Pdx-1 promoter area controlled the expression of a reporter fluorochrome, eGFP. We managed to generate a functional lentivirus, however, further analysis is needed in order to be able to use it in developmental studies. Finally, we tested the hypothesis that glucose affects the differentiation of hESCs towards pancreatic endoderm. Our preliminary results suggested that glucose does affect the differentiation potential of hESCs.
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Adrenal chromaffin cell function in high-altitude deer mice (Peromyscus maniculatus)Pranckevicius, Nicole 11 1900 (has links)
The deer mouse (Peromyscus maniculatus) inhabits a broad altitudinal range from sea level to over 4300m, where they experience continuous hypoxia. Typically hypoxia activates the sympathetic nervous system; however this could become maladaptive in high-altitude residents if it is maintained over chronic periods. We hypothesized that high-altitude deer mice might have altered the physiology of adrenaomedullary chromaffin cells (AMC) in the adrenal gland to avoid chronic activation of the sympathetic response. Highland mice had lower plasma adrenaline levels compared to lowland populations of Peromyscus mice, both before and after acclimation to hypobaric hypoxia. This did not correspond to any apparent changes in AMC Ca2+-signalling dynamics. Instead a profound blunting of catecholamine storage was found in highland AMCs that appeared to underlie the reduction in adrenaline release / Thesis / Master of Science (MSc)
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Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and functionTunn, Ruth Elizabeth January 2012 (has links)
Two-pore channels (TPCs, gene name Tpcn) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca2+) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA knockdown studies have implicated TPCs in fundamental cellular processes, including secretion, of insulin in pancreatic islets, and differentiation, of skeletal myoblasts and osteoclasts. Investigations of Tpcn1 and Tpcn2 mRNA expression have indicated widespread tissue distribution, but a lack of suitable antibodies has impeded study of the endogenous proteins. In this study, an anti-TPC1 antibody was purified from immune sera and used in immunoblotting investigations to demonstrate TPC1 protein expression in a wide range of mouse tissues, with highest expression levels observed in kidney, liver and adipose tissue. Endogenous mouse TPC1 was demonstrated to be glycosylated, with apparent differences in the extent of glycosylation in different tissues based on the indicated molecular weight before and after treatment with a deglycosylating enzyme, which may have implications for the functional regulation of channel activity. Given the increasing prevalence of type 2 diabetes and obesity, an understanding of the molecular basis of glucose homeostasis and adipose tissue formation and function is an important scientific goal. Tpcn KO mice have been developed; in both Tpcn1 KOs and Tpcn2 KOs, impaired pancreatic β-cell Ca2+ signalling and reduced insulin secretion from the whole pancreas were demonstrated. However, the whole-animal phenotype has not been extensively researched. In this study, intraperitoneal glucose tolerance tests were conducted in Tpcn KO mice. These indicated that glucose homeostasis was not significantly affected in Tpcn2 KOs or Tpcn1/2 double KOs (DKOs), and only mildly impaired in Tpcn1 KOs, despite the defects previously observed at the cellular and tissue level. In addition, body composition was investigated in Tpcn1 KO, Tpcn2 KO and Tpcn1/2 DKO animals using magnetic resonance spectroscopy and time domain-nuclear magnetic resonance. Single Tpcn KOs were found to have lower adipose tissue levels as a percentage of body composition, while Tpcn1/2 DKOs were shown to have increased bodyweight but normal body composition. To investigate potential roles for TPCs in adipose tissue formation, Tpcn expression during adipogenesis was investigated using an in vitro multipotent mesenchymal stem cell line model of adipogenic differentiation. Tpcn2 mRNA levels were demonstrated by quantitative PCR to be transiently increased during the early stages of adipogenic differentiation, and cyclic AMP (cAMP) was identified as the factor that induced this upregulation. Lentiviruses were developed to express fluorescently-tagged TPCs, and overexpression of TPC2 was demonstrated to partially overcome the requirement for the cAMP-inducing agent in the medium used for the induction of adipogenesis. Collectively, these data suggest that TPCs may play a role in the formation and/or function of adipose tissue.
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The role of two pore channels (TPCs) in pancreatic beta cell stimulus-secretion couplingHeister, Paula Maria January 2012 (has links)
This thesis presents an investigation into the role of the recently identified two pore channels (TPCs) in β-cell stimulus-secretion coupling. TPCs are the receptors for calcium mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP) located in the membrane of acidic intracellular calcium stores. It is proposed that they are responsible for the ATP-sensitive potassium channel (Katp channel) independent pathway of stimulus-secretion coupling; and that this pathway is not subordinate to the KAT? channel dependent pathway; but an alternative explanation of stimulus-secretion coupling in its own right. The first section of this thesis presents a characterisation of sub-membrane cal- cium signals observed in primary mouse β-cells in response to glucose and the membrane-permeable acetoxymethyl ester form of NAADP (NAADP-AM) using the non-ratiometric fluorescent calcium indicator fluo-4 and total internal reflection (TIRF) microscopy. These are compared to global cytosolic calcium changes observed with epifluorescence microscopy. Factors affecting the shape and time course of re- sponses are investigated, and pharmacological tools used to provide evidence for the role of intracellular calcium release from acidic stores mediated by NAADP. Having characterised the calcium responses of β-cells using TIRF; the second part of the thesis examines the effects of knocking out TPC2 (single KO), or both TPC1 and TPC2 (DKO) on these responses; after an initial assessment of pancreatic islet and β-cell morphology using electron microscopy. Gender differences in β-cell responses to glucose and NAADP are assessed in both wild type and knockout animals. Finally, the third section presents the discovery of elementary calcium release events in pancreatic β-cells. The current project visualises what are likely the triggering events for the global calcium signals examined in sections one and two. They take the form of localised calcium release in response to NAADP-AM and glucose; akin to sparks and puffs observed by stimulation with cADPR and IP3. Optical quantal analysis demonstrates the quantal nature of the events and estimates the size of the unitary calcium release unit (CRU) for NAADP. .
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