The Notch receptor family plays a key role in development and disease. In cancer, Notch can act either as an oncogene or as a tumour suppressor, and possibly as a cancer stem-cell factor. Whereas most research has focused on downstream signalling events, little is known about the cell surface organisation of Notch and its ligands. The extracellular part of Notch consists mainly of 36 epidermal growth factor-like domains (EGF-domains), many of which bind calcium. Studies have shown that tandem repeats of calcium-binding EGF domains form a rigid linear arrangement; however, the lack of calcium binding in EGF6, EGF10 and EGF22 led to the hypothesis that these might be sites of flexibility. This thesis addresses the effect of these domains on the organisation of the extracellular region of Notch and provides further insight into the calcium-binding properties of Notch. NMR residual dipolar coupling (RDC) measurements of these regions are presented, together with the X-ray crystallographic data obtained in collaboration. The crystal structure of the human Notch-1 construct EGF4-7 shows a tilt angle of 90° at the EGF5-6 interface which is much larger than the tilt angles of 10-20° observed for the EGF11-13 crystal structure. RDC measurements demonstrated an angle of ~70° in solution. The crystal structures of EGF21-23 and EGF20-23 showed a rod-shaped interface for the EGF21-22 domain, in which a cis-proline forms the packing interaction to a tyrosine at the β-turn in the major β-sheet of EGF22. These two interfaces are novel and demonstrate the possibility of interface formation without Ca²⁺. Crystallisation was unsuccessful for the EGF8-11 construct. However, RDC measurements indicate interdomain motion between EGF9 and EGF10 demonstrating a flexible interface. These data establish new information on the structural organisation and calcium-binding properties of the extracellular region of Notch and identify flexible and rigid interfaces within multiple tandem repeats of EGF domains. This information will be invaluable in constructing models of Notch-ligand complexes for testing in future functional experiments.

No description available.

The intracellular Ca2+ handling system in a cardiac myocyte is of crucial importance. It regulates the contraction and relaxation of the myocyte during the excitation-contraction (EC) coupling. A normal intracellular Ca2+ handling system keeps the contraction of the heart orderly, which represents a powerful force to pump blood to the whole body. However, disarrayed or remodelled cellular structure associated with the intracellular Ca2+ handling system at the subcellular level, such as loss of T-tubule network in diseased conditions, may promote abnormal cardiac EC coupling, leading to genesis of cardiac arrhythmias impairing cardiac mechanical functions. Up to date, it is still incompletely understood how the intracellular Ca2+ handling system is altered by changes in subcellular structures of Ca2+ handling systems. In this thesis, biophysically detailed computational models for the intracellular Ca2+ handling system of a cardiac cell were developed, providing a powerful platform to investigate the spatio-temporal complexity associated with the intracellular Ca2+ handling, and its role in generating abnormal cardiac EC coupling. First, a well-validated single cell model was used to investigate how the diastolic and systolic Ca2+ concentration responded to alterations in the model parameters related to the Ca2+ handling system, from which the mechanisms underlying the rate-dependence of EC coupling were analysed. Then, a novel single cell model, with a 2D presentation of the spatial structures of subcellular Ca2+ handling and membrane action potential, of a sheep atrial myocyte was developed for simulating the abnormal intracellular Ca2+ regulation system due to the loss of T-tubules during atrial fibrillation. Variant scenarios of T-tubule loss were considered to investigate the role of the T-tubule in affecting the intracellular Ca2+ regulation. Furthermore, membrane currents' alterations due to the electrical remodelling arising from atrial fibrillation were considered together with the loss of T-tubule. Three typical types of abnormal Ca2+ cycling phenomenon, namely intracellular Ca2+ alternans, spontaneous Ca2+ sparks and intracellular Ca2+ waves were observed in AF conditions. The relationship between T-tubule loss, AF-remodelling and the genesis of delayed afterdepolarizations (DADs) was also investigated. It was shown that the loss of T-tubule in AF condition played an important role in disturbing the Ca2+ regulation system, which increases the risk for a cell to generate impaired contraction.

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD+) to produce poly (ADP-ribose) (PAR) polymers on target proteins or itself. As a result, hyper-activity of PARP1 may lead to necrosis by excessively depleting ATP pool which results in mitochondrial energetic collapse. On the other hand, it is known that Ca2+ overload induces necrosis, but much still remains unknown about how Ca2+ overload-induced necrosis is regulated in cells. In this study, we show that ATR, besides its hallmark regulatory role in DDR, also plays a role in NDDR by suppressing ionomycin-induced necrosis. Ionomycin as a Ca2+ ionophore can dramatically raise the intracellular level of Ca2+, leading to necrosis. We found that this Ca2+ overload-induced necrosis occurs without inducing DDR in cells. Instead, the hyper-poly(ADP-ribosyl)ation (PARylation) activity of activated PARP1 could be a reason leading to necrosis, as NAD+ supplied to media can rescue ionomycin-induced necrosis. In vitro PARylation assay also demonstrates that PARP1 hyper-activation is Ca2+ dependent. In cells, ATR-PARP1 interaction happened after ionomycin treatment. Furthermore, ionomycin treatment induces more full-length PAR polymers formed in ATR-deficient cells than in ATR-proficient cells. The interaction of kinase-dead ATR and PARP1 dramatically decreased as compared to wild-type ATR. Therefore, ATR plays a novel role in NDDR wherein it is able to suppress Ca2+ overload-induced PARP1-mediated necrosis. Ca2+ overload-induced cell death is a major cause of many human medical conditions and diseases, such as brain injury, stroke and ischemia et al. Our ongoing studies will help to define the molecular mechanisms of the anti-necrosis activities of ATR, which may support ATR as a new clinical target for therapeutic treatment of those diseases.

Mitochondria plays a crucial role in cells by maintaining energy metabolism and directing cell death mechanisms by buffering calcium (Ca2+ )from cytosol. Therefore, the Ca2+ overload of mitochondria due to the upregulated cytosolic Ca2+ , observed in many neurological disorders is hypothesized to be a key pathway leading to mitochondrial dysfunction and cell death. In particular, Ca2+ homeostasis disruptions due to Alzheimer’ s disease (AD)-causing presenilins (PS1/PS2) and oligomeric forms of β-amyloid peptides Aβ commonly found in AD patients are presumed to cause detrimental effects on the mitochondria and its ability to function properly. We begin by showing that Familial Alzheimer’s disease (FAD)-causing PS mutants affect intracellular Ca2+ ([Ca2+]i) homeostasis by enhancing the gating of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channels on the endoplasmic reticulum (ER), leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data in conjunction with a computational model of mitochondrial bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+ ([Ca2+ ]m). We find that as a result of exaggerated [Ca2+]i in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates of proton pumping due to diminished membrane potential (Ψm) along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially cell death. Next, the model for the mitochondria is expanded to include the mitochondrial uniporter (MCU) that senses Ca2+ in the microdomain formed by the close proximity of mitochondria and ER. Ca2+ concentration in the microdomain ([Ca2+] mic) depends on the distance between the cluster of IP3R channels (r) on ER and mitochondria, the number of IP3R in the cluster (nIP3R), and open-probability (Po) of IP3R. Using the same experimental results for Ca2+ release though IP3R due to FAD-causing PS mutants, in conjunction with a computational model of mitochondrial bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore the difference in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cell expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to nIP3R. Furthermore, we observe diminished inner mitochondrial Ψm, [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/ Ca2+ -exchanger affect the latency of PTP to open in PTPh. Intracellular accumulation of oligomeric forms of Aβ are now believed to play a key role in the early phase of AD as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aβ oligomers. To study the effect of intracellular Aβ on Ca2+ signaling and the resulting mitochondrial dysfunction, we employed data-driven modeling in conjunction with total internal reflection fluorescence (TIRF) microscopy (TIRFM). High resolution fluorescence TIRFM together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the IP3R. Achieving this requires a close agreement between Ca2+ signals from computational models and TIRFM experiments. However, we found that elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel opening and closing during x and between puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10 ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature. Once a close agreement between experiment and model is achieved, we use multiscale modeling in conjunction with patch-clamp electrophysiology of IP3R and fluorescence imaging of whole-cell Ca2+ response, induced by intracellular Aβ42 oligomers to show that Aβ42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the ER by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and [H2O2]. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aβ42 cytotoxicity by EGTA in our live cells experiments. Finally, our modeling study was extended to other pathological phenomena such as epileptic seizures and spreading depolarizations (SD) and their effects on mitochondria by incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients to the neuron. By examining the dynamics as a function of potassium and oxygen we can account for a wide range of neuronal hyperactivity from seizures, normoxic SD, and hypoxic SD (HSD) in the model. Together with a detailed model of mitochondria xi and Ca2+ -release through the ER, we determine mitochondrial dysfunction and potential recovery mechanisms from HSD. Our results demonstrate that HSD causes detrimental mitochondrial dysfunction that can only be recovered by restoration of oxygen. Once oxygen is replenished to the neuron, organic phosphate and pH gradients along the mitochondria determine how rapid the neuron recovers from HSD.

m-3M3FBS is a new compound that has been used as a phospholipase C (PLC) activator. The effect of m-3M3FBS on cytosolic free Ca2+ concentrations in human gastric cancer cells (SCM1) is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended SCM1 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 1-50 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. This Ca2+ influx was inhibited by phospholiapase A2 inhibitor aristolochic acid , store-operated Ca2+ channel blockers nifedipine ¡B econazole and SK&F96365; and protein kinase C inhibitor GF109203X. Phorbol 12-myristate 13-acetate ([PMA] a protein kinase C activator) had no effect on m-3M3FBS-induced [Ca2+]i rise. In Ca2+-free medium , pretreatment with m-3M3FBS abolished thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) - induced [Ca2+]i rise. Conversely, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors TG or BHQ partly inhibited m-3M3FBS -induced Ca2+ release. The inhibition of PLC with U73122 did not alter mMIRC. Collectively, in SCM1 cells, mMIRC by causing PLCindependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-protein kinase C-sensitive store-operated Ca2+ channels.

The effect of the natural essential oil carvacrol or the anesthetic propofol on cell viability, cell cycle distribution, reactive oxygen species (ROS), intracellular free Ca2+ concentrations ([Ca2+]i) and caspase-3-associated apoptosis in human normal cells or non-normal cells is unclear. Human gingival fibroblasts (HGF), human oral cancer cell line (OC2) and human glioblastoma cell line (DRTBG-05MG, HGB) were used in this study. Cell viability was measured by detecting reagent water soluble tetrazolium salt-1 (WST-1). Apoptosis was detected by Annexin V/propidium iodide (PI) staining, cell cycle distribution was detected by PI staining, and ROS was detected by membrane-permeable probe dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) staining. Apoptosis, cell cycle distribution and ROS were analyzed by flow cytometry. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Caspase-3 expression was detected by western blotting. Carvacrol at 200-800 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 1,000 £gM carvacrol almost killed all OC2 or HGB cells, but in HGF cells, 200-800 £gM carvacrol did not significantly kill cells and 1,000 £gM carvacrol decreased only about 63% of cell viability. Similarly, propofol at concentrations between 300 and 600 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 700 £gM propofol almost killed all OC2 or HGB cells, but in HGF cells, 300-600 £gM propofol did not significantly kill cells and 700 £gM propofol decreased about 62% of cell viability. In OC2 or HGB cells, carvacrol (200 £gM, 400 £gM or 600 £gM) or propofol (300 £gM, 400 £gM or 500 £gM) induced apoptosis, increased ROS production, evoked cell cycle arrest and activated caspase-3. The caspase-3 inhibitor (DEVD-CHO) partially decreased the apoptotic effect induced by carvacrol or propofol. On the other hand, in OC2 or HGB cells, carvacrol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner and the signal was reduced by removal of extracellular Ca2+. In HGF cells, carvacrol at 1000 £gM did not induce immediate [Ca2+]i rises in Ca2+-containing or Ca2+-free medium. Similarly, propofol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner in OC2 or HGB cells, but not in HGF cells. This cytotoxic effect was not reversed in carvacrol-treated groups, but was partially reversed in propofol-treated groups when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA-AM) in OC2 or HGB cells. The apoptotic effect of propofol was also partially decreased by BAPTA-AM treatment in OC2 and HGB cells. In OC2 and HGB cells, carvacrol- or propofol-induced Ca2+ signal was not altered by L-type voltage-gated Ca2+ channel blocker nifedipine, store-operated Ca2+ channel blocker econazole or SK&F96365) and protein kinase C (PKC) activator phorbol myristate acetate (PMA), but was inhibited by PKC inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol- or propofol-induced [Ca2+]i rises. Incubation with carvacrol or propofol also abolished TG or BHQ-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol- or propofol-induced [Ca2+]i rises. Together, first, in HGF cells, carvacrol (200-800 £gM) or propofol (300-600 £gM) did not induce [Ca2+]i rises and cell death. Second, in OC2 or HGB cells, carvacrol induced [Ca2+]i rises and cell death that might involve ROS- and caspase-3-associated apoptosis. Third, in OC2 or HGB cells, propofol induced [Ca2+]i rises and cell death that might involve ROS-, Ca2+- and caspase-3-associated apoptosis. Lastly, in OC2 or HGB cells, carvacrol or propofol induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, non store-operated Ca2+ channels.

Abstract The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+-sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 mM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+-induced [Ca2+]i increase by 67%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+-free medium, the Hg2+-induced [Ca2+]i increase was nearly abolished by pretreatment with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+-induced Ca2+ release was not altered by inhibition of phospholiase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 mM Hg2+ did not alter cell proliferation rate, but 10 mM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.

No description available.

Thèse (M.Sc.)--Université Laval, 2006. / Sur la p. de t. 2 et + est en suscrit. Bibliogr.: f. [72]-76. Présenté aussi en version électronique.