Structural and molecular determinants of the sensitivity of alpha4beta2 nicotinic acetylcholine receptors to the allosteric ligand desformylflustrabromine

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. By binding to a site of the receptor distinct from the neurotransmitter binding site, allosteric modulators alter the response of the receptors to their agonists. There are two major locations of allosteric modulator binding sites. One is in subunit interfaces of the extracellular N-terminal domain. The other is in the transmembrane domain close to the channel gating machinery. This thesis focuses on a positive allosteric modulator of the human α4β2 nAChR, desformylflustrabromine (dFBr), which was found to exert its potentiating effects on this receptor by binding to a site in the transmembrane region of the α4 subunit. α4β2 nAChRs are the most abundant nAChR type in the brain, where they modulate a range of brain functions such as mood, cognition, nociception and reward. This receptor subtype has been shown to be sufficient and necessary for the rewarding and reinforcing properties of nicotine. In addition, α4β2 nAChRs have been implicated in aging-related cognitive dysfunction, Alzheimer’s and Parkinson’s diseases, mood disorders and a rare type of family epilepsy. dFBr is a positive allosteric modulator of the α4β2 and α2β2 nAChRs that displays selectivity against all other nAChRs. Using functional mutagenesis and structural modelling, the molecular basis for the selective potentiation of α4β2 nAChRs has been identified. The potentiating binding site of dFBr is located in the top-half of a transmembrane cavity between the M3 and M4 helices of the α4 subunit. α4Y309, α4F312 and α4L617 influence dFBr potentiation in accord with a role in dFBr binding. Alanine substitutions of these residues annulled dFBr potentiation and experiments using MTSET showed that the residues in this putative site are accessible to MTSET and that dFBr competes with MTSET for the access to the cavity. These residues map to a highly conserved intra-subunit cavity in the pentameric ligand gated ion channel (pLGIC) family. In addition, the effector system for the potentiating effects of dFBr was also identified. The post-M4 region (C-terminal) and the Cys loop residues F167 and F170 of the α4 subunit play central roles in transducing dFBr binding to potentiation of the ACh responses of the α4β2 nAChR. Whilst the residues that contribute to the dFBr binding site in the α4 are conserved across all nAChR subunits, except for α7, the post-M4 region is not. It is this region that determines the selective potentiating effects of dFBr on α4β2 nAChR. This finding, together with recent data on the effect of propofol in bacterial and invertebrate evolutionary related pLGICs, suggest that for highly conserved transmembrane domain allosteric binding sites, the effector machinery associated with these sites, rather than the binding sites, define the receptor selectivity of the modulators

The Infrared Ca II triplet as metallicity indicator

From observations of almost 500 RGB stars in 29 Galactic open and globular clusters, we have investigated the behaviour of the infrared Ca II triplet (8498, 8542 and 8662 \AA) in the age range 13$\leq$Age/Gyr$\leq$0.25 and the metallicity range $-2.2\leq$ [Fe/H] $\leq$+0.47. These are the widest ranges of ages and metallicities in which the behaviour of the Ca II triplet lines has been investigated in a homogeneous way. We report the first empirical study of the variation of the CaII triplet lines strength, for given metallicities, with respect to luminosity. We find that the sequence defined by each cluster in the Luminosity-$\Sigma$Ca plane is not exactly linear. However, when only stars in a small magnitude interval are observed, the sequences can be considered as linear. We have studied the the Ca II triplet lines on three metallicities scales. While a linear correlation between the reduced equivalent width ($W'_V$ or $W'_I$) versus metallicity is found in the \citet{cg97} and \citet{ki03} scales, a second order term needs to be added when the \citet{zw84} scale is adopted. We investigate the role of age from the wide range of ages covered by our sample. We find that age has a weak influence on the final relationship. Finally, the relationship derived here is used to estimate the metallicities of three poorly studied open clusters: Berkeley 39, Trumpler 5 and Collinder 110. For the latter, the metallicity derived here is the first spectroscopic estimate available.Comment: 52 pages, 16 figures, accepted for publication in Astronomical Journa

Assessing Long-Term Excavation Damage in Switzerland’s Bedretto Adit: A Field Investigation in the Northern Section

The Bedretto adit, in the Alps of Switzerland, was excavated in 1972–1982 as an auxiliary adit for the main Furka Base Tunnel. The adit is 5218 m long, being excavated mostly in the Rotondo Granite with drill and blast methods to create a horseshoe cross-section. Much of the tunnel has remained unlined and such long observation periods are seldom available since tunnels typically are lined shortly after construction and mines are often closed or backfilled. Previous research in 2004 characterized the depth of failure (DoF) around the adit and found on average a depth of 1.1 ± 0.03 m. A site investigation was carried out in 2017, aimed to measure the DoF through 3D light detection and ranging scanned sections again. The new 3D scans covered a longer section of the tunnel with each scan, up to 6 m in length, compared to the previous laser scans with a single circumferential pass. The results were compared with the data from 2004 and with empirical and numerical prediction curves for Excavation Damage Zone depths. The findings indicate that between 2004 and 2017, the measured DoF generally extended, on average, by 0.1 m, implying that failure has progressed with time. Furthermore, the analysis showed that the volume of failure from 2004 extended up to 1.9 m3/m, and this volume increased to 3.2 m3/m in 2017 on average. The VoF was used as a means to overcome the challenge of comparing past to present scan results, since the exact location of the 2004 measurements were not known. Significant variations in the measured DoF at sections of the adit near faults and below a glacier indicate that these external factors can significantly influence the rock mass behaviour. When comparing the DoF to empirical excavation damage zone prediction intervals, the level of damage at the Bedretto adit is comparable with the outer excavation damage zone. This is a region of isolated micro-cracking, and it conceptually suggests that the continued spalling in the Bedretto adit is the results of crack propagation from initial excavation induced damage that leads to interaction even after 41 years since excavation works ceased

Kinematics of the Galactic Globular Cluster System: New Radial Velocities for Clusters in the Direction of the Inner Galaxy

HIRES on the Keck I telescope has been used to measure the first radial velocities for stars belonging to eleven, heavily-reddened globular clusters in the direction of the inner Galaxy. The question of kinematic substructuring among the Galactic globular cluster system is investigated using an updated catalog of globular cluster distances, metallicities and velocities. It is found that the population of metal-rich globular clusters shows significant rotation at all Galactocentric radii. For the metal-rich clusters within 4 kpc of the Galactic center, the measured rotation velocity and line-of-sight velocity dispersion are similar to those of bulge field stars. We investigate claims that the metal-rich clusters are associated with the central Galactic bar by comparing the kinematics of the innermost clusters to that of the atomic hydrogen in the inner Galaxy. The longitude-velocity diagram of both metal-rich and metal-poor clusters bears a remarkable similarity to that of the gas, including the same non-circular motions which have traditionally been interpreted as evidence for a Galactic bar, or, alternatively, a non-axisymmetric bulge. However, uncertainties in the existing three-dimensional Galactocentric positions for most of the clusters do not yet allow an unambiguous discrimination between the competing scenarios of membership in a rigidly rotating bar, or in a bulge which is an oblate isotropic rotator. We conclude that the majority of metal-rich clusters within the central 4 kpc of the Galaxy are probably associated with the bulge/bar, and not the thick disk. (ABRIDGED)Comment: 18 pages, including 7 of 13 postscript figures. Figures 1-6 available at http://astro.caltech.edu/~pc. Accepted for publication in the Astronomical Journa

VI Photometry of Globular Clusters NGC6293 and NGC6541: The Formation of the Metal-Poor Inner Halo Globular Clusters

We present VI photometry of the metal-poor inner halo globular clusters NGC6293 and NGC6541 using the planetary camera of the WFPC2 on board HST. Our color-magnitude diagrams of the clusters show well-defined BHB populations, consistent with their low metallicities and old ages. NGC6293 appears to have blue straggler stars in the cluster's central region. We discuss the interstellar reddening and the distance modulus of NGC6293 and NGC6541 and obtain E(B-V) = 0.40 and (m-M)_0 = 14.61 for NGC6293 and E(B-V) = 0.14 and (m-M)_0 = 14.19 for NGC6541. Our results confirm that NGC6293 and NGC6541 are clearly located in the Galaxy's central regions (R_{GC} < 3 kpc). We also discuss the differential reddening across NGC6293. The interstellar reddening value of NGC6293 appears to vary by Delta E(B-V) ~ 0.02 -- 0.04 mag within our small field of view. The most notable result of our study is that the inner halo clusters NGC6293 and NGC6541 essentially have the same ages as M92, confirming the previous result from the HST NIC3 observations of NGC6287.Comment: AJ, accepte

A Far-UV Variability Survey of the Globular Cluster M80

We have searched for variable sources in the core region of M80, using far ultra-violet data taken with the Advanced Camera for Surveys on board the Hubble Space Telescope. We found three sources that exhibit strong signs of variability in our data. Among these is source TDK1, which we believe to be an RR Lyrae star that reached maximum brightness during our observations. The light curve shows a >3 mag FUV brightening over the course of ~5 hours, with an estimated peak brightness of ~16.7 mag, followed by a decrease to ~20 mag. Archival optical data obtained with WFPC2 confirm that TDK1 is variable in all wavebands. TDK1's SED is reasonably fit by a star with temperature T(eff)=6700K and radius R=4.2R(sun), consistent with the suggestion that it is an RR Lyrae. Based on the photometric and variability characteristics of the other two variables, we suggest that TDK2 is likely to be an SX Phoenicis star with ~55 minutes period, and TDK3 is likely another RR Lyrae. Finally, we briefly discuss the FUV counterparts to two previously known variables in M80, the classical nova T Sco and the dwarf nova DN1.Comment: 12 pages, 9 figures and 3 tables. Accepted for publication in MNRAS

The Chemical Enrichment History of the Fornax Dwarf Spheroidal Galaxy from the Infrared Calcium Triplet

Near infrared spectra were obtained for 117 red giants in the Fornax dwarf spheroidal galaxy with the FORS1 spectrograph on the VLT, in order to study the metallicity distribution of the stars and to lift the age-metallicity degeneracy of the red giant branch (RGB) in the color-magnitude diagram (CMD). Metallicities are derived from the equivalent widths of the infrared Calcium triplet lines at 8498, 8542, and 8662 A, calibrated with data from globular clusters, the open cluster M67 and the LMC. For a substantial portion of the sample, the strength of the Calcium triplet is unexpectedly high, clearly indicating that the main stellar population of Fornax is significantly more metal-rich than could be inferred from the position of its RGB in the CMD. We show that the relative narrowness of the RGB in Fornax is caused by the superposition of stars of very different ages and metallicities. The metallicity distribution in Fornax is centered at [Fe/H]= -0.9, with a metal-poor tail extending to [Fe/H] ~= -2. While the distribution to higher metallicities is less well determined by our observations, the comparison with LMC data indicates that it extends to [Fe/H] ~ -0.4. By comparing the metallicities of the stars with their positions in the CMD, we have derived the complex age-metallicity relation of Fornax. In the first few Gyr, the metal abundance rose to [Fe/H] ~ -1.0 dex. The enrichment accelerated in the past ~ 1-4 Gyr to reach [Fe/H] ~ -0.4 dex. More than half the sample is constituted of star younger than ~ 4 Gyr, thus indicating sustained recent star formation in Fornax. These results indicate that the capacity of dwarf spheroidal galaxies to retain the heavy elements that they produce is larger than expected. (Abridged)Comment: 36 pages, 19 figures, to appear in Astronomical Journal, January 200

Structures in surface-brightness profiles of LMC and SMC star clusters: evidence of mergers?

The LMC and SMC are rich in binary star clusters, and some mergers are expected. It is important to characterize single clusters, binary clusters and candidates to mergers. We selected a sample of star clusters in each Cloud with this aim. Surface photometry of 25 SMC and 22 LMC star clusters was carried with the ESO Danish 1.54 m telescope. 23 clusters were observed for the first time for these purposes. We fitted Elson, Fall and Freeman (1987, EFF) profiles to the data, deriving structural parameters, luminosities and masses. We also use isophotal maps to constrain candidates to cluster interactions.} {The structural parameters, luminosities and masses presented good agreement with those in the literature. Three binary clusters in the sample have a double profile. Four clusters (NGC 376, K 50, K 54 and NGC 1810) do not have companions and present as well important deviations from EFF profiles. The present sample contains blue and red Magellanic clusters. Extended EFF profiles were detected in some blue clusters. We find evidence that important deviations from the body of EFF profiles might be used as a tool to detect cluster mergers.Comment: 16 pages and 8 figures. Accepted by A&

Role of the Cys loop and transmembrane domain in the allosteric modulation of α4β2 nicotinic acetylcholine receptors

​Allosteric modulators of pentameric ligand gated ion channels (pLGICs) are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors (nAChRs) and the α4β2-selective positive modulators 17β-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pLGICs. Previous studies have shown that the binding site for potentiating βEST is in the C-terminal (post-M4 region) of the α4 subunit. Here, using homology modelling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top-half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and F170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Y309) and F167, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating βEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop

VLT Observations of Turnoff stars in the Globular Cluster NGC 6397

VLT-UVES high resolution spectra of seven turnoff stars in the metal-poor globular cluster NGC 6397 have been obtained. Atmospheric parameters and abundances of several elements (Li, Na, Mg, Ca, Sc, Ti, Cr, Fe, Ni, Zn and Ba) were derived for program stars. The mean iron abundance is [Fe/H] = -2.02, with no star-to-star variation. The mean abundances of the alpha-elements (Ca, Ti) and of the iron-peak elements (Sc, Cr, Ni) are consistent with abundances derived for field stars of similar metallicity. Magnesium is also almost solar, consistent with the values found by Idiart & Th\'evenin (2000) when non-LTE effects (NLTE hereafter) are taken into account. The sodium abundance derived for five stars is essentially solar, but one object (A447) is clearly Na deficient. These results are compatible with the expected abundance range estimated from the stochastic evolutionary halo model by Argast et al. (2000) when at the epoch of [Fe/H] $\sim$ -2 the interstellar medium is supposed to become well-mixed.Comment: to appear in A&