Over the past decade there has been a growing body of evidence, obtained from studies employing a wide variety of pharmacological, biochemical and biophysical techniques, suggesting that G protein coupled receptors (GPCRs) exist not as monomeric entities but rather as dimers or other higher order oligomeric arrays. To further the accumulation of knowledge pertaining to this research area, the work presented in this thesis has made use of one such particular biophysical technique called bioluminescence resonance energy transfer (BRET). In order to utilise this system GPCRs were modified at their carboxyl terminal tail with either the anthozoan enzyme Renilla luciferase or the fluorescent protein eYPP. Through this expedient, if the differentially tagged GPCRs are in close proximity when co-expressed within mammalian cells, upon addition of the bioluminescent molecule coelenterazine, there is a non-radiative exchange of energy between the Renilla and eYFP, resulting in a fluorescent emission from eYFP. The technique can be used to monitor interactions in real time, in living cells and does not require any biochemical manipulations/treatments such as are associated with more traditional approaches to this line of enquiry (e.g. co-immunoprecipitation). Using this technique, it was demonstrated that the β2-AR was closely associated when expressed within HEK 293T cells, as were the σ-opioid and K-opioid receptors since all gave robust signals in energy transfer experiments. Contrary to some previous reports however, it was not seen to be the case that the presence of ligand was capable of modulating the magnitude of the energy transfer signal. This indicated that for these GPCRs the binding of ligand did not result in any alteration in the dimerization status of the receptor. It was further shown, through monitoring the energy transfer at varying levels of receptor expression, that energy transfer was favourable between homomers of the K-opioid receptor. At similar receptor densities, energy transfer between coexpressed K-opioid receptor and TRHr was seen to be considerably less favourable, requiring far higher receptor expression levels to be achieved before meaningful levels of energy transfer could be detected. These results strongly indicated that closely related GPCR types had a greater propensity for mutual interaction than did more distantly related ones. Many of these results were confirmed using a modified version of BRET, designated BRET2, which conferred an additional sensitivity to detection of protein-protein interactions. Using BRET2 a previously ill-defined result obtained with traditional BRET, that suggested that the β2-AR might interact with the 6-opioid-receptor, was confirmed. An additional purpose of the work described herein was to explore the potential of GPCR dimerization as a means of providing a novel ligand detection assay suitable for application to industrial high-throughput screening programmes. Since the experiments concentrating on GPCR oligomerization failed to provide such an assay, it was decided that the ability of GPCRs to recruit β-arrestin as part of the process of desensitisation should be evaluated as a possible alternative. Using a cell line stably expressing the GPCR CCR2 the ability of this receptor to recruit various fluorescent proteins conjugated to β-arrestin2 (β-arrestin-red NFP and β-arrestin-cyan NFP) from the cytosol in response to receptor activation was demonstrated. The β-arrestin2 was localized into endocytic vesicles and remained tightly associated with the internalised receptors after sequestration had occurred. This behaviour was in accordance with other previous reports for GPCRs that, like CCR2, possessed serine and threonine clusters within their carboxyl terminal tails. If adapted to a FRET based format this particular protein-protein interaction could form the basis of a ligand screening method for agonists that would be equally applicable to most GPCRs. It was further shown that β-arrestin2-red NFP had a higher affinity for CCR2 than did β-arrestin1- GFP through the monitoring of the respective kinetics and extent of translocation when the constructs were co-expressed within the same cells. As an alternative strategy for the detection of ligands, a constitutively active mutant of β 2-AR (CAM β 2-AR) was modified C-terminally with the bioluminescent enzyme Renilla luciferase. This CAM 2-AR was structurally destabilized to a high degree so that only modest expression levels could be obtained upon expression of the Renilla modified receptor construct (CAM β 2-AR-Rluc) in HEK 293T cells. Upon prolonged exposure to various antagonist ligands, a two to three fold upregulation of the receptor construct could be detected via light output from the luciferase. From parallel competition binding experiments it was also demonstrated that, for each of these ligands, the EC50 for upregulation highly correlated with the dissociation equilibrium constant (Ki). This strongly indicated that it was the presence of the ligand within the receptor binding pocket that alone accounted for the observed upregulation effects. In a similar manner, it was demonstrated that agonist compounds were also capable of mediating a similar degree of upregulation. The increase in receptor density of CAM β2-AR in response to the presence of ligand was subsequently shown to be dependent on the constitutively active nature of the receptor. In an additional experiment co-transfection of CAM β 2-AR-Rluc along with a GFP conjugated version of the βib- adrenoceptor into HEK 293T cells and subsequent monitoring of the upregulation of either construct in response to selective ligands confirmed the necessity for pharmacological specificity in mediating the upregulatory effect. Finally, to show that this assay method would be suitable as a means of detecting ligands in a high- throughput screening format, the ability of β2-AR to be upregulated was assessed in the presence of a wide variety of compounds, only a proportion of which possessed pharmacological specificity for the β2-AR. When tested in this manner it was seen that only compounds that were specific for β2-AR were capable of mediating an upregulatory effect.

No description available.

No description available.

The principal aim of this Thesis is the synthesis and characterisation of a range of novel liquid crystals designed to exhibit the twist-bend nematic phase (NTB), in order to enhance our understanding of the relationships between molecular structure and the observation of NTB behaviour. Moreover, the inclusion of chiral fragments allowed the effects of molecular chirality on the structure of the NTB phase, specifically the chiral twist-bend nematic phase, N*TB, to be studied. In Chapter 3, a series of non-symmetric odd-membered liquid crystal dimers are prepared and the terminal chain length m is varied. A change in the local molecular structure from intercalated to bilayer is seen on increasing m, but this has no apparent effect on the stability of both the nematic and twist-bend nematic phases, which show a regular dependence on m. A novel twist-bend smectic phase is reported. Chapter 4 investigates the effects of branching this terminal chain on phase behaviour. It is evident that in dimers with a shorter spacer, branching destabilises the N and NTB phases while stabilising smectic behaviour, but in longer homologues smectic behaviour is also destabilised. Chapter 5 explores the effect of molecular bulk on phase behaviour, specifically the stability of the NTB phase, by the incorporation of a pyrene moiety. This group supresses crystallisation such that stable, low temperature NTB phases are formed despite the bulky group. Chapters 6 and 7 study the inclusion of chiral moieties in bent-shaped, odd-membered dimers: specifically, 2-methylbutyl, 1-methylheptyl (6) and lactic esters (7). New examples of the rarely observed chiral twist-bend nematic phase are seen. Phase behaviour is investigated and compared to achiral and racemic analogues. Chapter 8 describes the mesogenic behaviour of molecular complexes assembled by hydrogen bonding between both achiral and chiral stilbazole-based and benzoic acid-based fragments. A selection of the complexes exhibit the N(*)TB phase despite only one or neither component being mesogenic.

Thesis (M.S. in chemistry)--Washington State University, August 2009. / Title from PDF title page (viewed on Aug. 10, 2009). "Department of Chemistry." Includes bibliographical references (p. 53-55).

Thesis (M.S.)--University of Louisville, 2004. / Department of Chemistry. Vita. "May 2004." Includes bibliographical references (leaves 55-59).

Conversion of 1-Methylpentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹]undecane- 8,11-dione into the corresponding mono(ethylene ketal) followed by Wolff-Kishner reduction resulted in a mixture of two isomers (i.e., 1- and 7-methyl-8-[2',-(1',3',dioxolano)]pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹] undecane. Hydrolysis of each isomer in turn resulted in 1- and 7- methyl pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹ ]undecan-8-ones (i.e.,"methylated PCU-8-ones"), respectively. "Titanium-promoted reductive dimerization of each of the methylated pentacycloundecane (PCU)-8-ones afforded mixtures of "methylated PCU alkene dimers". Individual isomers have been isolated from these mixtures via column chromatography by using silver nitrate impregnated silica gel as adsorbent followed by fractional recrystallizations of individual chromatography fractions. Structures of three isomerically pure methylated PCU alkene dimers (C₂₄H₂₈) have been established unequivocally by application of single crystal X-ray crystallographic methods.

This thesis reports laser spectroscopic studies of gas-phase transition metal dimers using laser ablation/reaction with free jet expansion and laser-induced fluorescence (LIF) spectroscopy technique. Themolecules studied in this work are palladium dimer (Pd2) and vanadium dimer (V2). Many compounds formed from these transition metals are important and functional catalysts in chemical reactions. Therefore, it is of great significance to start from the fundamental level to understand the properties and characteristics of the metal bonding and also the behavior of these metals when reacting with other chemicals. The electronic transitions of Pd2and V2in the visible region were studied. Gas-phase Pd2and V2moleculeswereproduced by laser ablation of palladium and vanadium metal rod, respectively. For the Pd2molecule, eleven vibrational bands were recorded and analyzed, and have been assigned to the 〖[17.1]〗^3 □_g□ X^3 □_u^+ transition system. The bond length and vibrational frequency of the ground X^3 □_u^+ state were determined to be 2.47 Å and 211.38 cm-1, respectively. This is the first experimental investigation of the electronic transitions of Pd2.For the V2molecule,six vibrational bands were observed and assigned to a new 〖[19.6] 〗^3 □_u^□□ X^3 □_g^□ transition system. Molecular constants for the 〖[19.6] 〗^3 □_u^□ excited state were obtained from high-resolution LIF spectra. The electronic structure of the Pd2andV2molecules was discussed in detail using molecular orbital energy level diagrams, which is important for understanding the nature of chemical bonding in these dimers. Comparison of the transition metal dimers studied in this work with other dimers is also presented. / published_or_final_version / Chemistry / Master / Master of Philosophy

Thesis (M.S.)--University of North Texas, Aug., 2007. / Title from title page display. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 131-143). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.