From the Outside In: Incorporating the use of EFT into traditional psychotherapeutic approaches and its impact on therapeutic alliance

This thesis examines the effect of the use of Emotional Freedom Techniques (EFT) on the therapeutic alliance. Sixteen therapists who had both traditional psychotherapy training and had been trained to use EFT were interviewed in an exploration of their beliefs about whether the use of EFT enhanced, threatened or had any effect on the therapeutic alliance in their work with clients. There was no existing research in this field and due to the essential nature of the therapeutic alliance in any therapeutic modality (Bordin, 1994; Horvath, 2001; 2018) the necessity for it seemed clear, when thinking about how EFT might be incorporated into traditional psychotherapeutic work. The research arose out of an episode in the researcher’s clinical work when the therapeutic alliance appeared to have been ruptured by the introduction of EFT. The methodology used for this qualitative research was Framework Analysis. This allowed the researcher to analyse the results, developing themes that emerged across all participants and simultaneously dissecting some aspects of the interviews in greater depth. The results indicate that the use of EFT appears to enhance the therapeutic alliance. However, there were also indications that the therapists participating in this study found it difficult to define the term ‘therapeutic alliance’ and struggled to put their conceptualisation of the phenomenon into words

An Assessment of Tarong Bottom Ash for Use on Agricultural Soils

Coal combustion by-products (CCBs), including fly ash and bottom ash, present a waste disposal problem in Australia due to a continuing demand for coal fired power. Pozzolanic Enterprises handle ash produced by the Tarong Energy coalfired power station in SouthWest Queensland, which produces approximately 1,200,000 tonnes per annum of CCBs. This comprises roughly 1,100,000 tonnes of fly ash and 100,000 tonnes of furnace bottom ash. The volume and unique properties of the Tarong bottom ash present a significant opportunity for agronomic use. Of particular interest is the ability of Tarong bottom ash to markedly improve the water holding capacity of soils. Given Australia's rural environment is currently enduring a 'one-in-one- hundred-year' drought a study of the ability of Tarong bottom ash to improve water holding capacity is timely. This paper details physical and chemical properties relevant to agronomic use and water holding capacity of ash/soil blends along with some results from initial field trials

Phosphorylation of Spinophilin Modulates Its Interaction with Actin Filaments

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission

Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating

CFTR, the protein defective in cystic fibrosis, functions as a Cl− channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [α32P]8-N3ATP or [γ32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0°C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30°C, with extensive washing, also at 30°C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30°C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore

Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35°C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(ΔH‡) were determined from Eyring plots. ΔH‡ was 117 ± 6 and 69 ± 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 ± 6 and 73 ± 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. ΔH‡ for reversal of the channel opening step, estimated from closure of ATP hydrolysis–deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (ΔS‡), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. ΔS‡ appears large for opening but small for normal closure. The large ΔH‡ and ΔS‡ (TΔS‡ ≥ 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small ΔS‡ for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface

Distinct Mg2+-dependent Steps Rate Limit Opening and Closing of a Single CFTR Cl− Channel

The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels remain controversial. In this work, the contributions made by ATP and Mg2+ ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg2+] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg2+-dependent step that followed binding of both ATP and Mg2+. Once a channel had opened, sudden withdrawal of all Mg2+ and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg2+ ions alone could close it, and the closing rate increased with [Mg2+] over the micromolar range (half maximal at ∼50 μM [Mg2+]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg2+ ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP