HEADS OR TAILS: LIPID INHIBITORS OF THE GLYCINE TRANSPORTER, GLYT2

Membrane proteins are influenced by the dynamic lipid membrane environment, which can impart stability, mediate protein interactions, and provide highly selective contacts essential for function. Membrane proteins can also bind endogenous lipid ligands or are able to be allosterically modulated by lipids, many of which are thought to access their specific binding sites via the cell membrane. N-arachidonyl glycine (NAGly) is a bioactive lipid that is found in its highest concentrations within the spinal cord and may play an important role in endogenous regulation of glycinergic neurotransmission and pain perception through inhibition of the glycine transporter, GlyT2. In addition to NAGly, a number of lipid inhibitors of GlyT2 have been identified. These compounds are comprised of a long flexible unsaturated acyl tail conjugated to an amino acid or amino acid derivative head group. The aims of my study were two-fold; first to identify new, more potent, lipid inhibitors and develop a structure activity relationship for these compounds; and second, to elucidate the molecular mechanisms of inhibition. Wild type and mutant GlyT2 transporters were expressed in Xenopus laevis oocytes with glycine transport and the subsequent inhibition of transport measured using two-electrode voltage clamp electrophysiology, and radiolabelled uptake of glycine. A library of 55 N-acyl amino acids with varying head and tail groups were synthesised and tested at both GlyT2 and the closely related glycine transporter, GlyT1. Two distinct groups of compounds were tested: the first group maintaining a glycine head group and altering the lipid tail; and the second conjugating the [C18 ω9] oleoyl tail to amino acids with varying properties. I found the lipid constituent of the acyl-glycine analogues is essential for specific interactions and the mechanism of inhibition and is not merely a non-selective, sticky adjunct. There was an ideal chain length, with an order of potency C18 > C16 > C14, and stringently defined double bond conformation and position. Conservative differences between compounds are sufficient to impart or remove inhibitory activity which validates highly specific binding to a subtype specific, allosteric pocket. While changing the tail did not greatly alter potency, analogues where the head group was altered significantly influenced apparent affinity. Acyl amino acids containing an aromatic or positively charged side chain conferred the highest apparent affinity, with C16 ω3 L-Lys possessing the highest potency (10.7 nM). 12 compounds inhibited GlyT2 < 100nM, and one of these inhibitors, oleoyl D-Lys, is also metabolically stable and produces analgesia in a rat model of neuropathic pain. Mutagenesis of extracellular loop 4 (EL4), and transmembrane helices TM5 and TM8 suggest that the allosteric binding site is comprised of a cluster of aromatic residues which may strongly coordinate aromatic or positively charged head groups of the most potent analogues. Additionally, changing the properties of a membrane facing residue alters the otherwise slow washout of lipids. From these results, in addition to dynamic docking studies, it is proposed that acyl amino acids may first diffuse into the lipid bilayer and interact with regions of GlyT2 at the protein-membrane interface. Acyl amino acids then access their final binding site formed by aliphatic and aromatic residues from TM5, TM8, and EL4. It has previously been shown that EL4 undergoes important conformational changes in this family of transporters, where EL4 shifts into the outward facing vestibule to occlude the extracellular side and continue the transport cycle. Acyl amino acids may therefore inhibit GlyT2 by stabilising EL4 in a conformation that does not favour transport. The combination of structure-activity studies with molecular insights provides key information on the mechanism of inhibition which will drive further generation of GlyT2 inhibitors for the treatment of neuropathic pain

MAGNA CARTA AND ACCESS TO JUSTICE IN FAMILY PROCEEDINGS

We had a surprise in January 2012 when a practising QC was parachuted straight into the Supreme Court (as the Judicial Committee of the House of Lords became in 2009). But if there were expressions of discontent they were definitely sub rosa since the man in question, Jonathan Sumption QC, was then, and has since proved himself to be, pre-eminently qualified for the position. He is one of our foremost medieval historians, a Fellow of Magdalen College who taught History, before leaving to pursue a career at the Bar, where he rose to dizzy heights. His as yet uncompleted history of the Hundred Years’ War has received the highest praise. So it perhaps was not surprising that in this octocentenial year he should have been asked by the Friends of the British Library (an audience I warrant as challenging as this) to speak to them about Magna Carta on 9 March 2015. If I may say so, his address ‘Magna Carta then and now’ is a masterpiece. It completes the destruction of the hermeneutical myth originated by Sir Edward Coke which had been commenced by William McKechnie in his landmark essay published in 1905.

A Decision Model for Choosing among Photovoltaic Technologies to Generate Electricity at Grid-Connected Air Force Facilities: A Value-Focused Approach

The United States is currently consuming fossil fuels faster than natural processes can replace them. Our nation\u27s leaders recognize that a diverse energy portfolio including renewable energy is the key to maintaining our economy, security, and the environment. The federal government is by far the greatest energy consumer; thus, our nation\u27s leaders have directed federal agencies to strive to increase the use of renewable energy at federal facilities. Solar electricity technologies, in the form of photovoltaics, have great potential in the renewable energy mix. Although a major strategy should be integrating photovoltaics into the design of new facilities, an important early consideration should be the installation of photovoltaic modules in open areas and/or module retrofits onto existing structures. This research developed a model based on decision makers\u27 value systems to quantify and rank several photovoltaic technologies. The goal of the model was to determine what alternatives would most align with Air Force energy and environmental objectives. After working with subject matter experts at three bases, a comprehensive hierarchy was developed. This hierarchy was then used to find the best alternatives at one base. It was found that photovoltaic technologies may indeed successfully compete with grid-supplied electricity when utilizing a value-focused approach

Taxonomy and pathogenicity of rusts from Allium species in the U.K

Comparison of seven isolates of rust from leek (Allium porrum), three from chives (Allium schoenoprasum), one from A. scorodoprasum and one from A. babingtonii on the basis of telial and uredinial morphology showed there to be three morphologically distinct species. Application of these criteria to herbarium material confirmed these findings and showed the rust on leeks in the U.K. to be the same species found on European leeks, garlic and certain wild Allium spp. from the Mediterranean. It is suggested that the following names be adopted, viz. Puccinia allii (DC.) Rud. for the rust on leeks, Puccinia mixta Fuck. for the rust on chives and Uromyces ambiguus (DC) Lev. for the rust on A. babingtonii. Infection studies on isolates of each rust supported the morphological evidence, and showed the three species to have different, extensive but overlapping host ranges within the genus Allium. In the ampeloprasum complex, A. kurrat accessions were highly susceptible to leek rust whereas some accessions of A. ampeloprasum and A. babingtonii had high levels of quantitative resistance. There was no evidence of 'hypersensitive-type' resistance in the complex. Tests within one leek cultivar (Musselburgh) showed older plants to be more resistant than seedlings in at least two quantitative components. However leaf tissue appeared to become more susceptible to infection with age, except in the leaf tips, which did not change in susceptibility over time. Inoculation of 16 leek cultivars with leek rust isolates from different geographical areas, and subsequent analysis during the disease cycle of several components of resistance (viz; latent period, pustule density and pustule length) showed that some cultivars performed consistently better against all isolates. However, in most cases there was a considerable and complex cultivar-isolate-component interaction. There was no evidence of physiologic specialisation in the isolates, but low levels of specialisation could have been hidden by the high level of variation in the experiments. Comparison of field cultivars of garlic with equivalent virus-free material using an isolate of leek rust gave inconclusive results, and further study of the rust-garlic-virus interaction is recommended. Major trends in the infection/resistance studies included a high level of environmentally-dependent variation and a lack of 'hypersensitive-type' resistance, even in host species quite distantly related to the normal host

Ligand isotope vibrational studies of metal(II) complexes with particular reference to heterocyclic N-oxides

Includes bibliographical references.A critical examination of the characteristic N-O vibrational frequencies of aromatic N-oxides questions the widely held assignment of αN-O (840cm⁻¹) and YN-O (47oon- 1). The present investigation supports the assignments of αN-O (470cm⁻¹) and γN-O (280cm⁻¹), with the assignment of the band at 840cm⁻¹ as being vring coupled with νN-O

From Murphy’s Christian Physicalism to Lowe’s Dualism

Nancey Murphy argues that God created us as physical beings without immortal souls. She supports this Christian physicalism by arguing that neuroscience can better explain minds in terms of physical information processing than dualists can in problematic nonphysical terms. We reply that Murphy overestimates neuroscience and underestimates dualism. She doesn’t show how neuroscience can explain the mind’s characteristic qualia, unity, privacy, or causality. We argue that Lowe’s dualism can better explain minds, often with experimental support and in testable ways. Murphy’s physicalism thus serves to highlight the value of Lowe’s dualism today

Fatty acids in veterinary medicine and research

Fatty acid regulation is an essential process for all animals. A number of studies have shown that diet affects the levels/availability of fatty acids in the body but increasingly evidence shows that disease states can alter the amounts within the body too. Fatty acid levels and availability have been altered by a number of diseases, disorders and reactions including inflammatory responses, heart disease and heart failure and wound repair. They are also essential during the growth and development stages of animals. The amount of research into the consequences of different fatty acid intake and levels in various disease states and during development has increased in both humans and animals. This review presents an overview of the research undertaken to date and highlights the importance, uses and benefits of understanding the roles of fatty acids in both the healthy animal and under differing disorders and diseases

Endocrine regulation of adipose tissue thermogenesis in the fetal and neonatal sheep

Leptin is produced primarily by white adipose tissue but also by brown adipose tissue (BAT) and the placenta. It has a major physiological role in the control of energy balance. BAT is critically important for the initiation of non-shivering thermogenesis in precocial newborns through the uncoupling protein, UCP1 unique to BAT. This is particularly important in neonatal lambs and infants in which levels of UCP1 peak at birth concomitant with a peak in plasma leptin and a surge in Cortisol. Other mitochondrial proteins may contribute to this effect e.g. VDAC which is located in the outer mitochondrial membrane. The aim of this thesis was to: 1. investigate the role of the late gestation Cortisol surge on BAT development 2. investigate the effect of route of delivery on subsequent neonatal plasma leptin concentrations 3. assess the effect of leptin administration to neonatal lambs on thermoregulation Late gestation fetuses were infused with Cortisol or saline or underwent adrenalectomy or sham operation. BAT was sampled at 129 and 144 days of gestation, respectively. UCPl abundance was significantly increased in the animals receiving Cortisol treatment compared to controls and was significantly reduced in adrenalectomised animals. Plasma leptin was analysed from lambs delivered vaginally or near term by caesarean section (CS). Cord plasma leptin decreased significantly after birth, an adaptation that was delayed by CS delivery. Acute and chronic administration of leptin to neonatal lambs improved thermoregulation by preventing a decline in body temperature. Chronic leptin treatment over 7 days (lOOp-g daily) promoted the loss of UCP1 mRNA and protein, but had no deleterious effects on body temperature. Polyclonal antibodies were developed against mitchondrial voltage-dependent anion channel (VDAC). The postnatal ontogeny of VDAC was found to be similar to that of UCP1 and cytochrome c, with abundance peaking around one day of age. VDAC was found in high abundance in organs with high metabolic requirements such as heart, muscle and BAT. These results suggest that VDAC is involved in ensuring BAT maintains a maximal rate of thermogenesis after birth. In conclusion, I have shown for the first time that leptin has an important role in thermogenesis during the transition from fetal to neonatal life. This is attenuated in CS animals, possibly linked to a reduced sympathetic nervous system activity. The rapid loss of UCP1 mRNA, which occurs within the first few days of life, appears to be modulated by leptin, possibly stimulating the development of white adipose tissue and generation of body heat through mechanisms other than nonshivering thermogenesis in BAT. VDAC may be important during this period in ensuring adequate substrate delivery to BAT. Intact adrenal glands are also necessary for the increase in UCP1 abundance during late gestation, an effect mediated in part by Cortisol. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text