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We have observed details of the internal motion and dissociation channels in photoexcited carbon disulfide (CS2) using time-resolved x-ray scattering (TRXS). Photoexcitation of gas-phase CS2 with a 200 nm laser pulse launches oscillatory bending and stretching motion, leading to dissociation of atomic sulfur in under a picosecond. During the first 300 fs following excitation, we observe significant changes in the vibrational frequency as well as some dissociation of the C–S bond, leading to atomic sulfur in the both 1D and 3P states. Beyond 1400 fs, the dissociation is consistent with primarily 3P atomic sulfur dissociation. This channel-resolved measurement of the dissociation time is based on our analysis of the time-windowed dissociation radial velocity distribution, which is measured using the temporal Fourier transform of the TRXS data aided by a Hough transform that extracts the slopes of linear features in an image. The relative strength of the two dissociation channels reflects both their branching ratio and differences in the spread of their dissociation times. Measuring the time-resolved dissociation radial velocity distribution aids the resolution of discrepancies between models for dissociation proposed by prior photoelectron spectroscopy work.

TMEM16A is a Ca2+-activated Cl- channel (CaCC) encoded by the TMEM16A gene, expressed in vascular smooth muscle cells (VSMCs). TMEM16A has a proposed role in depolarising the membrane potential (Vm) in response to activation of the Gq-protein coupled receptor (Gq-PCR) signalling cascade. Therefore, TMEM16A may constitute a key contractile mechanism in VSMCs. Recent elucidation of the TMEM16A channel structure has revealed that the pore region is partially exposed to the plasma membrane. It is therefore hypothesised that TMEM16A may be sensitive to the lipid composition of the plasma membrane. This thesis explores the lipid regulation of cloned TMEM16A channels, and the role of lipids in the control of native TMEM16A currents in VSMCs, and the importance of this in murine arterial (aortic) tone. The main findings of this thesis are: 1, The TMEM16A pharmacological modulator 2-(4-chloro-2-methylphenoxy)-N-[(2- methoxyphenyl)methylideneamino]-acetamide (Ani9) is currently the most potent and efficacious modulator of TMEM16A available. This study demonstrated that Ani9 was selective for TMEM16A in VSMCs. Studies involving Ani9 and a heterozygous knock-out of TMEM16A were used to confirm that TMEM16A is functionally expressed in VSMCs, and that TMEM16A has a key role in vascular contraction. 2, Plasma membrane levels of the intracellular signalling lipid phosphatidylinositol 4,5- bisphosphate (PIP2) are dynamically controlled during activation of Gq-PCR. Whether these changes affect TMEM16A was unknown. Here, it was found that PIP2 increased TMEM16A channel activity. This effect was especially pronounced at physiologically relevant intracellular Ca2+ concentrations ([Ca2+]i) and membrane potentials (Vm). This revealed a novel pathway in the Gq-PCR signalling cascade. 3, The dietary polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) was found to inhibit cloned and native TMEM16A channels. Consistent with this observation, DHA reduced agonist-induced contraction of isolated aortic rings. This observation provided a mechanistic basis for the reported vasodilator properties of DHA. To conclude, the research in this thesis has demonstrated that TMEM16A is highly sensitive to both intracellular lipids (PIP2) and extracellular lipids (DHA). This could form the basis for future studies investigating the TMEM16A pore region, in order to explore whether the TMEM16A pore region could be exploited as a target for pharmacological intervention.

1. Total carbohydrate (CHO) and ingested glucose oxidation was measured in five obese subjects with normal glucose tolerance after an oral load of 100g naturally-labelled [13C]glucose using indirect calorimetry and mass spectrometry respectively. 2. CHO utilization rate (107 ± 14 mg/min in the post-absorptive state) increased 30 min after the glucose load to reach a plateau (245±25 mg/min) between 90 and 120 min. It then decreased to basal values at 330 min. Cumulative CHO oxidation over 480 min was 66±7 g and the CHO oxidized above basal levels was 26 ± 7g. 3. Enrichment of expired carbon dioxide with 13c began at 45 min and maximum values were observed between 210 and 300 min. At 480 min, cumulative oxidation of the ingested glucose was 24± 2 g. 4. Compared with controls, the obese subjects exhibit an impairment of CHO utilization which precedes glucose intolerance. This impairment can be explained by an increased availability of free fatty acids which favours lipid oxidation at the expense of ingested [13C]glucose oxidation

1. Carbohydrate (CHO) oxidation was measured simultaneously in a group of five normal subjects after an oral load of 100 g naturally-labelled [13C]glucose, using indirect calorimetry and mass spectrometry. 2. CHO utilization, calculated from the results of indirect calorimetry, increased 30 min after the glucose load to reach a peak at 90 min. It then decreased to reach basal values at 380 min. Cumulative total CHO oxidation at 480 min was 83±8g, and CHO oxidized above basal levels, 37±3 g. 3. Enrichment of expired carbon dioxide with 13C began at 60 min and maximum values were observed at 270 min. At 480 min, cumulative CHO oxidation measured by use of [13C]glucose was 29 g. The difference from calorimetric values can be attributed in part to the slow isotopic dilution in the glucose and bicarbonate pools. 4. Thus, approximately 30% of the glucose load was oxidized during the 8 h after its ingestion and this accounts for a significant part of the increased CHO oxidation (37 g), as measured by indirect calorimetry

Background and Purpose Ca2+-activated Cl- channels (CaCCs) are gated open by a rise in intracellular Ca2+ concentration ([Ca2+]i), typically provoked by activation of Gq-protein coupled receptors (GqPCR). GqPCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we determined whether PIP2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. Experimental Approach Patch-clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP2 content, was used to define the mechanism of action of PIP2 on TMEM16A and TMEM16B channels. Key Results A water-soluble PIP2 analogue (diC8-PIP2) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by ~0.2-fold. The effects of diC8-PIP2 on TMEM16A currents were especially pronounced at low [Ca2+]i. In contrast, diC8-PIP2 modulation of TMEM16B channels did not vary over a broad [Ca2+]i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co-expression of TMEM16A or TMEM16B with a Danio rerio voltage-sensitive phosphatase (DrVSP), which degrades PIP2, led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co-expression of a phosphatidylinositol-4-phosphate 5-kinase that catalyses PIP2 formation. Conclusions and Implications PIP2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy.