Sensorimotor processes within the spinal cord are profoundly influenced by descending systems from the brain yet the neurotransmitter phenotypes of these systems remains enigmatic. Using tract tracing methods, this study identifies the neurochemical content of axons descending from the sensorimotor cortex and sites within the anatomically diverse reticular formation. There are several classes of neurons in spinal lumbar segments targeted by these descending systems which contribute to recruiting adaptively appropriate motor patterns. This study investigates the neuronal targets of descending systems and the neurochemical content of their inputs. Immunofluorescent tissue containing labelled neuronal processes were examined and reconstructed interneurons were defined according to their neuronal geometry and morphology across laminar boundaries.

Kinesin-12, Kif15 is a molecular motor involved in bipolar spindle assembly. Kif15 function is regulated through autoinhibition of its C-terminal tail and binding to the microtubule-associated protein Tpx2. Previous studies have reported Kif15 to function as a tetramer as well as a dimer. In this study, a cross-linking mass spectrometry (XL-MS) protocol and analysis workflow was developed to study the structural organisation of Kif15. Using XL-MS studies, it was found that Kif15 adopts a parallel tetramer conformation, which is autoinhibited by its C-terminal leucine zipper. Next, we show that this autoinhibited conformation is stabilised by the binding partner Tpx2. We also show that there is a shift in the binding interface between Kif15 and Tpx2 when microtubules are present and absent. In the presence of microtubules, Tpx2 mainly binds to the leucine-zipper of the Kif15 motor, whereas in the absence of the microtubules, this binding is exclusively localised to the fourth coiled-coil. We also reveal that Tpx2 adopts a dimeric conformation at physiological ionic strength. Finally, to understand the function of Kif15 in-vivo, we have developed putative Kif15 knock-out cell lines and developed a cross-linking protocol to cross-link Kif15 in cells.

The dynamics of membrane proteins is an understudied area due to the difficulties in production and manipulation of samples. Interactions of the G-protein coupled receptor, rhodopsin, with an allosteric reagent were systematically investigated through absorbance spectroscopy. Folding mechanisms in particular are not understood for membrane proteins, due to the fact that even partial unfolding often leads to aggregation. There are few model systems for exploring membrane protein folding, but methods for obtaining large quantities of non-aggregating unfolded states of rhodopsin have previously been established. In this thesis the previous work establishing rhodopsin as a model system was extended with the use of isotope labelled methionines for NMR spectroscopy of unfolded states. The misfolding rhodopsin mutant, P23H, was compared with wild-type using this system, and found to have significant differences. The same mutant was also found to be destabilised by normally benign cysteine labelling, which has implications for concurrent research. The first membrane protein to have been fully unfolded and refolded is bacteriorhodopsin, to which sensory rhodopsin II (pSRII) is an analogue. Computational work suggests that pSRII has a folding mechanism somewhat like both bacteriorhodopsin and rhodopsin. The kinetics of unfolding and refolding pSRII were established using experiments with low sample requirements, and thoroughly analysed. Several analytical methods were applied to find underlying processes in unfolded states, revealed through NMR spectroscopy.

Two functions of the upper limb are reaching and maintaining balance. To successfully perform these roles requires a mechanism for detecting and compensating for body sway. Here I investigate this possible mechanism by using vestibular stimulation to evoke responses in both the upper and lower limbs. I demonstrate that the evoked arm movements are scaled to the degree of sensed whole-body motion, and only operate when reaching within an earth-fixed reference frame. These findings suggest that vestibular signals contribute to maintaining reach accuracy during unexpected body motion which would otherwise take the limb off-target. I also show that the arm responds to vestibularly sensed body motion for balance. When firmly grasping a stationary support, upper limb forces are coordinated with ground reaction forces to produce a counteractive whole-body sway response. In contrast, during light grasp (< 1N grip force) the arm does not actively engage in balance. Instead, it simply acts as a passive sensor to provide feedback of body motion which improves balance. Finally, even though postural control during stance has been successfully modelled as an inverted pendulum, my results suggest that the nervous system does not transform light touch feedback into a signal of rotation about the ankle joint.

Major common complications of diabetes such as myocardial infarction arise from the onset of vascular disease. However, there is also evidence of a direct impairment of cardiac contractile function in diabetic individuals in the absence of atherosclerosis and hypertension, termed diabetic cardiomyopathy (DCM). This is characterised by early diastolic dysfunction that progresses to systolic dysfunction and heart failure through a pathological remodelling process. The earliest identified mechanism underlying this disease is the onset of metabolic perturbations such as cardiac insulin resistance. However, currently there are no specific treatments available, partly due to the lack of an appropriate experimental model with which translational research could be performed. iPSC-CM are a recently developed technology, whereby human dermal fibroblasts can be reliably harvested, dedifferentiated into a pluripotent form, and then differentiated into cardiomyocytes. These cells have an established intracellular calcium handling system and contractile capacity, however are generally considered to be at a foetal stage of development. A key aim of this project was to characterise the metabolic phenotype of these cells, in order to assess their potential suitability as the basis of a novel cellular model of DCM. Specifically, it was investigated if these cells exhibited robust insulin stimulated glucose uptake through insulin sensitive intracellular trafficking of the glucose transporter GLUT4, as impairment of this response is a central feature of any diabetic model. After adaptation of a [3H]-2-deoxyglucose uptake assay to a 96-well plate format, and optimisation of experimental factors, it was determined that iPSC-CM could not display robust insulin (or IGF-1) stimulated glucose uptake. Inhibition of the spontaneous contractile capacity of these cells did not induce a response upon subsequent insulin stimulation. iPSC-CM were found to express and activate central insulin signalling molecules such as Akt and Erk1/2, and also possess elements of the GLUT4 trafficking machinery such as the SNARE proteins Syntaxin 4 and SNAP23. However, the critically limiting factor identified was an approximate 10-fold lower expression of GLUT4 in iPSC-CM compared to primary adult cardiomyocytes, accompanied by strong expression of GLUT1. This data was supported by the finding that inhibition of GLUT4 had no impact on glucose uptake in iPSC-CM, whereas inhibition of GLUT1 significantly reduced uptake by ~50%. This phenotype suggests that iPSC-CM are also at a foetal-like stage of development with regards to their metabolic capacity, and are currently not suitable for modelling DCM. Subsequently, initial interventions based upon the literature were implemented in order to try and increase iPSC-CM GLUT4 content. However, neither increasing metabolic reliance upon fatty acid (rather than glucose) nor exposure to triiodothyronine were successful. In contrast, Lipofectamine 2000 mediated transfection of a customised GLUT4 plasmid facilitated a reliable 3-5 fold increase in iPSC-CM GLUT4 content. This increased basal glucose uptake, however did not induce an insulin response. It was concluded that a further increase in expression levels may be required. Finally, it was demonstrated that iPSC-CM are highly amenable to lentiviral mediated infection, and initial steps were taken towards the generation of a virus targeting the overexpression of GLUT4. Additionally, SNARE proteins are essential in facilitating insulin stimulated GLUT4 expression at the plasma membrane. Therefore they represent a possible mechanism by which cardiac insulin resistance could occur in disease states such as DCM. On account of this, the expression of a wide range of SNARE protein isoforms was assessed in cardiac lysates generated from 2 diabetic mouse models (db/db and high fat diet induced). The expression of SNAP29 and VAMP5 were found to differ in lysates from the high fat diet model, although the role of these proteins in GLUT4 trafficking is unclear. In contrast, in the more severe diabetic db/db model GLUT4 protein content was found to be significantly reduced, but SNARE protein content was unaffected. Finally, there is also an established link between glycemic control and both the risk of developing and subsequent prognosis for myocardial infarction (MI). There is a line of evidence suggesting that cardiac insulin sensitivity may also be highly relevant in this disease context. Accordingly, it was demonstrated that cardiomyocytes isolated from a clinically relevant 8-12 weeks post-MI rabbit model exhibited impaired insulin stimulated glucose uptake. This strengthens the association between MI and cardiac metabolic parameters. However, insulin stimulated phosphorylation of Akt, GLUT4 levels, and SNARE protein expression were unaffected post-MI. Therefore future work must identify both the underlying mechanism and clinical relevance of this finding.

Few results have been published about the composition of individual phospholipids in liver cell nuclei. Nuclei have been isolated in either 1% citric acid or 2.2M sucrose. Rigorous tests by chemical and microscopic methods showed them to be virtually free from contamination. The chemical composition of these nuclei has been compared. Evidence from lipid analyses, surface area measurements of lipid films, and electron microscopy is consistent with the view that citric acid-isolated nuclei are bounded by a single unit-membrane, whereas in sucrose-isolated nuclei the typical double membrane is preserved. The composition of glycerophosphatides in various tissues has been measured by paper chromatography of their water-soluble deacylation products. Both phospholipids and fatty acids are similarly distributed in nuclear, mitochondrial and microsomal fractions of rat liver. Only cardiolipin seems to have a unique site in the cell - namely in the mitochondrion. A survey of various tissues indicated that a particular phospholipid pattern is fairly widespread. The 32P uptake by phospholipids of rat liver cell fractions has been studied at different times in vivo. Whereas large differences have been observed in the uptake of label by individual phospholipids, the pattern of labelling is similar in each cell fraction. There was no significant change in the uptake of 32P into phospholipids of regenerating liver over normal or sham-operated controls in any cell fraction under the conditions chosen for investigation. No specific phospholipid appeared to be concerned in cell division. In the intestinal mucosa of the rat, phosphatidic acid was the most highly labelled phospholipid after a thirty minute isotopic exchange period. During the absorption of triglyceride, the labelling of this compound did not change significantly, but the uptake of isotope into phosphatidylcholine increased five-fold. This was probably due to the increased requirement of phosphatidylcholine for stabilisation of the chylomicrons during fat absorption.

Face perception is important in a variety of human social interactions, allowing us to keep track of individuals’ identities, recognise emotional expressions and intentions and make judgements about variables such as age, ethnicity and health. While early research assumed that face recognition strategies were universal, more recent studies have shown that East Asian and White Caucasian observers use different looking strategies to recognise faces, with East Asian participants focusing more on the centre of the face, which has been interpreted as representing a configural processing strategy, while White Caucasian observers fixate more on the eyes and mouth, which has been interpreted as representing a more featural processing strategy. Debate continues over the reasons behind this difference, with some researchers arguing that it represents an extension of more holistic cognition in the more collectivist East Asian cultures, and more analytic cognition in individualist Western cultures. The Sapir-Whorf hypothesis suggests that cognition is bound by language, and there have been studies showing changes in response patterns in tasks conducted by bilingual participants in their different languages. Others argue that these differences in face processing are driven instead by different salient diagnostic features of faces of different ethnicity. In this thesis, I present the results of five studies examining the role of culture and facial appearance in determining the looking strategy of East Asian and White Caucasian observers. In Chapter 2, we attempted to use a Navon task to prime featural or configural processing in Malaysian Chinese observers engaged in a face recognition and description task of East Asian and White Caucasian faces. While the Navon task failed to elicit a change in either looking or description strategy, it was noted that the features fixated on most were not the features described most frequently. Further, the race of the face impacted on the looking strategy used to recognise faces, with participants fixating more on Caucasian hair than Asian hair, suggesting that the different diagnostic features may drive differences in looking strategies. It was also casually observed that observers with stronger Asian accents made more configural descriptions. In Chapter 3, I investigate the strategies used by Malaysian Chinese and White Caucasian observers when recognising and describing East Asian and White Caucasian faces. A linguistic/cultural priming paradigm was used in an attempt to induce featural or configural processing in observers. In Study 1, the East Asian observers’ eye movements were impacted by the race of the faces, making more fixations on Caucasian hair and eyes than on Asian hair and eyes. Again, patterns of looking and description were very different. Also, the description patterns differed by language, with participants making more descriptions of hair when speaking English and more descriptions of noses when speaking Chinese, suggesting that descriptions may be constrained by language. In Study 2, White Austrian Caucasian observers again showed very different description and fixation patterns. Observers again showed different fixation patterns for Asian and Caucasian faces, fixating more on Caucasian hair than Asian hair, suggesting that fixation pattern may be driven by the diagnostic features of the faces. Observers made more descriptions in German than in English, but did not show a difference in the pattern of describing different facial features depending on either the language spoken or the race of face, suggesting that the more similar German and English languages have similar constraints. Asian observers have been previously shown to direct more attention to contextual information in images of scenes than Caucasian observers, possibly due to a more holistic/configural cognitive style. Since it is known that faces are processed in a different way to other stimuli, in Chapter 4, I report the results of two studies investigating the impact of linguistic/cultural priming on participants’ eye movements and descriptions when describing street scenes. The Malaysian Chinese participants made more fixations on, and descriptions of, nonfocal than focal objects in Asian street scenes and when speaking Chinese, but not when describing European scenes in English. The White Austrian Caucasian observers did not show any difference in fixation or description patterns depending on linguistic condition, other than making more descriptions overall in German than in English. This suggests that, in a non-face description task, linguistic/cultural priming was successful in eliciting cultural “frame shifting” in Malaysian Chinese participants speaking English and Chinese, but not in Austrian Caucasian participants speaking the culturally more similar English and German. We conclude that culture/language does impact on description patterns in face and scene stimuli, possibly reflecting the constraints of different languages. Further, an impact of linguistic/cultural priming was found on fixation patterns in street scene stimuli. However, in face perception tasks, race of face, but not cultural/linguistic condition, impacted on fixation patterns. We conclude that, while language and culture may have an impact on cognition, and place constraints on descriptions, the diagnostic features of faces appear to primarily determine the fixation patterns on face stimuli.

Chapter 1 introduced the old and new measurement of endothelial function, and vascular health. The first part of this thesis focussed on the “old” flow-mediated dilation measurement, introduced in 1992, which relies on brachial artery vasodilation after a hyperaemic stimulus (e.g. after occlusion of a pneumatic cuff), and the susceptibility of the FMD measurement to variability. In Chapter 2 we sought for factors that might help to improve the reproducibility of the FMD measurement. We performed an analysis including 672 participants with repeated FMD. Overall we found an acceptable reproducibility, with 33% of the FMD measurements showing an excellent-to-moderate reproducibility. We identified several factors that independently increased the variation of the FMD, including the presence of hypertension, a lower resting FMD%, a larger baseline artery diameter, a longer time between subsequent measurements, and less laboratory experience with the measurement. Future studies should take these factors into consideration, as certain measures may lower variability of the FMD or more subjects should be included in individual studies when variation relates to non-modifiable factors. Unfortunately, we also found that a large proportion of our study population demonstrated a moderate-to-poor reproducibility, despite all included studies adhered to expert-consensus guidelines. In Chapter 3, we described the relation between adherence to the expertconsensus guidelines and reproducibility of the FMD. In this meta-analysis, we combined data from twenty-seven studies, comprising 48 study groups, with a total of 1537 subjects. Adherence to expert guidelines was inversely related to the measurement error and adopting the guidelines (with specific notification of the use of a stereotactic probe-holder, continuous diameter recording and the use of automated wall-detection and analysis software) was crucial for improving the reproducibility of the FMD. The second part of this thesis introduced the “new” carotid artery reactivity (CAR) measurement, which depends on carotid artery vasomotor responses following sympathetic stimulation (e.g. after a cold pressor test, CPT). The carotid artery appears to mirror coronary responses to CPT, as it shows dilation in healthy participants, whilst those at risk demonstrate a constriction. In this thesis, we sought to understand the relation of CAR with risk factors, and the similarity with the coronary responses. An attempt was made to unravel the underlying physiological mechanism of CAR. Finally, the prognostic value of CAR was examined in peripheral arterial disease patients, to further investigate to clinical potential of the CAR test. In Chapter 4 we explored the relation of CAR with cardiovascular risk, followed by assessing the similarity in response to sympathetic stimulation between the carotid artery and coronary arteries. We first compared CAR between 50 young and 44 older participants to assess relationships between CAR and traditional cardiovascular risk factors. We found that CAR was lower in participants with ≥2 risk factors, compared to those with lesser risk factors. Secondly, we compared left anterior descending (LAD) artery velocity with carotid artery diameter in a subgroup of 33 participants, to assess similarity between coronary and carotid artery responses. We found that CAR correlated well with coronary artery velocity. This implies that CAR is related to increased CV risk and may represent a surrogate measure for coronary vascular health. In Chapter 5, the physiological mechanism underlying the CAR was further explored, by examining carotid artery responses to different sympathetic stimuli (e.g. the cold pressor test [CPT] and the lower body negative pressure test [LBNP]), exploring the role of α1-receptors, (nor) epinephrine receptors contributing to vasoconstriction, and assessing similarity between carotid and coronary arteries. First, 10 participants underwent both sympathetic tests in randomized order, whilst concurrently measuring CAR and coronary artery velocity. We found distinct carotid artery responses to different tests of sympathetic stimulation (e.g. dilation in response to CPT, and constriction following LBNP). Second, when measurements were repeated following α1-receptor blockade by Prazosin, we found α1-receptors partly contributed to CPT-induced responses. Finally, we found agreement between carotid and coronary artery responses, during both types of sympathetic nerve stimulation as well as during α1-receptor blockade. These data indicate strong similarity between carotid and coronary responses to sympathetic tests and the role of α1-receptors. Since the CAR test was newly introduced, the prognostic value of the test remained unknown and this question is highly relevant to understand its clinical utility. Therefore, in Chapter 6, we examined whether CAR predicts (cardiovascular) events in patients with peripheral arterial disease. A total of 172 PAD patients were included, and we recorded cardiac and cerebrovascular events, mortality and clinical progression to percutaneous transluminal angioplasty or loss of patency during a 12-months follow-up. We found that patients with carotid constriction showed a four-fold higher risk for cardiovascular events and two-fold increased risk for clinical deterioration, even after adjustment for other risk factors. This indicates that CAR provides a simple, novel strategy to predict CV events and progression in PAD patients, which has stronger prognostic value than current techniques. Chapter 7 summarizes, discusses, and explains the findings of these studies, and aims to provide recommendations and implications. We discuss future prospects, and provide perspective for future vascular health assessment.

Introduction: The first beats of the developing heart resemble peristaltic contractions with electrical activity arising in the precursor to the sino-atrial node and propagating through the heart tube. The purpose of this thesis was to investigate channelrhodopsin-2 (ChR-2) based optogenetic stimulation of induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with the intention of using the technique to investigate the functional effects of directional activation on immature cardiomyocytes. Methods: Commercially supplied iPSC-CMs (Axiogenesis) were made to express ChR-2 (H134R) via an adeno-associated viral vector (AAV1.CAG.hChR2(H134R)-mCherry.WPRE.SV40). Cell function was assessed before and after pacing using in house software to measure contraction kinetics and strength-duration curves to determine cell excitability. α-actinin staining allowed for analysis of sarcomere length, inhomogeneity and alignment. Results: Commercially supplied iPSC-CMs had significantly shorter sarcomeres than their adult counterparts (1.766 ± 0.017 μm vs 2.028 ± 0.079 μm respectively, P=0.0125 unpaired t-test). A viral load of 2500GC/cell produced ChR-2 expression sufficient for optical control of these cells 5 days post transfection. The chronaxie of photostimulated cells (1.53 ± 0.32 ms) did not differ significantly from that of electrically stimulated cells (2.17 ± 0.44 ms, P=0.2434 unpaired t-test). Prolonged photostimulation of a single site led to a loss of stimulation:contraction coupling after 2.22 ± 1.26 hours and almost complete loss of contractile function after 24 hours. Alternating stimulation between multiple sites allowed cells to be paced for 96 hours and allowed directional electrical activation to be imposed on immature heart cells. 96 hours of directional pacing produced no changes in cell structure or cell excitability but produced a significant reduction in contraction upstroke time (72.03 ms vs 48.85 ms P=0.023 one-way ANOVA) 24 hours after the termination of stimulation. Conclusions: This project has demonstrated that replicating the directional electrical activity of the developing heart can induce a modest change in contractile function that may represent a step towards functional maturation of commercially available iPSC-CMs.

This case completes the trilogy and attempts to answer the open questions raised in the A and B Cases. It offers a retrospective of the events since the IBM-Lenovo merger in 2005 until August 2012. The main focus is on the period between the global financial crisis and mid-2012. The case describes the frequent changes at the top management level and highlights the leadership issues involved in making Lenovo a global leader in the PC industry. An industry and market overview reveals that while Lenovo was attempting to deal with internal issues during the post-merger integration phase it lost market share to competitors. A series of strategic changes, organizational restructurings, and changes in organizational culture paved the way for a new era in Lenovo's history, marked by strong financial performance, product innovation, and promising growth. / Series: WU Case Series