The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Biochemical and epidemiological investigations of non-steroidal anti-inflammatory drug usage and related side effects in equids

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in equine veterinary practice. These drugs exert their effect by inhibiting cyclooxygenase (COX) enzymes, which control prostaglandin production, a major regulator of tissue perfusion. Two isoforms of COX enzymes exist: COX-1 is physiologically present in tissues, while COX-2 is up-regulated during inflammation and has been indicated as responsible for the negative effects of an inflammatory response. Evidence suggests that NSAIDs that inhibit only COX-2, preserving the physiological function of COX-1 might have a safer profile. Studies that evaluate the effect of NSAIDs on COX enzymes are all performed under experimental conditions and none uses actual clinical patients. The biochemical investigations in this work focus on describing the effect on COX enzymes activity of flunixin meglumine and phenylbutazone, two non-selective COX inhibitors and firocoxib, a COX-2 selective inhibitor, in clinical patients undergoing elective surgery. A separate epidemiological investigation was aimed at describing the impact that the findings of biochemical data have on a large population of equids. Electronic medical records (EMRs) from 454,153 equids were obtained from practices in the United Kingdom, United States of America and Canada. Information on prevalence and indications for NSAIDs use was extracted from the EMRs via a text mining technique, improved from the literature and described and validated within this Thesis. Further the prevalence of a clinical sign compatible with NSAID toxicity, such as diarrhoea, is reported along with analysis evaluating NSAID administration in light of concurrent administration of other drugs and comorbidities. This work confirms findings from experimental settings that NSAIDs firocoxib is COX-2 selective and that flunixin meglumine and phenylbutazone are non-selective COX inhibitors and therefore their administration carries a greater risk of toxicity. However the impact of this finding needs to be interpreted with caution as epidemiological data suggest that the prevalence of toxicity is in fact small and the use of these drugs at the labelled dose is quite safe.

Autonomous formation flying: unified control and collision avoidance methods for close manoeuvring spacecraft

The idea of spacecraft formations, flying in tight configurations with maximum baselines of a few hundred meters in low-Earth orbits, has generated widespread interest over the last several years. Nevertheless, controlling the movement of spacecraft in formation poses difficulties, such as in-orbit high-computing demand and collision avoidance capabilities, which escalate as the number of units in the formation is increased and complicated nonlinear effects are imposed to the dynamics, together with uncertainty which may arise from the lack of knowledge of system parameters. These requirements have led to the need of reliable linear and nonlinear controllers in terms of relative and absolute dynamics. The objective of this thesis is, therefore, to introduce new control methods to allow spacecraft in formation, with circular/elliptical reference orbits, to efficiently execute safe autonomous manoeuvres. These controllers distinguish from the bulk of literature in that they merge guidance laws never applied before to spacecraft formation flying and collision avoidance capacities into a single control strategy. For this purpose, three control schemes are presented: linear optimal regulation, linear optimal estimation and adaptive nonlinear control. In general terms, the proposed control approaches command the dynamical performance of one or several followers with respect to a leader to asymptotically track a time-varying nominal trajectory (TVNT), while the threat of collision between the followers is reduced by repelling accelerations obtained from the collision avoidance scheme during the periods of closest proximity. Linear optimal regulation is achieved through a Riccati-based tracking controller. Within this control strategy, the controller provides guidance and tracking toward a desired TVNT, optimizing fuel consumption by Riccati procedure using a non-infinite cost function defined in terms of the desired TVNT, while repelling accelerations generated from the CAS will ensure evasive actions between the elements of the formation. The relative dynamics model, suitable for circular and eccentric low-Earth reference orbits, is based on the Tschauner and Hempel equations, and includes a control input and a nonlinear term corresponding to the CAS repelling accelerations. Linear optimal estimation is built on the forward-in-time separation principle. This controller encompasses two stages: regulation and estimation. The first stage requires the design of a full state feedback controller using the state vector reconstructed by means of the estimator. The second stage requires the design of an additional dynamical system, the estimator, to obtain the states which cannot be measured in order to approximately reconstruct the full state vector. Then, the separation principle states that an observer built for a known input can also be used to estimate the state of the system and to generate the control input. This allows the design of the observer and the feedback independently, by exploiting the advantages of linear quadratic regulator theory, in order to estimate the states of a dynamical system with model and sensor uncertainty. The relative dynamics is described with the linear system used in the previous controller, with a control input and nonlinearities entering via the repelling accelerations from the CAS during collision avoidance events. Moreover, sensor uncertainty is added to the control process by considering carrier-phase differential GPS (CDGPS) velocity measurement error. An adaptive control law capable of delivering superior closed-loop performance when compared to the certainty-equivalence (CE) adaptive controllers is finally presented. A novel noncertainty-equivalence controller based on the Immersion and Invariance paradigm for close-manoeuvring spacecraft formation flying in both circular and elliptical low-Earth reference orbits is introduced. The proposed control scheme achieves stabilization by immersing the plant dynamics into a target dynamical system (or manifold) that captures the desired dynamical behaviour. They key feature of this methodology is the addition of a new term to the classical certainty-equivalence control approach that, in conjunction with the parameter update law, is designed to achieve adaptive stabilization. This parameter has the ultimate task of shaping the manifold into which the adaptive system is immersed. The performance of the controller is proven stable via a Lyapunov-based analysis and Barbalat’s lemma. In order to evaluate the design of the controllers, test cases based on the physical and orbital features of the Prototype Research Instruments and Space Mission Technology Advancement (PRISMA) are implemented, extending the number of elements in the formation into scenarios with reconfigurations and on-orbit position switching in elliptical low-Earth reference orbits. An extensive analysis and comparison of the performance of the controllers in terms of total Δv and fuel consumption, with and without the effects of the CAS, is presented. These results show that the three proposed controllers allow the followers to asymptotically track the desired nominal trajectory and, additionally, those simulations including CAS show an effective decrease of collision risk during the performance of the manoeuvre.

The influence of British rule on elite Indian menswear: the birth of the Sherwani

‘The Influence of British Rule on Elite Indian Menswear: The Birth of the Sherwani’ is a study of the influence of politics on fashion and the resulting development of new garments. This research is designed to demonstrate the effect on elite Indian menswear of the two centuries of British rule in India. It is an effort to understand how the flowing garments worn by elite Indian men in the 18th century gradually became more tailored and fitted with the passage of time. The study uses multiple sources to bring to light lesser known facts about Indian menswear, the evolution of different garments and especially of the sherwani. The sherwani is a knee-length upper garment worn by South-Asian men, and is considered to be India’s traditional menswear. My study highlights the factors responsible for the birth of the sherwani and dispels the myth that it was a garment worn by the Mughals. Simultaneously, this study examines the concept and value of ‘tradition’ in cultures. It scrutinises the reasons for the sherwani being labelled as a traditional Indian garment associated with the Mughal era, when in fact it was born towards the end of the 19th century. The study also analyses the role of the sherwani as a garment of distinction in pre- and post-independence India.