BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

Surgical intervention can result in endothelial denudation, driving growth factor-stimulated vascular smooth muscle cell (VSMC) migration towards the intima, leading to luminal narrowing and restenosis. Clinically approved PGI₂ analogues, including beraprost, activate the cyclic adenosine monophosphate (cAMP) signaling pathway to inhibit VSMC migration in vitro. This pathway is a potential therapeutic target, however the downstream proteins involved in the inhibitory effects of cAMP on migration remain unknown. The aims of this study were to determine the signalling pathways involved in inhibiting VSMC migration through cAMP downstream mediators, protein kinase A (PKA) and the more recently characterised exchange protein activated by cAMP (Epac), and delineate the mechanisms involved. In human saphenous vein VSMCs, Epac activation using an Epac analogue inhibited VSMC migration. Therapeutic concentrations of beraprost (1 nM) also resulted in an inhibition of VSMC migration. The use of fluorescence resonance energy transfer (FRET) confirmed 1 nM beraprost activated Epac, but not PKA. Epac is a guanine nucleotide exchange factor (GEF) for Rap1 thus Rap1 siRNA was used to inhibit the Epac pathway. This blocked the inhibitory effects of beraprost on VSMC migration. Epac1 was localised to the leading edge of migrating VSMCs. Another G-protein, RhoA, was investigated since it is essential for cell migration and is involved in several processes including actin regulation. Epac signaling inhibited PDGF-induced RhoA activation and disassembled F-actin at the leading edge, where Epac1 was previously located. This indicates that beraprost activated the Epac pathway, which inhibited RhoA to decrease VSMC migration. The clinical relevance of this study has discovered the mechanisms of Epac's inhibitory action on VSMC migration and this pathway could be targeted therapeutically to reduce restenosis. In the future the potential use of beraprost on a drug eluting stent might be beneficial to prevent restenosis formation following surgical intervention.

This paper studies the predictive performance and in-sample dynamics of three regime switching models for Swedish macroeconomic time series. The models discussed are threshold autoregressive (TAR), Markov switching autoregressive (MSM-AR), and smooth-transition autoregressive (STAR) regime switching models. We perform recursive out-of-sample forecasting to study the predictive performance of the models. We also assess the in-sample dynamics correspondence to the forecast performance and find that there is not always a relationship. Furthermore, we seek to explore if these unrestricted models yield interpretable results regarding the regimes from an macroeconomic standpoint. We assess GDP-growth, the unemployment rate, and government bond yields and find evidence of Teräsvirta's claims that even when the data has non-linear dynamics, non-linear models might not improve the forecast performance of linear models when the forecast window is linear.

Smooth tracking and efficient reaching for moving objects require the ability to predict the velocity and trajectory of the object. This skill is important to be able to perceive human action and object motion in the world. This thesis explores early visual motor development in full term and preterm infants. Study I showed that horizontal eye tracking develops ahead of vertical (full term infants at 5, 7 and 9 months of age). The vertical component is also more affected when a second dimension is added during circular pursuit. It is concluded that different mechanisms appear to underlie vertical and horizontal eye movements Study II-IV compared the development of the ability to visually track and reach for moving objects in very preterm infants born <32 gestational weeks to healthy infants born at term. The development of horizontal smooth pursuit at 2 and 4 months of corrected age was delayed for the preterm group (Study II). Some infants were catching up whereas others were not improving at all. A question raised by the results was whether the delay was caused by specific injuries as a result of the prematurity. However, the delays persisted when all infants with known neonatal complications and infants born small for gestational age were excluded (Study III), indicating that they were caused by prematurity per se. At 8 months corrected age preterm and full term infants were equally good at aiming reaches and successfully catching a moving object. Nevertheless, the preterm group used a bimanual strategy more often and had a more jerky and circuitous path than the full term group (Study IV). In summary, preterm infants showed a delayed visual motor development compared to infants born at term. The results of these studies suggest that there is additional diffuse damage to the visual motor system that is not related to neonatal complications as diagnosed today. Measuring smooth pursuit could potentially be a new method for early non-invasive diagnosis of impaired visual function.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The 1-adrenoceptors (1-ARs) serve as an interface between the sympathetic nervous system and the cardiovascular system where they are mediators of systemic arterial blood pressure, initiators of positive inotropy, and regulators of cellular growth responses. There are three subtypes: 1A-, 1B-, and 1D-ARs. This dissertation research investigated the trafficking properties of the 1-ARs at the cellular level as well as physiological relevance of the 1-ARs at the tissue level. In vitro studies using transiently transfected 1-AR/GFP subtypes revealed distinct basal localization patterns and different agonist-mediated activation and desensitization properties. The 1A- and the 1B-AR/GFP subtypes displayed agonist-mediated receptor redistribution, in which rate and degree of redistribution differed. Additionally, redistribution of either of these two receptor subtypes required arrestin-1, a protein often associated with receptor internalization. In contrast, the 1D-AR/GFP did not require arrestin-1 for maintaining the basal receptor orientation pattern. Although these data increase our knowledge of trafficking properties of the 1-AR subtypes, it is of equal importance to determine the role(s) that each subtype contributes to cardiovascular function. The lack of subtype-selective 1-AR pharmacological agents prompted the use of genetically manipulated mouse models with a systemic overexpression of a constitutively active 1B-AR. Echocardiographic analysis of transgenic hearts indicated both an enlarged left ventricular chamber in the absence of hypertrophy and a depressed cardiac function. From isolated transgenic hearts, experimental results suggested a role for the 1B-AR in attenuating the inotropic responses. However, experiments using isolated thoracic aortae from transgenic animals suggested that the 1B-AR does not participate in vascular smooth muscle contractile responses. Additional studies investigated the role of 1D-AR in cardiovascular function by using animals systemically lacking the 1D-AR subtype. Experimental data suggested an 1D-AR participation in vascular smooth muscle function since the deficiency of the 1D-AR subtype affected vasoconstriction in the coronary arteries but not inotropy in the heart. The data presented in this dissertation research suggest subtype specific differences of 1-ARs in cellular localization, signal regulation, and trafficking. Additionally, the data provide an investigation into the physiologic significance of both the 1B- and the 1D-ARs in cardiovascular tissue.

In a rapidly changing visual environment, the delay between perception and action might impair the probability of survival of a prey or the efficiency of a predator. In order to compensate for delays associated with sensory-motor processing, primates often make predictions about future events and initiate anticipatory movements. To prepare an anticipatory movement, an estimation of when and where to a target is likely to move is necessary. Such an internal representation is often termed 'expectation'. The aim of this thesis was to investigate the gradual changes of a subject's expectation at the behavioral and electrophysiological levels. Anticipatory smooth pursuit was used in order to study temporal and directional changes in expectation. We found that temporal uncertainty strongly modulated the latency and the velocity of anticipatory movements suggesting that monkeys could estimate the hazard rate of target motion onset in order to decide when to initiate an anticipatory movement. In addition, we have shown that monkeys could use prior directional information in order to voluntarily initiate anticipatory responses in the direction of expected target motion. This prior directional information significantly affected the latency and velocity of these movements. Finally, we have shown that the majority of recorded supplementary eye field (SEF) neurons encoded expected target motion direction. The presence of a directional cue induced an increase of activity in the preferred direction of the neuron. Moreover, a large sub-population of neurons encoded the direction of future anticipatory movement. These results suggest that the SEF could be involved in the cognitive control of anticipatory pursuit eye movements when prior temporal and directional information is provided.

Impaired mass transfer characteristics of blood borne vasoactive species such as ATP in regions such as an arterial bifurcation have been hypothesized as a prospective mechanism in the aetiology of atherosclerotic lesions. Arterial endothelial (EC) and smooth muscle cells (SMC) respond differentially to altered local hemodynamics and produce coordinated macro-scale responses via intercellular communication. Using a computationally designed arterial segment comprising large populations of mathematically modelled coupled ECs & SMCs, we investigate their response to spatial gradients of blood borne agonist concentrations and the effect of micro-scale driven perturbation on the macro-scale. Altering homocellular (between same cell type) and heterocellular (between different cell types) intercellular coupling we simulated four cases of normal and pathological arterial segments experiencing an identical gradient in the concentration of the agonist. Results show that the heterocellular calcium (Ca2+) coupling between ECs and SMCs is important in eliciting a rapid response when the vessel segment is stimulated by the agonist gradient. In the absence of heterocellular coupling, homocellular Ca2+ coupling between smooth muscle cells is necessary for propagation of Ca2+ waves from downstream to upstream cells axially. Desynchronized intracellular Ca2+ oscillations in coupled smooth muscle cells are mandatory for this propagation. Upon decoupling the heterocellular membrane potential, the arterial segment looses the inhibitory effect of endothelial cells on the Ca2+ dynamics of underlying smooth muscle cells. The full system comprising hundreds of thousands of coupled nonlinear ordinary differential equations simulated on the massively parallel Blue Gene architecture. The use of massively parallel computational architectures shows the capability of this approach to address macro-scale phenomena driven by elementary micro-scale components of the system.

Numerous macroscale models of arteries have been developed, comprised of populations of discrete coupled Endothelial Cells (EC) and Smooth Muscle Cells (SMC) cells, an example of which is the model of Shaikh et al. (2012), which simulates the complex biochemical processes responsible for the observed propagating waves of Ca2+ observed in experiments. In a 'homogenised' model however, the length scale of each cell is assumed infinitely small while the population of cells are assumed infinitely large, so that the microscopic spatial dynamics of individual cells are unaccounted for. We wish to show in our study, our hypothesis that the homogenised modelling approach for a particular system can be used to replicate observations of the discrete modelling approach for the same system. We may do this by deriving a homogenised model based on Goldbeter et al. (1990), the simplest possible physiological system, and comparing its results with those of the discrete Shaikh et al. (2012), which have already been validated with experimental findings. We will then analyse the mathematical dynamics of our homogenised model to gain a better understanding of how its system parameters influence the behaviour of its solutions. All our homogenised models are essentially formulated as partial differential equations (PDE), specifically they are of type reaction diffusion PDEs. Therefore before we begin developing the homogenised Goldbeter et al. (1990), we will first analyse the Brusselator PDE with the goal that it will help us to understand reaction diffusion systems better. The Brusselator is a suitable preliminary study as it shares two common properties with reaction diffusion equations: oscillatory solutions and a diffusion term.

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.