Cardiovascular toxicity is a leading cause of drug attrition at the preclinical and clinical stages of drug development. The specific mechanisms of drug-induced cardiovascular toxicities, however, are not well understood and can occur via direct interactions of the drug with cardiomyocytes or indirectly through interactions with other components of the cardiovascular system. Indirect drug effects can target non-cardiomyocyte cells such as fibroblasts, macrophages, vascular smooth muscle cells and endothelial cells. Evidence suggests interactions between these cell types are essential to the metabolism, growth, contractile performance and rhythmicity of the myocardium (Brutsaert, 2003). Preclinical and clinical drug safety tests primarily focus on cardiomyocytes, ignoring the other cellular components of the myocardium. High content biology in combination with ATP content as a measure of cytotoxicity was used to assess the sensitivities of endothelial and fibroblast cells from different vascular beds to known structural cardiotoxins. This assay principle has previously been successful in the detection of structural cardiotoxicity in human embryonic stem cell derived cardiomyocytes (hESC-CM’s) (Pointon et al., 2013). The endothelial cells displayed no significant difference in their compound responses, while the fibroblasts showed variation; imatinib was more potent in the cardiac fibroblasts and lapatinib more potent in the dermal fibroblasts. When the sensitivities of the cells from the myocardium (human cardiac fibroblasts (hCFs), human cardiac microvascular endothelial cells (hCMECs) and hESC-CMs) were globally compared the non-myocytes were sensitive to structural cardiotoxicity at the acute time point of 6 hour (h), whereas the hESC-CMs display toxicity only after 72 h. The conclusions from this work are that non-myocyte cells play a role in drug-induced cardiovascular toxicity since early sensitivity is displayed, however current in vitro models lack the complexity required to investigate the translation of this in vivo. Multiple types of cardiac microtissue models were developed and their responses to reference structural cardiotoxins and inotropes evaluated using ATP content and video-based edge monitoring of contractility, respectively. Compound responses highlighted the promotion of maturity in the cardiac tri-cultured microtissue. Further investigations into gene expression and calcium (Ca2+) handling suggested SR function and Ca2+ handling maturity had been promoted and that the ca2+ handling protein S100A1 plays a critical role in this maturity. These findings show adult cardiac non-myocyte cells can be used to promote contractile maturity of hESC-CM’s in vitro when co-cultured as a three dimensional (3D) microtissue. The induction of maturity was very much multi-parameter dependent requiring a) both non-myocyte cell types, b) cells of cardiac origin and c) a 3D culture environment. More advanced human relevant in vitro models that better reconstitute the in vivo cellular physiology of the heart could allow improved in vitro to in vivo correlation in future drug safety screens.

Successful development of mammalian oocytes, eggs and embryos relies on the production of ATP by their mitochondria, through metabolism of pyruvate and fatty acids. Imaging of lipid droplets in mammalian eggs has proven difficult due to the invasive, unspecific and unquantitative nature of fluorescent lipophilic stains. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy can be used to image lipid droplets in live mouse oocytes and pre-implantation embryos, in a label-free, chemically-specific manner. CARS enables visualisation of lipid droplet distributions, and quantitation of droplet size, number and spatial distribution, notably whilst maintaining their developmental viability. CARS also allows examination of the type of lipids comprising lipid droplets, through means of hyperspectral imaging. It is shown that the chemical composition of these droplets differ in oocytes matured in media supplemented with saturated and unsaturated fatty acids. Correlation of CARS measurements with simultaneous two-photon fluorescence (TPF) microscopy of conventionally used lipid dyes demonstrates only partial correlation, and shows their lack of specificity and unpredictable staining patterns. Dynamic monitoring of lipid metabolism in eggs allows determination of the extent of fatty acid oxidation occurring in mouse oocytes and embryos. Investigation into how inhibition of fatty acid metabolism affects the mitochondrial redox state, membrane potential and ATP level allows further understanding of why fatty acid metabolism is significant for egg and pre-implantation embryo development. Starvation and fatty acid-feeding of oocytes shows that the lipid droplet distribution reflects their level of lipid metabolism, while the detrimental effects of palmitic acid are shown to involve its action at the endoplasmic reticulum SERCA pumps.

Most of the time we are not passively viewing scenes but want to extract behaviourally relevant information. In addition, objects do not often occur in isolation outside the visual scientist’s laboratory but are embedded in complex visual scenes. If the brain is to be adaptive, it needs to process visual information with regards to its context. Thus perception is not purely determined by the specific input to the retina but depends on the surrounding scene, objects, attention, memory, prior knowledge, expectations and predictions. Traditionally, the visual system in the human brain has been viewed as having a hierarchical organisation with signals travelling in one direction: input from the eyes arrives at "lower" order areas, which then transmit their computations to "higher" order areas. As one moves up the hierarchy, visual areas code more complex and more abstract information, and after the final processing stage, the system gives an output. However, in reality things are not so simple. In fact, in the primary visual cortex (V1), which is one of the first visual processing stages in the brain, external stimuli constitute less than 10% of the total input. The rest of the input originates from internal connections, either within V1 itself or via signals arriving from "higher" areas, back down to V1. In this way, "higher" areas can tell "lower" ones about the bigger picture and the neighbouring elements. This internal processing in the brain is the mechanism which provides context and enriches the information reaching us from the external world. The signals arriving to V1 from the retina are referred to as feedforward, while the signals going in the opposite direction, from higher areas back to V1, are called feedback. Each neuron responds to its preferred stimulus in a specific region of the visual field, called the receptive field. Feedforward signals act on the central region of a neuron’s receptive field, while feedback signals act on a larger surround region and thus are able to inform the centre about the surrounding context. However, it is not well established which aspects of the surrounding scene define these contextual interactions. This thesis investigated the influence of the scene surround on feedback to V1. We aimed to establish how the scene surround contributes to informative feedback signals. An introduction about what is already known regarding the function of feedback and the information it transmits is provided in Chapter 1. I give an overview of the previous studies which highlight the various contextual roles of feedback, such as perceptual grouping, contour and object completion, expectation, attention and prediction, as well as being the mechanism allowing visual imagery. Chapter 2 aimed to address whether feedback provides coarse or fine-grained information about the surrounding scene. Since during normal viewing both feedback and feedforward signals are present, we investigated feedback signals in isolation by using a partial occlusion paradigm to remove meaningful feedforward input in a specific region of the scene. We filtered the scene surrounding the occluded region into a fine-grained and a coarse version. We also varied how much information was shared between the fine-grained and coarse version of the same scene. This was done to investigate whether the information feedback carried was tightly tuned to the spatial scale of the surrounding scene, or whether the information it contained was similar across the two types of the scene surround. We found that the feedback contained signals about both coarse and fine-grained surrounds, but there was also some overlap between these feedback signals. In addition, we found that the feedback information did not correspond to a direct "filling-in" of the missing feedforward input, suggesting that feedback and feedforward signals represent the scene in different ways. In Chapter 3 we took a closer look at the amount of meaningful scene surround that is necessary to elicit informative feedback signals. The results showed that increasing the amount of scene information in the surround resulted in more meaningful feedback signals. We confirmed our earlier finding that the feedback information in the occluded region is dissimilar to the corresponding feedforward input when the feedforward region is isolated from the scene surround. Adding the scene surround to the feedforward stimulus increased this feedback/feedforward similarity. Overall, these findings point to the notion that feedback signals combine with feedforward input under normal visual processing. Isolated feedforward input in the absence of the surround provides V1 neurons with impoverished information. Neighbouring elements of the scene or its overall global structure can be sources of context. In Chapter 4 we explored which regions of the scene surround contribute the most to the contextual feedback signals arriving at V1 – is this limited to only local neighbouring regions or does the feedback directly contain information about the overall global image structure, taking into account distant retinotopic regions as well? In the first experiment, we used simple global structures made up of four Gabor elements and showed that such simplistic shapes failed to induce contextual feedback into the occluded region. However, in the presence of feedforward information, we saw that feedback from the local surround combined with identical feedforward input to give rise to different activity patterns in that feedforward region. This suggests that feedback may be recruited differentially depending on whether feedforward stimulation is present or absent. In the second experiment, we used natural scenes and tested whether contextual feedback can originate from a distant retinotopic region in the situation when the local scene surround was not informative. We manipulated scene information in a distant retinotopic region (in the opposite hemisphere) while keeping the local neighbouring surround information the same. The results showed a lack of meaningful feedback in the occluded region, and that feedback from the distant surround had a negligible effect on the identical feedforward information, in contrast to the finding obtained previously with the local surround. These findings suggest that feedback preferentially originates from nearby regions and provides context to disambiguate local feedforward elements. Therefore context about the global scene structure may arise from a series of local surround interactions. Chapter 5 summarises these findings and discusses the overarching themes regarding the content of feedback and its role in full visual processing. At the end, I propose some future research directions.

Osteoarthritis (OA) is a chronic and highly prevalent degenerative disease of the synovial joint leading to cartilage destruction and bone remodelling. The current management of end-stage OA is joint replacement, however, this procedure is not suitable for a subset of patients hence there is a growing need for alternative treatments and technologies to address this limitation. One such approach to this problem is the application of cell-based therapies that regenerate areas of damaged cartilage. Recently discovered articular cartilage progenitor cells (CPC) have been hallmarked as a potential cell source for repair and/or regeneration of damaged articular cartilage. Initial focus was on the characterisation of human CPC isolated from healthy donors and compared with OA derived CPC and patient matched OA Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Comparison of all cell types showed similar morphology and proliferative capacity. In addition, all cell types isolated showed positive expression of the putative mesenchymal stem cell makers; CD-90, CD-105 and CD-166 while lacking expression of CD-34. All cell types investigated showed successful osteogenic, chondrogenic and adipogenic differentiation, hence providing evidence of the mesenchymal stem cell properties of isolated CPC. A gene profiler array was used to identify the expression of Wnt pathway genes from RNA isolated from CPC cell lines originating from healthy and OA cartilage. Interestingly, the expression of Dkk-1 was observed to have the highest up-regulation in OA-derived CPC. The role of Dkk-1 was further studied in a number of CPC and chondrocyte cell lines from healthy and OA cartilage. It was found that normal CPC cell lines showed homogenously low expression and secretion of Dkk-1, however, OA-derived CPC cell lines exhibited a heterogeneous expression and secretion of Dkk-1. In a pellet culture model of chondrogenic differentiation, CPC cell lines secreting high levels of Dkk-1 failed to undergo chondrogenic differentiation, measured by diminished expression of chondrogenic differentiation markers, Type II collagen, ACAN and Sox-9 at both molecular and protein levels. Immunolocalisation of Dkk-1 in OA osteochondral plugs showed peri-cellular expression in chondrocytes located in all zones and around migratory endothelial cells invading articular cartilage where there was a quantifiable increase of blood vessel invasion. This later observation was further studied through a series of experiments to investigate the role of Dkk-1 in relation to endothelial cell migration and angiogenesis using an in vitro model of angiogenesis and migration/invasion assays. A novel finding emerged from these studies, which provides evidence for a pro-angiogenic and pro-migratory role of Dkk-1 and to a lesser extent Dkk-2 in human endothelial cell lines. A novel in vitro Transwell co-culture model was developed to study the interaction between chondrocytes and endothelial cells mimicking the osteochondral interface. A novel finding from these studies included the observation that normal or OA-derived chondrocytes appeared to induce an endothelial to mesenchymal transformation (EndMT) of the co-culture endothelial cells. This was assessed by a loss of the endothelial cobble stone morphology and a down-regulation of key factors implicated in endothelial cell phenotype, including VE-cadherin, Tie-2, e-NOS, PDGF-AA and PECAM-1. As endothelial cells lost their phenotype they adopted a spindle morphology and expressed mesenchymal cell markers including: Lumican, Snail, α-SMA, Vimentin and MMPs. Interestingly, this was also associated with an increase in Dkk-1 expression. To confirm a role for Dkk-1 in this process endothelial cells were cultured in the presence of Dkk-1 and were found to undergo EndMT when compared to the control. In summary, this thesis has uncovered several interesting differences in CPC phenotype. In addition, my results suggest that Dkk-1 has potential as a biomarker of OA pathology. This thesis highlights further the complex role of the Wnt Pathway and in particular Dkk-1 may play a role in the pathogenesis of osteoarthritis.

With the current crisis of organ shortage and an increasing number of dialysis patients,studies directed at ameliorating such a substantial organ discrepancy are of considerable importance to the transplant community. The use of extended criteria donation has helped to compensate the disparity of organs however, we are still a long way from achieving satisfactory targets. Still there are many organs from older donors that are discarded primarily on the basis of chronological age. It is here that biological age may display a crucial role in allowing the transplant team to characterize donor organs with greater accuracy. Indeed both biological and chronological age are very closely related and ECD criteria are based very much on the latter, albeit with other clinical variables. However the biomarker of ageing CDKN2A which is suitably represented by Baker and Sprott’s criteria, displays closer variabilities with post-operative transplant function, at least up to one year. Telomere length known as the “Gold standard” biomarker of ageing does not display as robust a role in predicting organ function as CDKN2A. The classification of organs represented in this text from category I-IV serves merely as a guide to future studies and is yet to be validated in larger clinical trials. It is however a simple and rapid assessment tool (Gingell-Littlejohn et al PLOS One 2013). Relatively advanced cellular senescence was displayed in the mutant AS/AGU rat kidney when compared to the parent AS strain. This was exploited in a unique animal model to study the effects of ischaemia reperfusion injury on the mutant kidney, hence mimicking to a certain degree the transplant related injuries in ECD kidneys. Although ischaemic times in the model were moderate in nature, there was nonetheless a difference in the tolerance to IR injury between parent and mutant strain as evidenced by increased p16 and p21 staining in AS/AGU rats. Such a model therefore is exclusive in that interventions to improve ECD renal function post-transplantation can accurately and conveniently be represented and studied at a pre-clinical level. Anti-ischaemic compounds have been the subject of much debate over the years, however a single “Holy Grail” compound able to completely abolish the injurious effects of IR injury has never been elicited. mTOR inhibitors however, display several cellular effects and act as potent immunosuppressants. They (AZ-6) have also been shown to partially mediate the detrimental effects of IR injury on the native kidneys of AS rats in a specifically designed animal model as shown. Further studies encompassing transplanted kidneys from mutant AS/AGU rats exposed to such a promising agent would be of undoubted importance to the clinical field of transplantation, potentially leading to immeasurable economic and patient benefits.

White adipose tissue is not just a storage organ. It is now recognised as an endocrine organ. It secretes many substances known as adipokines, which are thought to link obesity with type 2 diabetes (T2D). One of the most important adipokines is adiponectin. It is a peptide hormone consisting of 244 amino acids with molecular weight of 30 KD. It circulates in plasma in high concentrations (3-30 4g/ml). Adiponectin polymerises to form many bigger forms. Those are low molecular weight (LMW); middle molecular weight (MMW) and high molecular weight (HMW). The HMW adiponectin is the active form of the hormone. The concentrations of most adipokines are increased in obese people. Adiponectin is unusual in that its concentration is lower in obese people. Consequently its concentration is decreased in some related metabolic disorders. Its concentrations decrease in cardiovascular diseases, diabetes, dyslipidemia and insulin resistances. It is well known that exercise increases insulin sensitivity, also adiponectin was reported to regulate insulin. The effect of exercise on the adiponectin concentrations in plasma is controversial, but the extent to which the exercise regulates the interstitial adiponectin concentrations is not fully examined. The main site of adipokines secretion is adipose tissue. Therefore the study of these substances at the site of their production has a special interest. Recently, microdialysis techniques have been extended to become important in the measurement of substances in the extracellular fluid of many tissues such as subcutaneous adipose tissue. In particular, it has been used for measurement of adipokines. This thesis includes three studies. The first study was aimed at examining the effect of one hour of moderate exercise at 50% of maximum oxygen consumption v on adiponectin concentration in dialysate samples taken from subcutaneous abdominal adipose tissues (SCAAT). 15 healthy young female volunteers, age 22.8 ± 3.0 years (mean ± SD) participated in this experiment divided into two groups depending on their body mass index (BMI), a lean group BMI 22.2 ± 1.6 kg/m2 (mean ± SD) and an overweight group BMI 27.7 ± 1.9 kg/m2 (mean ± SD). The samples were collected using CMA 66 M. Alzwayi iii microdialysis catheters with membrane cut off 100 KD. Fitness assessment was done for all volunteers about one week before the main trials. The main trials were done on two consecutive days, a rest day and an exercise day. Each day lasted for 4 to 6 hours. On the first day the microdialysis catheter were inserted in abdominal subcutaneous tissue 4 cm lateral to the umbilicus on the left side. Dialysate samples were collected every 30 - 45 minutes. On the exercise day volunteers exercised for one hour at 50% 2 . V O max. All samples were analysed for adiponectin concentrations using Mercodia ELISA technique. The principle findings of this study were that CMA 66 microdialysis catheters worked effectively for two consecutive days for fluid recovery. Adiponectin concentrations were very low and varied, in same volunteer from time to time, and between volunteers. However, the statistical analysis showed no significant difference in adiponectin concentrations between lean and overweight groups. Adiponectin concentrations in the first two samples on the first day of the insertion were significantly higher than the first two samples on the second day of the insertion. Finally, adiponectin concentrations in dialysate samples recovered by 100 KD microdialysis catheters were very low. Therefore, the effect of the exercise was not clear. The second study aimed to compare the adiponectin concentrations in plasma and dialysate samples. Six healthy male volunteers age 32.8 ± 13.1 years and BMI 25.9 ± 3.3 kg/m2 (mean ± SD), were recruited for this study. The experiment was run for two consecutive days using the same microdialysis catheters CMA 66. Dialysate samples were collected as before. 2 ml of blood samples were collected using a cannula inserted into the anticubital vein. Samples were taken every hour for a period of five hours each day. The plasma and dialysate samples were analysed for adiponectin using the Mercodia kits. Adiponectin concentrations in plasma samples were 256 and 1791 times higher than the adiponectin concentrations in dialysate samples. The conclusion of the two studies was that the CMA 66 microdialysis catheter with cut off 100 KD membranes only recovers a small part of the total adiponectin present. M. Alzwayi iv Therefore a third study was designed to use plasma samples. The aim of the study was to investigate the effect of acute exercise at 50% 2 . V O max on HMW adiponectin, total adiponectin, interleukin (IL)-6, tumour necrosis factor alpha (TNF-α), insulin and glucose concentrations directly after the exercise, one hour after and 48 hours. 13 young healthy female volunteers age 24.3 ± 2.7 years and BMI 21.9 ± 2.2 kg/m2 (mean ± SD) contributed in this study. The volunteers were invited for five visits. Their fitness was measured on the first visit. Then they came for two main trials rest day and exercise day, which they were randomly assigned. The main trails lasted for two hours. Three blood samples were collected each day using same cannulated system in the second study. The volunteers followed 48 hours after each trial, one blood sample were collected each day. The 8 plasma samples were analysed for: total adiponectin and insulin concentrations via Mercodia ELISA kits, HMW adiponectin, IL-6 and TNF-α concentration via R&D systems and glucose concentration using the glucose oxidase colorimetric method. The results showed no statistical difference in total or HMW adiponectin, TNF-α and glucose concentrations under the effect of moderate exercise at 50% 2 . V O max either directly or 48 (p value > 0.05). IL-6 concentrations increased about two fold one hour after the exercise above the resting level (P value < 0.05). IL-6 concentrations return to the basal level 48 hour latter. Insulin concentrations show a decrease one hour after the exercise finished. The number of volunteers was small and the change was close to significance. A one way ANOVA returned a P value of < 0.05, but a two way ANOVA with repeated measures returned a P value of > 0.05. In conclusion, the acute exercise at 50% 2 . V O max changes IL-6 concentrations but it has no effect on adiponectin concentrations in dialysate or plasma samples. Low adiponectin concentration is related to obesity, insulin resistance and T2D. Therefore, increase in adiponectin concentration probably lies in weight loss and the exercise may play role, even if it has little direct action on adiponectin concentration.

A typical absence is a non-convulsive epileptic seizure that is a sole symptom of childhood absence epilepsy (CAE). It is characterised by a generalised hyper-synchronous activity (2.5-5 Hz) of neurons in the thalamocortical network that manifests as a spike and slow-wave discharge (SWD) in the electroencephalogram. Although CAE is not a benign form of epilepsy, its physiological basis is not well understood. In an attempt to make progress regarding the mechanism of SWDs, I built a large-scale computational model of the thalamocortical network that replicated key cellular and network electric oscillatory behaviours. Model simulation indicated that there are multiple pathological pathways leading to SWDs. They fell into three categories depending on their network-level effects. Moreover, all SWDs had the same physiological mechanism of generation irrespective of their underlying pathology. They were initiated by an increase in NRT cell bursting prior to the SWD onset. SWDs critically depended on the T-type Ca2+ current (IT) mediated firing in NRT and higher-order thalamocortical relay cells (TCHO), as well as GABAB synaptic receptor-mediated IPSPs in TCHO cells. On the other hand, first-order thalamocortical cells were inhibited during SWDs and did not actively participate in their generation. These cells, however, could promote or disrupt SWD generation if they were hyperpolarised or depolarised, respectively. Importantly, only a minority of active TC cells with a small proportion of them bursting were necessary to ensure the SWD generation. In terms of their relationship to other brain rhythms, simulated SWDs were a product of NRT sleep spindle (6.5-14 Hz) and cortical δ (1-4 Hz) pacemakers and had their oscillation frequency settle between the preferred oscillation frequencies of the two pacemakers with the actual value depending on the cortical bursting intensity. These modelling results are discussed in terms of their implications for understanding CAE and its future research and treatment.

Introduction: Over the past decades, several labelling techniques have been used in an attempt to track stem cells using magnetic resonance imaging (MRI). However, very few of these were able to definitely determine the precise location of stem cells within a living organism and monitor throughout a long term period, without loss or diffusion of the signal. A novel MRI cell tracking method described in 2005 proposed that reporter genes that could effectively increase the iron content of a target cell would allow a stronger contrast when imaged via MR. Being a fundamental part of the iron metabolism, transferrin receptor 1 (TfR 1) and ferritin heavy chain 1 (Fth 1) were naturally suggested to have the potential to increase the iron load of cells when overexpressed. More recently, there has been some interest in the reporter gene MagA, which is a known iron transporter found in magnetotactic bacteria. Aim: To evaluate the suitability of using TfR 1, Fth 1 and MagA as potential magnetic reporter genes for MRI-based cell tracking. Methods: Several cell and stem cell lines were transduced with a 2nd generation HIV-based lentiviral system containing one or more magnetic reporters. Viral transduction resulted in genome incorporation of bicistronic construct(s) with TfR 1 gene alongside a gene encoding a green fluorescent reporter (GFP) and/or Fth 1 and MagA gene alongside a red fluorescent reporter (RFP). This allowed for identification and monitoring of positive cells with complementing imaging modalities: MRI and fluorescence based methods. Transgenes were evaluated for integration stability over passages and their influence on iron homeostasis was assessed; also, integration and/or overexpression were confirmed at the mRNA and protein level. Finally, the influence of magnetic reporters on intracellular iron retention and MRI contrast capacity was tested both in vitro and in a model organism, the chick embryo. Results: After analysing all three potential magnetic reporters, TfR 1 was found to be the most promising, as its overexpression induced an adjustment of iron homeostasis in Chinese hamster ovary K1 cells, leading to higher intracellular iron accumulation relative to controls. The same adjustment was found in mouse mesenchymal stem cells (mMSC), but only when TfR 1 was overexpressed in conjunction with Fth 1, also leading to an increase in iron retention capacity. However, a limitation was found when overexpressing Fth 1 in mMSC, as permanent iron supplementation was needed in order to keep these cells viable. In contrast with previous studies, MagA gene integration posed some restrictions in certain cell lines studied. The results presented here show that while some cell types are able to stably maintain MagA expression over several passages, others fail to survive and die shortly after transduction, suggesting that a potential toxic effect may be originating from MagA gene integration. From the surviving cells, two were compared side by side and contradictory results were obtained, demonstrating that MagA would only be a suitable magnetic reporter for some cell types. Conclusion: The results obtained with this project are of relevance for reporter gene-based MRI cell tracking as they show that no single magnetic reporter is capable of generating detectable MRI contrast for a global cohort of cell lines. On the contrary, overexpression of endogenous genes or integration of foreign genes should be performed with caution and analysed on a case by case basis. Finally, for some cell type and magnetic reporter gene combinations, this study suggests that MRI could be a promising method for the longitudinal monitoring of engrafted cells, especially when the cells have been cultured in media supplemented with low concentrations of iron.