Vascular, especially cerebrovascular, dysfunction may be a critical factor in ageing and dementia. Cerebrovascular impairment due to risk factors such as ageing, stroke, smoking, diabetes and cerebral hypoperfusion has a deterious impact on the normal supply of basic nutrients such as oxygen and glucose to the brain; their absence leads inevitably to neuronal death. The cerebral white matter lesions found in most forms of dementia are reportedly the result of chronic cerebral hypoperfusion. However the temporal and spatial evolution of damage remains unclear. Furthermore, any decrease in the integrity of the blood-brain barrier (BBB) has been hypothesised to be a precocious attack on white matter. The “milieu interieure” the most protected in the body, namely the extracellular fluid of the brain, is no longer maintained homeostatically. The cumulation of these various pathophysiological processes alters cerebral function and it has been postulated that, in the most extreme instances, the outcome of this cascade of nefarious events leads to dementia. This thesis examines the supposition that chronic cerebral hypoperfusion could be responsible for the time-related development of white and grey matter pathology and investigates the relationships between the disturbances in the integrity of the BBB and white matter pathology. Three studies addressed these aims. In the first, chronic cerebral hypoperfusion, induced in male Wistar rats by bilateral common carotid artery occlusion (BCCAo), was chosen as the model to study changes in axons, myelin, perikarya as well as microglial activation. The groups of rats that underwent BCCAo were examined at three hours as well as three, seven, 14 and 28 days after the induction of chronic cerebral hypoperfusion. The microscopic examination revealed that, after three hours post BCCAo, damage was detected only in axons and myelin. In contrast, no visible pathology to the neuronal perikarya or enhancement of activated microglia (compared to the sham group) was observable. Injury in both white and grey matter and enhancement of activated microglia was observed from three days post BCCAo and increased with time post BCCAo. The most severe damage to the white and grey matter and enhancement of microglial activation was detected at seven days post BCCAo. These results would indicate that white matter damage precedes grey matter pathology and the enhancement of activated microglia. In the second study, the integrity of the BBB at three hours (when only white matter pathology was found according to the results of the first study) and seven days post BCCAo (when more severe damage to the white and grey matter was shown) was assessed by the use of MRI on T1-weighted image acquisitions with gadolinium as a tracer for BBB permeability. White matter integrity was measured by MTR maps from MTI acquisitions in four brain structures (corpus callosum, caudatoputamen, the external and internal capsules). No differences in white matter integrity were detected between the BCCAo and sham group at three hours and seven days. No differences in signal enhancement of gadolinium were detected three hours post BCCAo. However, a significant signal enhancement of gadolinium was detected at seven days post BCCAo in the caudatoputamen and in the external capsule. Furthermore, immunohistochemistry revealed a significant enhancement of activated microglia seven days post BCCAo compared to the sham group. This functional and immunohistochemical finding, when taken together, might indicate that chronic cerebral hypoperfusion is not in itself responsible for BBB permeability. Rather, the damage to the white matter caused by cerebral hypoperfusion may be responsible for the dysfunction of the BBB over time. Another point of interest was the evidence that the enhancement of activated microglia may play a critical role in the increased permeability of the BBB. The final study in this thesis aimed to investigate the possible pathway and proteins potentially implicated in white matter damage and BBB permeability. To address this question, protein levels and the expression of genes involved in the apoptotic and nonapoptotic hypoxic pathways were compared to the sham groups (at three hours and seven days after BCCAo), in three brain structures (cortex, corpus callosum and caudatoputamen). The levels of HIF-1α, MMP-2, Caspase-3 and VEGF were unchanged compared to the sham group after BCCAo. However, VEGF mRNA expression was found to be significantly different to the sham group seven days post BCCAo in all the three structures examined. An overexpression of HIF-1α and a significant level of Caspase-3 would indicate the activation of the apoptotic pathway. However, neither of these criteria were met and these negative results suggest that the apoptotic pathway is not implicated in the mechanisms that lead to white matter pathology after cerebral hypoperfusion. Finally, the significant expression of VEGF mRNA, compared to the sham group seven days post BCCAo, may contribute to the time-relate increased permeability of the BBB. The results presented within this thesis provide a body of evidence to support the hypothesis that chronic cerebral hypoperfusion is - at least – causal to the damage to different components of the white matter which precedes either early ischaemic changes to the perikarya or enhancement of activated microglia following BCCAo. The increased permeability of the BBB, which can be related to the significant over-expression of VEGF mRNA (compared to the sham group seven days post BCCAo), does not appear to be primarily responsible for white matter pathology, because the MRI investigations indicated that BBB integrity was not affected after three hours of BCCAo. The increased permeability of the BBB, observed seven days post BCCAo with MRI, seems to be the consequence of increased brain damage; thereafter, there is a time-dependent relationship between increasing BBB permeability and increasing brain pathology. Overall, the studies reported herein, strengthen the initial working hypothesis. The conclusion – and direction for future studies – would be that minimising white matter pathology and protecting components of the BBB represent potential targets to decrease then incidence of neuropsychological function or to obtund the cerebral dysfunction in patients who suffer from chronic cerebral hypoperfusion.

Lacunar stroke accounts for one quarter of all ischaemic stroke and in the long term carries a greater risk of death and disability than was previously realised. Much of our current knowledge originated from neuropathological studies in the 1950s and 1960s. In the last thirty years, brain computed tomography (CT) and magnetic resonance imaging (MRI) have revolutionised our understanding of lacunar stroke and associated features of cerebral small vessel disease (SVD), namely white matter lesions (WML), enlarged perivascular spaces (EPVS) and brain microbleeds (BMB). The purpose of the projects which led to the writing of this thesis was to improve understanding of imaging characteristics of cerebral SVD. We aimed to assess (i) clinical and imaging features which might explain misclassification of lacunar infarcts as cortical infarcts and vice versa, (ii) the proportion of symptomatic lacunar infarcts progressing to lacunar cavities and associations of cavitation, (iii) completeness of reporting of lacunar lesions in the lacunar stroke literature, (iv) definitions and detection of lacunar lesions amongst SVD researchers, (v) the relationship between WML and carotid stenosis, (vi) clinical and imaging associations of EPVS and, (vii) observer variability in the assessment of EPVS and BMB, in order to develop visual rating scales. Section one describes neuroimaging of lacunar stroke. To investigate features which might explain clinical stroke subtype misclassification (‘clinical-imaging dissociation’), I used data from a stroke study. The main factor associated with clinical-imaging dissociation was diabetes, and in patients with acute lacunar infarction, proximity of the lacunar infarct to the cortex, age, diabetes and left hemisphere location. To investigate the proportion of symptomatic lacunar infarcts progressing to cavities, I used data from two stroke studies. A fifth of patients with acute lacunar ischaemic stroke showed definite cavitation on follow-up imaging at a median of 227 days; cavitation was associated with increasing time to follow-up. To assess completeness of reporting of lacunar lesions in the lacunar stroke literature, I reviewed 50 articles from three journals with a stroke focus. There was marked variation in terminology and descriptions of imaging definitions of lacunar lesions. To assess lacunar lesion definitions and detection amongst SVD researchers, I used an online survey consisting of case-based and non-case-based questions. There was marked variation in definitions and descriptions. Cavitated lesions were detected with the highest degree of confidence. Section two describes neuroimaging of associated features of cerebral SVD. Using data from two stroke studies, I examined the relationship between WML and ipsilateral carotid artery stenosis. There was no association between carotid stenosis and WML. I tested the association of EPVS with WML and lacunar stroke subtype using data from a stroke study. Total EPVS were associated with age and deep WML; basal ganglia (BG) EPVS were associated with age, centrum semiovale (CS) EPVS, cerebral atrophy and lacunar stroke subtype. Quantification of observer variability in EPVS rating was assessed on 60 MRI scans selected from a stroke study and an ageing cohort. Intrarater agreement was good and interrater agreement was moderate. Main reasons for interrater disagreement included the visualisation of very small EPVS and the presence of concomitant WML and lacunar lesions. Observer variability in BMB rating was quantified using MRI scans from a stroke study. Interrater agreement was moderate but improved following modification of the pilot rating scale (BOMBS; Brain Observer MicroBleed Scale), which had its main effect by differentiating ‘certain’ BMB from ‘uncertain’ BMB and BMB ‘mimics’. In conclusion, neuroimaging, particularly MRI, is a valuable tool for the investigation of lacunar stroke and associated features of cerebral SVD. With recent technological advances in both CT and MRI, neuroimaging will remain central to future SVD studies, hopefully leading to a much improved understanding of this important disease.

Lacunar strokes account for approximately a quarter of all ischaemic strokes and traditionally are thought to result from occlusion of a small deep perforating arteriole in the brain. Lacunar infarcts can be up to 2cm in diameter and are found in deep brain structures such as the thalamus and internal capsule. Despite their prevalence and specific accompanying clinical syndromes, the cause of lacunar stroke and its associated vascular pathology remain unclear. Many hypotheses as to the cause exist, which fall broadly into two categories; firstly, a direct occlusion via emboli or thrombus usually from a cardiac or large artery source, microatheroma (intrinsic lenticulostriate occlusion) or macroatheroma (parent artery occlusion) all operating primarily via ischaemia. Secondly, there could be an indirect occlusion resulting from vasospasm, endothelial dysfunction or other forms of endovascular damage (e.g. inflammation). Therefore the question of whether the resulting lesions are truly “ischaemic” or actually arise secondary to an alternative process is still under debate. To clarify the chain of pathological events ultimately resulting in lacunar stroke, in this thesis I firstly undertook a systematic assessment of human lacunar stroke pathology literature to determine the information currently available and the quality of these studies (including terminology). The majority of these studies were performed in patients who had died long after their stroke making it difficult to determine the early changes, and there were few patients with a clinically verified lacunar syndrome. Therefore I adopted alternative approaches. In this thesis, I systematically looked for all potential experimental models of lacunar stroke and identified what appears at present to be the most pertinent - the spontaneous pathology of the stroke-prone spontaneously hypertensive rat (SHRSP). However, the cerebral pathology described in this model to date is biased towards end stage pathology, with little information concerning the microvasculature (as opposed to the brain parenchyma) and confounding by use of salt to exacerbate pathology. Therefore, the aim of the experimental work in this thesis was to assess pathological changes within the cerebral vasculature and brain parenchyma of the SHRSP across a variety of ages (particularly young pre-hypertensive animals) and to look at the effects of salt loading on both the SHRSP and its parent strain (the Wistar Kyoto rat - WKY). Three related studies (qualitative and quantitative histology, immunohistochemistry and a microarray study of gene expression confirmed by quantitative PCR), revealed that the presence of inflammation (via significant changes in gene expression in the acute phase response pathway and increased immunostaining of activated microglia and astrocytes) plus alterations in vascular tone regulation, (via genetic alteration of the nitric oxide signaling pathway probably secondary to abnormal oxidative state), impaired structural integrity of the blood brain barrier (histological evidence of endothelial dysfunction and significantly decreased Claudin-5 staining) and reduced plasma oncotic potential (reduced albumin gene expression) are all present in the native SHRSP at 5 weeks of age, i.e. well before the onset of hypertension and without exposure to high levels of salt. We also confirmed previous findings of vessel remodelling at older ages likely as a secondary response to hypertension (thickened arteriolar smooth muscle, increased smooth muscle actin immunostaining). Furthermore, we found not only that salt exacerbated the changes see in the SHRSP at 21 weeks, but also that the control animals (WKY) exposed to a high salt intake developed features of cerebral microvascular pathology independently of hypertension (e.g. white matter vacuolation and significant changes in myelin basic protein expression). In conclusion, via the assessment of the most pertinent experimental model of lacunar stroke currently available, this thesis has provided two very important pieces of evidence: firstly that cerebral small vessel disease is primarily caused by a non-ischaemic mechanism and that any thrombotic vessel lesions occur as secondary end stage pathology; secondly that these features are not simply the consequence of exposure to raised blood pressure but occur secondary to abnormal endothelial integrity, inflammation, abnormal oxidative pathways influencing regulation of vascular tone and low plasma oncotic pressure. Patients with an innate susceptibility to increased blood brain barrier permeability and/or chronic inflammation could therefore have a higher risk of developing small vessel disease pathology and ultimately lacunar stroke and other features of small vessel disease. Research, addressing whether lacunar stroke patients should be treated differently to those with atherothromboembolic stroke is urgently needed.

Dricksvattnet ombord på Sjöfartshögskolans fartyg Calmare Nyckel har vid tidigare provtagning visat på undermålig kvalitet. Orsaken till detta har inte undersökts innan. I detta arbete undersöks och identifieras orsaken och ett underlag för förbättring av vattenkvaliteten har tagits fram. Underlaget omfattas också av en arbetsbeskrivning för ombyggnation av färskvattensystemet och ett underlag för installation av en färskvattenproducerande anläggning. För att kontrollera vattenkvaliteten ombord genomfördes vattenprover och en undersökning av färskvattensystemet med endoskop. Resultaten analyserades sedan och ett åtgärdsprogram för förbättring av vattenkvaliteten har rekommenderats. Det nuvarande färskvattensystemet undersöktes och outnyttjade anläggningsdelar identifierades. En arbetsbeskrivning för demontering av ovan nämnda anläggningsdelar togs fram. Sjöfartshögskolan hade förvärvat en begagnad färskvattenanläggning vilken undersöktes för att avgöra om den gick att använda ombord på Calmare Nyckel. Undersökningen visade att anläggningen inte gick att använda och arbetet fortsatte med att utveckla en offert på en färskvattenproducerande anläggning som Sjöfartshögskolan beställde 2009. Ett underlag för installation av den färskvattenproducerande anläggningen togs fram och innehåller montageritningar, arbetsbeskrivningar, materialbehov, plan för monteringsarbetet och resursbehov. Metoden och åtgärdsprogrammet kan i framtida undersökningar fungera som hjälpmedel vid behov av att förbättra vattenkvaliteten ombord på liknande fartyg. / Drinking water onboard Kalmar Maritime Academy’s ship Calmare Nyckel has on previous sampling demonstrated insufficient quality. The reason for this has not been investigated before. This study examines and identifies the cause for the substandard water quality and support material for improvement has been developed. The material also contains a job description for rebuilding of the fresh water system and documents regarding installation of a freshwater generator. Water samples and examination of the fresh water system with endoscope was performed to verify the water quality onboard the ship. The results were analysed and a plan for improvement of water quality was recommended. The current fresh water system was investigated and unused components were identified. A description for removing the mentioned components was made. Kalmar Maritime Academy had acquired a used freshwater generator which was examined to determine whether it could be used onboard Calmare Nyckel. The examination revealed that it could not be used and the study proceeded to develop a quotation on a freshwater generator that Kalmar Maritime Academy had ordered in 2009. Material for the installation of the freshwater generator was created and includes assembly drawings, job descriptions, material requirements, plan for the assembly work and resource needs. The method and plan may in future investigations be used as aids to the improvement of water quality onboard similar vessels.

The Paris-Edinburgh cell (PEC) is a widely used opposed-anvil device for neutron scattering. Since its development, it has been used to study a number of samples loaded as solids or liquids. However, studying gases at room temperature has not yet been possible. Up until now only a few gases could be loaded as liquids, in cryogenic conditions. Thus, it was impossible to study many gases and gas mixtures and also it was difficult to use gases as pressure-transmitting media (PTM). In order to overcome these limitations, a technique that would enable loading of gases into the PEC was required. The work described in this thesis was focused on the design and use of a gas-loading system for the PEC. The challenge of designing such a system comes from the fact that the gases need to be loaded into the gasket at sufficient density in order to achieve any significant pressure during further compression in the cell. This can be achieved by using a separate pressure vessel. Because the whole PEC is too large to be placed inside the vessel, a technique of loading gas into the anvils separated from the rest of the cell had to be devised. Designing the holder for the anvils, which would make this possible, presented a major challenge as it should allow the anvils to be transferred between the vessel and the PEC, with the gasket filled with high-pressure gas. Then it needs to allow further compression of the gasket inside the PEC. The developed system consists of a pressure vessel and a locking clamp for the anvils. The pressure vessel is a closed-end thick-walled cylinder with a top cover which has an opening for a piston. The vessel is placed on the table of a hydraulic press and the piston, sealed by a high-pressure reciprocating seal, is used to transmit the force from hydraulic ram onto the anvils which are held by the clamp and placed inside the vessel. One of the anvils is fixed to the clamp and the other one is supported by spring-loaded latches - the latches engage when the anvils are pushed towards each other. Thus, when the force is applied onto the anvils to compress the gasket, latches lock the anvils in their positions stopping them from retracting and maintaining the gasket compressed after the force is released. The clamp allows the gasket to be filled with the gas and then deformed to seal the compressed gas. The locking mechanism keeps the gasket compressed, which enables the clamp to be transferred from the vessel to the PEC. After the system was built and tested, it was installed at ISIS neutron source (Oxfordshire, UK), where it has been used in several experiments. The first experiment prepared with the gas-loading system was a neutron diffraction study of nitrogen at high pressure. Nitrogen was chosen as a sample material because its high-pressure structural phase diagram is well established. Nitrogen was loaded into the gasket using the gas loader and then it was compressed in increments to 6 GPa in the PEC. β and δ phases of solid nitrogen were clearly seen in the collected neutron diffraction data. The experiment proved the usability of the gas-loading system and verified its expected performance. The second experiment utilizing the gas-loading system was to study singlecrystal and powder samples of sodium chloride (NaCl) and squaric acid (H2C4O4). For these studies argon was used as a PTM, replacing the conventionally used methanol-ethanol mixture (ME). Up until this experiment the highest pressure reported in single-crystal neutron-scattering experiments was 12 GPa. This limit was set by the solidi cation pressure of ME. With argon as the PTM, the samples were compressed to 15 GPa without any damage to the crystals. Another advantage of replacing ME with argon is improved hydrostaticity. The highest pressure that ME remain hydrostatic to is 11 GPa. Compressing beyond this point causes sheer stress acting on the sample which affects the quality of the neutron scattering data manifested in the appearance of peak-broadening in the diffraction patterns. With use of argon, the powder samples have been compressed to 18 GPa while maintaining quasi-hydrostatic pressure conditions, resulting in clean and sharp diffraction patterns without any noticeable peak-broadening.

The tidal James River, a focus of VCU's Atlantic Sturgeon program, supports both commercial shipping and hydraulic dredging. These anthropogenic threats present documented but preventable sources of mortality to the endangered species. Using three separate VEMCO Positioning System (VPS) receiver arrays, spatial data of previously-tagged fish were collected. ArcGIS and Programita software were used to analyze fish spatial distributions in the presence and absence of potential threats, using additional data including automatic identification system (AIS) vessel locations, vessel passages compiled using camera footage, and dredge records provided by the US Army Corps of Engineers. The data showed a change in distribution associated with vessels that varied according to river width but not vessel type. Dredging was associated with differences in spatial distribution, but more clearly for adults than sub-adults. The responses of Atlantic Sturgeon provide information necessary to propose potential threat mitigations, including seasonal restrictions for both vessels and dredging.

<p>Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.</p><p>A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.</p><p>Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.</p><p>The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.</p> / Dissertation

This thesis is a personal journey examining the applications of Ritual Poetic Drama Within the African Continuum as developed by my mentor Dr. Tawnya Pettiford-Wates, to the profession of the dramatic artist whether they are actor, director, educator or producer, to the training of the dramatic artist as a means of empowering generative, self defined, self validating artists, and as a means of developing community specific dramas in the hopes of facilitating individual and personal revelation. Using the author’s personal experiences as evidence, it intends to affirm Ritual Poetic Drama Within the African Continuum as an emergent methodology for theatrical practitioners.

Knowledge of the microcirculatory system has added significant value to the analysis of tissue oxygenation and perfusion. While developments in videomicroscopy technology have enabled medical researchers and physicians to observe the microvascular system, the available software tools are limited in their capabilities to determine quantitative features of microcirculation, either automatically or accurately. In particular, microvessel density has been a critical diagnostic measure in evaluating disease progression and a prognostic indicator in various clinical conditions. As a result, automated analysis of the microcirculatory system can be substantially beneficial in various real-time and off-line therapeutic medical applications, such as optimization of resuscitation. This study focuses on the development of an algorithm to automatically segment microvessels, calculate the density of capillaries in microcirculatory videos, and determine the distribution of blood circulation. The proposed technique is divided into four major steps: video stabilization, video enhancement, segmentation and post-processing. The stabilization step estimates motion and corrects for the motion artifacts using an appropriate motion model. Video enhancement improves the visual quality of video frames through preprocessing, vessel enhancement and edge enhancement. The resulting frames are combined through an adjusted weighted median filter and the resulting frame is then thresholded using an entropic thresholding technique. Finally, a region growing technique is utilized to correct for the discontinuity of blood vessels. Using the final binary results, the most commonly used measure for the assessment of microcirculation, i.e. Functional Capillary Density (FCD), is calculated. The designed technique is applied to video recordings of healthy and diseased human and animal samples obtained by MicroScan device based on Sidestream Dark Field (SDF) imaging modality. To validate the final results, the calculated FCD results are compared with the results obtained by blind detailed inspection of three medical experts, who have used AVA (Automated Vascular Analysis) semi-automated microcirculation analysis software. Since there is neither a fully automated accurate microcirculation analysis program, nor a publicly available annotated database of microcirculation videos, the results acquired by the experts are considered the gold standard. Bland-Altman plots show that there is ``Good Agreement" between the results of the algorithm and that of gold standard. In summary, the main objective of this study is to eliminate the need for human interaction to edit/ correct results, to improve the accuracy of stabilization and segmentation, and to reduce the overall computation time. The proposed methodology impacts the field of computer science through development of image processing techniques to discover the knowledge in grayscale video frames. The broad impact of this work is to assist physicians, medical researchers and caregivers in making diagnostic and therapeutic decisions for microcirculatory abnormalities and in studying of the human microcirculation.

The function of a ceramic vessel is often evaluated in relation to its form. Vessels with complex forms can be challenging to analyze from this perspective and require a different approach. One such example is an overlooked yet long-lived specialized vessel type in the form of conjoined lúcuma fruits found throughout the ancient Andes. The main object of this study is a Nasca version of this vessel type in the Virginia Museum of Fine Art. This study explores the relationship between form and iconography, rather than function. It examines how Nasca potters adopted the conjoined lúcuma form vessel and adapted it to into their fertility iconography.