This is a thesis about how mollification can be used as a regularization method to reduce noise in ill-posed problems in order to make them well-posed. Ill-posed problems are problems where noise get magnified during the solution process. An example of this is how measurement errors increases with differentiation. To correct this we use mollification. Mollification is a regularization method that uses integration or weighted average to even out a noisy function. The different types of error that occurs when mollifying are the truncation error and the propagated data error. We are going to calculate these errors and see what affects them. An other thing worth investigating is the ability to differentiate a mollified function even if the function itself can not be differentiated. An application to mollification is a blood vessel problem in biometrics where the goal is to calculate the elasticity of the blood vessel’s wall. To do this measurements from the blood and the blood vessel are required, as well as equations for the calculations. The model used for the calculations is ill-posed with regard to specific variables which is why we want to apply mollification. Here we are also going to take a look at how the noise level affects the final result as well as the mollification radius.

Microglia, the resident macrophages of the central nervous system (CNS), are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about how microglia interact with the CNS vasculature, particularly under healthy steady-state conditions. Here, I provide the first extensive characterization of juxtavascular microglia in the healthy, postnatal brain and identify a molecular mechanism regulating the timing of these interactions during development. Using the mouse cerebral cortex, I show that microglia are intimately associated with the vasculature in the CNS, directly contacting the basal lamina in vascular sites that are devoid of astrocyte endfeet. I demonstrate a high percentage of microglia are associated with the vasculature during the first week of postnatal development, which is concomitant with a peak in microglial colonization of the cortex and recruitment to synapses. I find that as microglia colonize the cortex, juxtavascular microglia are highly motile along vessels and become largely stationary as the brain matures. 2-photon live imaging in adult mice reveals that these vascular-associated microglia in the mature brain are stable and stationary for several weeks. Further, a decrease in microglia motility along the vasculature is tightly correlated with the expansion of astrocyte endfeet along the vasculature. Finally, I provide evidence that the timing of these microglia-vascular interactions during development is regulated by the microglial fractalkine receptor (CX3CR1). Together, these data support a model by which microglia use the vasculature as a scaffold to migrate and colonize the developing brain and the timing of these associations is modulated by CX3CR1. This migration along the vasculature becomes restricted as astrocyte vascular endfoot territory expands and, upon maturation, vascular-associated microglia become largely stationary.

“Blood vessel mimics” (BVMs) are tissue-engineered constructs that serve as in vitro preclinical testing models for intravascular devices. The Cal Poly Tissue Engineering lab specifically uses BVMs to test the cellular response to stent implantation. PLGA scaffolds are electrospun in-house using the current “Standard Protocol” and used as the framework for these constructs. The performance of BVMs greatly depends on material and mechanical properties of the scaffolds. It is desirable to create BVMs with reproducible properties so that they can be consistent models that ultimately generate more reliable results for intravascular device testing. Reproducibility stems from the consistency of the scaffolds. Thus, scaffolds with consistent material and mechanical properties are necessary for creating reproducible BVMs. The aim of this thesis was to characterize the reproducibility of the electrospun PLGA scaffolds using fiber diameter measurements and compliance testing. Initial work in this investigation involved designing and testing several experimental electrospinning protocols to obtain smaller fiber diameters, which have been shown to elicit more ideal cellular responses. The most successful protocol in that regard was then analyzed for the reproducibility of fiber diameters and compared to the reproducibility of the Standard Protocol. After determining that the Standard Protocol produced scaffolds with more consistent fibers, a large-scale reproducibility study was performed using this protocol. In this expanded study, both fiber diameter and compliance were analyzed and used to characterize the scaffolds. It was established that the scaffolds demonstrated inconsistent mean fiber diameter and mean compliance. The current standard electrospinning protocol therefore does not create PLGA scaffolds with statistically reproducible properties. Future modifications should be made to the electrospinning parameters in order to reduce variability between the scaffolds and future studies should be performed to determine the acceptable range of properties.

Introduction: Vascular injury is the crucial initiating event in atherosclerosis and is universal following percutaneous coronary intervention. The cellular response to this injury largely determines vessel outcome. Endothelial progenitor cells (EPCs) and their progeny, late outgrowth endothelial cells (EOCs) are thought to play an important role in this process and characterising this role would be valuable in better understanding vascular injury and repair. Methods: The radial artery in the context of transradial cardiac catheterisation was examined as a model of vascular injury with characterisation of structural injury, longitudinal function and EPC populations. To examine the role of late outgrowth endothelial cells a method for GMP-compliant cell culture and labelling with F18Fluorodeoxyglucose was developed with a view to conducting a cell-tracking study of human administration. Results: Radial artery function was reduced following transradial cardiac catheterisation with recovery over a period of three months. There was no correlation between recovery of arterial function and EPC populations as defined by conventional surface markers. A research grade protocol for EOC culture was successfully translated to a GMP-compliant process producing a viable, phenotypically homogeneous EOC product. Cells were successfully labelled with F18Fluorodeoxyglucose and whilst proliferation was reduced, acute viability and function were not compromised. Conclusion: The radial artery in the context of transradial cardiac catheterisation is a useful model of vascular injury and repair although recovery of vascular function does not appear to be influenced by EPC populations. GMP-compliant culture and labelling of EOCs is feasible and will allow examination of the physiology of these cells in vivo in man.

The development of small diameter tissue engineered blood vessels (TEBVs) with low thrombogenicity, low immunogenicity, suitable mechanical properties, and a capacity to remodel to their environment could significantly advance the treatment of coronary and peripheral artery disease. Despite significant advances in the field of tissue engineering, autologous vessels are still primarily utilized as grafts during bypass surgeries. However, undamaged autologous tissue may not always be available due to disease or prior surgery. TEBVs lack long-term efficacy due to a variety of types of failures including aneurysmal dilations, thrombosis, and rupture; the mechanisms of these failures are not well understood. In vitro mechanical testing may help the understanding of these failure mechanisms. The typical mechanical tests lack standardized methodologies; thus, results vary widely. The overall goal of this study is to develop novel experimental and mathematical models to study the mechanical properties and failure mechanisms of TEBVs. Our results suggest that burst pressure tests, the current standard, are not sufficient to assess a TEBVs’ suitability as a coronary substitute; creep and/or cyclic loading tests are also required. Results from this model can help identify the most insightful experiments and quantities to be measured – ultimately reducing the overall number of experimental iterations. Improving the testing and characterization of TEBVs is critically important in decreasing the time necessary to validate the mechanical and functional responses of TEBVs over time, thus quickly moving TEBVs from the benchtop to the patient.

Compliance mismatch is a significant hurdle to long-term patency in small-diameter arterial bypass grafts. Vascular tissue engineering has the potential to produce compliant, non-thrombogenic small-diameter grafts. However, current engineered grafts are relatively non-compliant, resulting in intimal hyperplasia and graft occlusion when subjected to arterial pressures. This research investigates the mechanical and biological properties of engineered constructs based on a biodegradable synthetic elastomer, poly(glycerol sebacate) (PGS). Several methods for fabricating porous PGS scaffolds in a tubular geometry were developed and compared. Adult baboon vascular cells were cultured in the scaffolds under various in vitro experimental conditions, including variations in initial cell seeding density, the type of scaffold used for culture, culture time, scaffold material, and hydrostatic pressure, and properties of the resultant constructs were compared. Scaffold fabrication using heat-shrinkable mandrels and glass tubes coated with hyaluronic acid significantly decreased tolerances of wall thickness and mechanical properties, improved handling, and decreased culture time required to reach luminal cellular confluence compared to scaffolds made with other fabrication techniques. Altering scaffold material from PGS to poly(lactide-co-glycolide) (PLGA), a benchmark biomaterial, did not affect scaffold yield, porosity, or luminal cellular confluence. Extracellular matrix (ECM) deposition increased with SMC-only culture time, and ECM deposition and remodeling during culture influenced construct compliance. Compared to PLGA scaffolds, PGS scaffolds promoted elastin crosslinking by SMCs and elastic tissue properties but attenuated collagen deposition. Hydrostatic pressure promoted ECM synthesis and deposition by SMCs and decreased construct compliance. Collagen and crosslinked elastin content in constructs correlated positively with construct burst pressure, and a negative correlation dependent on scaffold type was found between collagen content and construct compliance at low pressures. The systematic investigation of culture conditions in this research provides insights into the control of engineered blood vessel properties. The central hypothesis of this work, that grafts engineered from PGS scaffolds and adult vascular cells under biomimetic in vitro culture conditions can possess compliance comparable to autologous vessels, is true at pressures below 60 mmHg and demonstrates potential for PGS-based vascular tissue engineering. Overall, this work provides tools for engineering tubular soft tissues based on porous PGS scaffolds.

<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

Esta dissertação apresenta o desenvolvimento e avaliação de um método para a segmentação de vasos sangüíneos em imagens de retina, em que se usa a transformada wavelet contínua bidimensional combinada com classificação supervisionada. A segmentação dos vasos é a etapa inicial para a análise automática das imagens, cujo objetivo é auxiliar a comunidade médica na detecção de doenças. Entre outras doenças, as imagens podem revelar sinais da retinopatia diabética, uma das principais causas de cegueira em adultos, que pode ser prevenida se detectada em um diagnóstico precoce. A abordagem apresentada consiste na geração de segmentações pela classificação supervisionada de pixels nas classes \"vaso\" e \"não vaso\". As características usadas para classificação são obtidas através da transformada wavelet contínua bidimensional usando a wavelet de Gabor. Resultados são avaliados nos bancos públicos DRIVE e STARE de imagens coloridas através da análise ROC (\"receiver operating characteristic\", ou característica de operação do receptor). O método atinge áreas sob curvas ROC de 0.9614 e 0.9671 nos bancos DRIVE e STARE, respectivamente, ligeiramente superiores àquelas apresentadas por outros métodos do estado da arte. Apesar de bons resultados ROC, a análise visual revela algumas dificuldades do método, como falsos positivos ao redor do disco óptico e de patologias. A wavelet de Gabor mostra-se eficiente na detecção dos vasos, superando outros filtros lineares. Bons resultados e uma classificação rápida são obtidos usando o classificador bayesiano em que as funções de densidade de probabilidade condicionais às classes são descritas por misturas de gaussianas. A implementação do método está disponível na forma de \"scripts\" código aberto em MATLAB para pesquisadores interessados em detalhes de implementação, avaliação ou desenvolvimento de métodos. / This dissertation presents the development and evaluation of a method for blood vessel segmentation in retinal images which combines the use of the two-dimensional continuous wavelet transform with supervised classification. Segmentation of the retinal vasculature is the first step towards automatic analysis of the images, aiming at helping the medical community in detecting diseases. Among other diseases, the images may reveal signs of diabetic retinopathy, a leading cause of adult blindness, which can be prevented if identified early enough. The presented approach produces segmentations by supervised classification of each image pixel as \"vessel\" or \"nonvessel\", with pixel features derived using the two-dimensional continuous Gabor wavelet transform. Results are evaluated on publicly available DRIVE and STARE color image databases using ROC (receiver operating characteristic) analysis. The method achieves areas under ROC curves of 0.9614 and 0.9671 on the DRIVE and STARE databases, respectively, being slightly superior than that presented by state-of-the-art approaches. Though good ROC results are presented, visual inspection shows some typical difficulties of the method, such as false positives on the borders of the optic disc and pathologies. The Gabor wavelet shows itself efficient for vessel enhancement, outperforming other linear filters. Good segmentation results and a fast classification phase are obtained using the Bayesian classifier with class-conditional probability density functions described as Gaussian mixtures. The method\'s implementation is available as open source MATLAB scripts for researchers interested in implementation details, evaluation, or development of methods.

The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research.

Thesis (Mech.E)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1980. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Bibliography: leaves 355-373. / by Martin J. Forbes. / Mech.E