Key Words: Thrombosis, Vascular Graft, Microfluidics, Wall Shear Stress / Vascular grafts are essential for the management of cardiovascular disease. However, the lifesaving potential of these devices is undermined by thrombosis arising from material and flow interactions on the blood contacting surface. To combat this issue, the use of antithrombogenic coatings has emerged as a promising strategy for modulating blood and graft interaction in vivo. Although an important determinant of graft performance, hemodynamics are frequently overlooked in the in vitro testing of coatings and their translatability remains poorly understood. We address this limitation with a microscale platform that incorporates vascular prosthesis and coatings with tuneable flow and surface conditions in vitro. As a proof of concept, we use the platform to test the thrombogenic performance of a novel class of lubricant infused (LIS) and antibody lubricant infused (anti-CD34 LIS) coated ePTFE vascular grafts in the presence of arterial wall shear stress, with and without the presence of endothelial cells. Our findings suggest lubricant infused coated ePTFE vascular grafts are thromboresistant under flow and may have potential for in vivo arterial grafting applications. It is moreover apparent that the microscale properties of the device could be advantageous for the testing and translation of novel antithrombogenic coatings or blood contacting prosthesis in general. / Thesis / Master of Applied Science (MASc)

A small shear sensor utilizing an array of carbon nanotubes to support a sensor head was developed for use in steady, high speed, 2D flow. The sensor is a non-intrusive, direct measurement device with a 2 x 2 mm square sensor head surrounded by a small gap on each side (~0.004 inches). The translation of the sensing element is due to the nanotubes bending when a shear force is applied to the sensor head. Displacements are measured by an interferometric technique using fiber-optics to measure the distance the sensor head travels by viewing a polished side of the head. The fiber-optical displacement sensor is bonded to a stationary substrate so that all measurements are relative to a fixed position. Arrays of carbon nanotubes were grown on bare 2 x 2 mm square silicon chips. The nanotubes were grown to heights of 75 microns with a thin layer of amorphous carbon on top. The silicon chips were then flipped, and the amorphous layer of carbon was bonded to bare 1 x 1 cm silicon substrates, making the bottom of 2 x 2 mm silicon chip the sensor head. The sensors were calibrated at Luna Innovations using a point-load technique. Four of the six sensors could not be successfully calibrated because they were fatally damaged during the last step of the calibration process. Wind tunnel tests were conducted on the one sensor that survived the calibration. An arrangement was designed and built from aluminum to test the performance of the sensor in the Virginia Tech Supersonic Wind Tunnel. Seven test runs were conducted in this cold-flow facility at a nominal Mach number of 2.4 and stagnation pressures ranging from 50 - 90 psia. Two test runs gave skin friction values 3 - 20% lower than those values predicted by indirect measurement techniques before the sensor was damaged. While these first results are encouraging, further studies are clearly needed. Due to distinct anomalies in the displacement data during test run 3, it was concluded that the sensor was damaged during this run. Possible explanations of the failure of this sensor are offered along with suggestions for future work. / Master of Science

An in vitro arterial flow bioreactor system for the generation of physiological flows in a biological environment was designed, constructed, and characterized. The design was based on models previously used to investigate the response of endothelial cells to shear. The model interfaces a bioreactor with flow elements to compose a flow loop that reproduces arterial flow conditions within the bioreactor. High-resolution (8.6 microns) time-resolved (4 ms) velocity field measurements within the bioreactor were obtained using Particle Image Velocimetry (PIV). Two physiological flows were considered, corresponding to medium human arteries at rest and exercise conditions: first, with an average Reynolds number of 150 and a Womersley parameter of 6.4, and second, with an average Reynolds number of 300 and a Womersley parameter of 9.0. Two cases were considered: first, using a smooth artery section, and second, with a confluent layer of human microvascular endothelial cells grown on the inner surface of the artery section. The instantaneous wall shear stress, time-averaged wall shear stress, and oscillatory shear index were computed from the velocity field measurements and compared for the cases with and without cells. These measurements were used to assess the value of the system for measurement of correlations between fluid dynamics and the response of biological tissue. It was determined that the flow present in such a system is not an accurate reproduction of physiological flow, and that direct measurement of the flow is necessary for accurate quantification of cellular response to fluid parameters. / Master of Science

Orientador: Willian Fernando Zambuzzi / Resumo: Mudanças nas forças tensionais do shear-stress estão associadas a um repertório de cascatas de sinalização celular, as quais modulam em conjunto o fenótipo vascular tornando o tecido endotelial susceptível a variações patofisiológica e, portanto, compreensão do repertório molecular neste cenário é necessária. Com este propósito, nós submetemos células endoteliais de veia umbilical humana (HUVEC) a um circuito de diferentes forças tensionais in vitro, considerando os grupos seguintes: 1. condição de fluxo de shear-stress fisiológico (nomeado Normo); 2. fluxo de shear-stress hipertenso (nomeado Hyper), e 3. células do grupo 2 foram retornadas para a condição Normo (nomeado Return). As amostras foram apropriadamente coletadas para seguir em diferentes metodologias. Nossos resultados mostraram um forte envolvimento de c-Src no controle da cascata de mecanotransdução modulando sinalização necessária para o fenótipo de adesão, sobrevivência (PI3K/AKT) e proliferação celulares. Além disso, c-Src parece desenvolver importante papel durante o remodelamento da Matriz Extracelular (MEC), cujo performance de matriz metaloproteinases (MMPs) mostrou mudanças significativas. Além disso, através de análise proteômica, mostramos um forte envolvimento de Heat Shock Protein 70 (HSP70) nas células estressadas de modo Hyper, reduzindo significativamente no grupo Return. Esse resultado levou-nos a investigar o proteassoma 20S como uma alternativa proteolítica intracelular para promover o turnover ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Shear-stress changes are associated with a repertory of signaling cascade, modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: one in a physiological blood flow shear-stress condition (named Normo), another in which these cells followed to a hypertensive blood flow shear-stress (named Hyper), and finally one that these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during Extracellular Matrix (ECM) remodeling, which showed significative changes. Additionally, proteomic analysis showed strong involvement of Heat Shock Protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in Return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative to promote the turnover of those proteins. Surprisingly, our data reveled significant over expression of sets of proteasome subunit α-typ... (Complete abstract click electronic access below) / Mestre

Orientador: Willian Fernando Zambuzzi / Resumo: As células endoteliais (ECs) e células musculares lisas (AoSMCs) são os principais componentes celulares do endotélio. As interações entre estes tipos celulares desempenham funções na homeostase e na estrutura vascular. Como uma interface entre o sangue e a parede do vaso, as ECs ocupam um local único diretamente exposto ao shear stress (SS), a força mecânica de atrito lateral produzido pelo fluxo de sangue na membrana apical da célula endotelial, que pode influenciar o comportamento de ambas ECs e AoSMCs. Geralmente, AoSMCs não sofrem diretamente às forcas de cisalhamento, no entanto, estas são diretamente expostas ao fluxo sanguíneo quando ocorre alguma injúria vascular, como por exemplo em algumas lesões ateroscleróticas ou por técnicas invasivas, como a angioplastia. As forças hemodinâmicas influenciam as propriedades funcionais do endotélio, porém estas não são profundamente compreendidas quanto aos mecanismos bioquímicos de respostas de células endoteliais e de musculatura lisa. Assim, a proposta desta dissertação foi estabelecer um modelo de cultivo in vitro que mimetize as forças tensionais de cisalhamento (shear stress), buscando compreender mecanismos celulares, bioquímicos e epigenéticos. Cultura de células primárias endoteliais e de musculatura lisa humanas foram obtidas da empresa LONZA e mantidas conforme recomendações do fabricante. Estas células foram mantidas rotineiramente em condições convencionais em incubadora de CO2. Para mimetizar o fluxo sanguíneo, esta... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

The effects of microbubbles injection in the boundary layer of a turbulent channel flow are investigated. Electrolysis demonstrated to be an effective method to produce microbubbles with an average diameter of 30 ??m and allowed the placement of microbubbles at desired locations within the boundary layer. Measurement of velocity fluctuations and the instantaneous wall shear stress were carried out in a channel flow facility. The wall shear stress is an important parameter that can help with the characterization of the boundary layer. This parameter can be obtained indirectly by the measurement of the flow pressure at the wall. The wall shear stress in the channel was measured by means of three different independent methods: measurement of the pressure gradient by a differential pressure transducer, Particle Image Velocimetry (PIV), and an optical wall shear stress sensor. The three methods showed reasonable agreement of the wall shear stress values for single-phase flow. However, differences as skin friction reductions were observed when the microbubbles were injected. Several measurements of wall-pressure were taken at various Reynolds numbers that ranged from 300 up to 6154. No significant drag reduction was observed for flows in the laminar range; however, a drag reduction of about 16% was detected for turbulent Reynolds numbers. The wall-pressure measurements were shown to be a powerful tool for the measurement of drag reduction, which could help with the design of systems capable of controlling the skin friction based on feedback given by the wall-pressure signal. The proposed measurement system designed in this work has capabilities for application in such diverse fields as multiphase flows, drag reduction, stratified flows, heat transfer among others. The synchronization between independent systems and apparatus has the potential to bring insight about the complicated phenomena involved in the nature of fluid flows.

Doctor of Philosophy / Department of Civil Engineering / Stacey E. Tucker-Kulesza / Surface soil erosion is a widespread problem that impacts the natural and built environment. Many disciplines, such as hydrology, soil science, agriculture, and geotechnical engineering, have investigated soil erosion. Although empirical equations to predict soil erosion exist, they are typically inaccurate, so several devices have been developed to quantify the erodibility of soil. The erosion function apparatus (EFA) was developed to predict the erosion potential of soil for evaluating bridge scour; however, it has been used for several other geotechnical applications. The main disadvantages of the EFA are that it is unable to directly measure the shear stress, it remains operator dependent, and it is time consuming as a standard test requires at least eight hours. Moreover, as erosion occurs, it affects the water quality and makes it difficult to observe the soil sample surface during the test, affecting the operator judgement. The research objective of this project is to instrument the EFA to address the limitations of the device. A stereo-photogrammetry system was developed to measure the soil surface roughness following an EFA test and reduce operator dependency. Turbidity sensors were added to provide a secondary measurement of erosion. The newly instrumented EFA was used to develop a new methodology for interpreting erosion results. Lastly, the new methodology and instrumentation were used to explore the influence of natural and engineered soil properties on soil erosion.

Sickle cell disease is a genetic disorder that affects 100,000 Americans and millions more worldwide. Although the sickle mutation affects one protein, which is only expressed in a single cell type, it has profound detrimental effects on nearly every organ system in the body. Young children with sickle cell disease have an 11\% chance of suffering a major stroke event by the age of 16, and a 35\% chance of developing ÒsilentÓ strokes that often result in significant learning and mental disabilities. Clinical investigations suggest that stroke development in people with sickle cell disease results from luminal narrowing of the carotid and cerebral arteries due to excess matrix deposition and fragmentation of the elastic lamina; however, the underlying cellular mechanisms that initiate arterial remodeling in sickle cell disease remain relatively unknown. Cathepsins K and V are members of the cysteine family of proteases and represent two of the most potent elastases yet identified in humans. Furthermore, the role of Cathepsins has been well established in other cardiovascular remodeling diseases, such as atherosclerosis. Due to the compelling histological similarities between vasculopathy in sickle cell disease and atherosclerosis, we tested the hypothesis that the unique inflammatory milieu, in conjunction with the biomechanical vascular environment of sickle cell disease upregulates cathepsin K and V activity in large artery endothelial cells, ultimately leading to arterial remodeling and stroke. Currently, there are few therapeutic options for the prevention of stroke in sickle cell disease; those that do exist carry significant health risks and side effects. Together, this body of work has generated a more mechanistic understanding of how the sickle milieu stimulates the endothelium to initiate arterial remodeling, which has enabled us to identify important pathways (JNK, NF$\kappa$B) downstream of inflammatory and biomechanical stimuli and validate new therapeutic targets within the JNK pathway to establish preclinical proof of efficacy for the prevention of arterial remodeling in sickle cell disease.

Hypercholesterolemia is a significant risk factor in the development of cardiovascular disease and is associated with chronic leukocyte adhesion in the microvasculature. While the underlying mechanisms behind this have yet to be determined, it may be possible that hypercholesterolemia impairs the fluid shear stress (FSS) inactivation of neutrophils through the rigidifying effect of cholesterol on membrane fluidity. FSS restricts surface expression of CD18 integrins through cathepsin B (ctsB) proteolysis, which minimizes neutrophil adhesivity. If hypercholesterolemia blocks FSS mechanotransduction, then the inhibition of CD18 cleavage may link pathologic blood cholesterol elevations with dysregulated neutrophil adhesion. We hypothesized that elevated cholesterol contributes to dysregulated neutrophil adhesion by impairing ctsB FSS-induced CD18 cleavage through membrane fluidity changes. In the first part of this study, we demonstrated that FSS-induced CD18 cleavage is a robust response of neutrophils and involves selective cleavage of macrophage 1-antigen (Mac1) through ctsB proteolysis. The second part of this study confirmed that ctsB regulates neutrophil adhesion through its proteolytic actions on Mac1, an important integrin involved in adhesion and chemotaxis. Specifically, ctsB accelerated neutrophil motility through an effect on Mac1 integrins during pseudopod retraction. Furthermore, by using a flow-based assay to quantify the mechanoregulation of neutrophil adhesivity, we demonstrated that FSS-induced ctsB release promoted neutrophil detachment from platelet-coated substrates and unstimulated endothelium. For the third part of this study, we linked cholesterol-related membrane fluidity changes with the ability of FSS to restrict neutrophil adhesion through Mac1. We also determined that pathologic cholesterol elevations associated with hypercholesterolemia could block FSS-induced Mac1 cleavage and were linked to disrupted tissue blood flow. This was accomplished using low-density lipoprotein receptor deficient (LDLR-/-) mice fed a high-fat diet. Ultimately, the results provided in the present study confirmed that cholesterol-related changes in membrane fluidity blocked the ability of ctsB to regulate neutrophil adhesion through FSS-induced Mac1 cleavage. This implicates an impaired neutrophil FSS mechanotransduction response in the dysregulation of neutrophil adhesion associated with hypercholesterolemia. Since dysregulated adhesion may be one of the earliest upstream features of cardiovascular disease associated with hypercholesterolemia, the present study provides a foundation for identifying a new mechanobiological factor in the pathobiology of microcirculatory dysfunction.

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