Biomedical Engineering - Theses

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    The dynamics and characterization of self-assembly in biopolymers and biosystems
    Jativa, Fernando ( 2020)
    Biological self-assembly is the foundation of the strength and elasticity which characterizes nature-derived biomaterials, including the cell’s architecture. Thus, an in-depth understanding of the mechanics behind this process can open the doors to various biomedical applications. For this purpose, this research employed novel experimental and characterization techniques to study self-assembly in biopolymers and biosystems. Initially, a droplet dissolution technique in liquid crystalline stages was used to analyze the process of self-assembly in two important biopolymers, silk and cellulose. Here, we report an effective and simple approach based on droplet dissolution in a liquid binary phase for the formation of silk fibroin transparent spheres as well as cellulose microbeads, both of which can span several hundred micrometers in diameter. The microstructure of the spheres formed at different ethanol concentrations was characterized by electron microscopy. High concentrations of ethanol caused droplets to be encased in a thin shell which collapses once it is taken out of the liquid phase. Generally, low ethanol concentrations produce transparent silk spheres and solid cellulose microbeads. This work on biopolymers demonstrates that controlled droplet dissolution self-assembling may be explored as a novel and effective way to tailor the microstructures of nature-derived biomaterials. The spheres generated in this manner have several different characteristics which can have multiple potential uses, such as templates for scaffolds, microcarriers, as well as photonics and nano-technological applications. The second part of this thesis investigated self-assembling in biosystems. Cell aggregates are an important tool in studying tissue remodelling, extracellular matrix formation, cell-cell interaction, and last but not the least, tissue-like biomechanical properties. A medium-throughput method was designed to characterize the mechanical properties of mesenchymal cell aggregates. This study was the first to present a precise and fast method to determine the Young’s modulus of mesenchymal cell aggregates, utilizing a step-by step aspiration technique. We were also able to recreate conditions that very closely resemble the in vivo environment, where the cells were found to be stretched, and the spheroids are soft and elastic Finally, potential applications of the self-assembled cell aggregates were explored in lung disease study and drug screening, specifically for Idiopathic Pulmonary Fibrosis (IPF). We demonstrated that the cell aggregates from IPF patients show an increase in stiffness, therefore mechanical testing of spheroids is an effective technique to study this disease. It was also found that a novel compound, PF670462, modulates the effect of TGF-beta and inhibits the fibrotic response of IPF cell aggregates. That is, this drug softens IPF spheroids and downregulates fibrogenic gene expression, therefore providing basis for the potential use of PF670462 in IPF treatment.
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    Role of ultrastructural alterations in diabetic cardiomyopathy
    Ghosh, Shouryadipta ( 2019)
    Cardiomyocytes inside the heart are densely packed with parallel columns of myofibrils and mitochondria. Growing evidence indicates a strong correlation between alterations in this sub-cellular ultrastructure and the alterations in energy metabolism during various pathological conditions of heart. The central hypothesis explored in this thesis is that changes in cardiac sub-cellular architecture in pathological conditions can also affect cardiac bioenergetics by interfering with various mechanisms of intracellular energy transport. Type 1 diabetic cardiomyopathy is an ideal candidate for a model disease state to understand this hypothesized interplay between ultrastructure and metabolism. It exhibits many common conditions which accompany heart failure, such as increased mitochondrial reactive oxygen species production and decreased reserve of creatine phosphate. In a preliminary study, 2D electron microscope images collected from control and streptozotocin induced type I diabetic rat hearts were analysed. It was found that diabetic cardiomyopathy leads to an increased mitochondrial fission and formation of large mitochondrial clusters. Further analysis showed that effective surface-to-volume ratio of mitochondrial clusters increases by 22.5% in diabetic cells. Subsequently, a compartmental model of cardiac energy transfer was developed. This simple model predicted that this increase in the surface-to-volume ratio can have a moderate compensatory effect by elevating the availability of adenosine triphosphate (ATP) in the cytosol when ATP synthesis within the mitochondria is compromised. Next, 3D electron microscope images from control animals were investigated. The analysis revealed that cardiac mitochondria are arranged non-uniformly in parallel columns of varying sizes. Following this, the compartmental model was extended to a reaction diffusion based 2D finite element model incorporating a realistic description of the observed sub-cellular ultrastructure. The new model predicted that rapid diffusion of creatine and creatine phosphate acts to maintain homogenous ATP distribution and uniform force dynamics in the control cardiomyocytes, despite the heterogeneous mitochondrial organization. Subsequently, 3D electron microscope images of cardiomyocytes from streptozotocin (STZ) induced type I diabetic rats were compared with controls. The analysis revealed that mitochondrial distribution along the transverse sections was significantly more heterogeneous in type I diabetes compared to control cells. Moreover, mitochondrial area fraction in the studied type I diabetic cells was higher than the control cells. Finally, 2D models of cardiac energy metabolism were created based on the electron microscope images collected from the control and diabetics cells. The results indicated that an increased fraction of mitochondria in diabetic cells can compensate for the reduced ATP synthesising capacity of diabetic mitochondria. The models also predicted that lower activity of mitochondrial enzymes in type I diabetes, coupled with the observed non-uniform mitochondrial distribution, can lead to large spatial variation in concentration of ATP and adenosine diphosphate (ADP). The heterogeneous metabolic landscape in the diabetic cell cross sections was also reflected in large spatial gradients of myofibrillar ATP consumption rate. This finding is important since ATP consumption rate correlates with the speed of muscle shortening. Different parts of a diabetic cell might contract at different rates, which can decrease the energy efficiency of the cell and also damage the cell structure. Thus, this thesis, combining image analysis with computational modelling, provides new insights into how the ultrastructure regulates the metabolism of the cardiomyocytes in disease and health.