The cardiac extracellular matrix (ECM) is a dynamic protein scaffold that is required to support cardiac function. Regular remodelling of the matrix involves protein turnover and deposition and is a highly regulated process. In disease states the normal balance of the ECM is disrupted and aberrant protein deposition and crosslinking can occur. This process, termed fibrosis, causes stiffening of the cardiac ECM, which in turn impairs organ function. Fibrosis is a hallmark of cardiovascular disease, is a progressive condition that can contribute to adverse clinical outcomes, and currently has no available treatments. One of the leading causes of cardiovascular disease is obesity and fibrosis is known to occur in this context. In order to investigate the development of fibrotic remodelling in the context of obesity I have developed a dietary obesity model in the fruit fly Drosophila melanogaster. Additionally, I developed a genetic overgrowth model as increased cardiac load is also known to trigger fibrotic remodelling. Dietary obesity models reveal altered ECM organization, as well as impaired cardiac contractility, while overgrowth models demonstrate a remarkable ability to appropriately scale heart morphology with increased body size. The overgrowth model does have extremely elevated expression levels of the crosslinking enzyme LOXL2, suggesting a major contributor to impaired function is increased crosslinking rather than altered protein deposition. However, inhibition of crosslinking caused only minor ECM organizational defects but was able to rescue the elasticity of the overgrowth model. Overall, this thesis raises intriguing questions for treatment of cardiovascular disease, where tissue dynamics are often overlooked in a clinical setting. / Thesis / Doctor of Science (PhD) / The cardiac extracellular matrix (ECM) is a protein scaffold that supports heart function. Cardiovascular disease often involves increased levels of ECM proteins, a condition called fibrosis, which causes increased tissue stiffness and functional impairment. There is no cure for fibrosis and developing treatments requires an understanding of how the ECM responds to disease. I developed a dietary obesity model and a genetically triggered overgrowth model to examine how the ECM responds to disease states. I found that obesity causes ECM reorganization and functional defects, but that overgrowth models scale their hearts remarkably well with increased body size. Overgrowth models were found to have elevated levels of matrix crosslinking enzymes, which contributed to a stiffer matrix in these individuals. This was rescued by inhibition of crosslinking. Overall, this thesis reveals a connection between cardiac ECM organization, tissue elasticity, and heart function, and how these are altered in disease.

Current methods for studying RNA binding proteins (RBPs) combine the use of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) to analyze RNA-protein interactions. An underexplored alternative approach is using site-specific incorporation of photoactivatable non-canonical amino acids (ncAAs) to enhance the crosslinking efficiency of many CLIP protocols. This thesis describes the incorporation of the photo-crosslinking unnatural amino acid p-azido-L-phenylalanine (AzF) into the Hepatitis C Virus (HCV) non-structural protein 3 helicase (NS3h) for photo-crosslinking and in vitro analysis of the potential binding sites found within the HCV RNA genome. From the five potential sites identified from the NS3h crystal structure for AzF incorporation, two sites, E503AzF and Q580AzF, allowed for nucleic acid photo-crosslinking with fluorescently labelled DNA substrates. We further tested if these mutations adversely affected NS3h and binding activity through a molecular beacon helicase assay and fluorescence polarization methods. We found that E503AzF unexpectedly had a faster unwinding rate than wild type (WT) NS3h and managed to have a similar binding affinity to the tested DNA substrate. Finally, we found that there was a 5-fold increase in the photo-crosslinking efficiency of nucleic acids for E503AzF NS3h mutant compared to our WT NS3h at 254 nm UV light. We are currently working on methods for our CLIP-based protocol to ensure quality RNA footprint generation and purification from photo-crosslinked NS3h. Other work contained in this thesis consists of using Prevotella sp. P5-125 Cas13b (PspCas13b), a clustered regularly interspaced short palindromic repeats (CRISPR) RNA-targeting system, which has been previously shown to knockdown viral RNA and mRNA through designable guide CRISPR RNA (crRNA). Here we incorporated the photo-crosslinking ncAA AzF into PspCas13b to irreversibly bind the crRNA in an attempt to enhance knockdown efficiency and longevity of viral and mRNA targets. We were able to design a crRNA that produced significant knockdown targeting the luciferase mRNA of a luciferase rennilla reporter system. When targeting an HCV subgenomic replicon luciferase reporter system, knockdown was not observed. Additionally, the WT PspCas13b had photo-crosslinking to the bound crRNA and requires further optimization for future use.

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Chronic wounds have a detrimental impact on patient quality of life, a significant economic cost, and often lead to severe outcomes such as amputation, sepsis or death. The elaborate interplay between bacteria, cutaneous cells, immune cells, growth factors, and proteases in chronic wounds has complicated the development of new therapies that could improve outcomes for chronic wound patients. Existing in vitro models of chronic wounds do not appreciably mimic the complexity of the wound environment. In this work, tissue-engineered skin was developed with the goal of creating an in vitro platform appropriate for testing potential clinical therapies for chronic wounds. The Lumen-X, a digital light processing bioprinter, was used to create tissue-engineered skin from a 7.5% (w/v) gelatin methacryloyl hydrogel laden with primary dermal fibroblasts. This dermal layer was developed with an emphasis on providing a favourable microenvironment for the fibroblasts in order to mimic their in vivo phenotype. An epidermal layer of human keratinocytes was formed on the hydrogel surface and stratified through culture at the air-liquid-interface. The maturation of the epidermis was thoroughly characterized with histology, immunohistochemistry, and trans-epithelial electrical resistance analyses which showed a degree of maturation suitable for wound healing studies. To verify the suitability of this tissue-engineered skin for studying healing in vitro, sharp tweezers were used to create physical wounds in the epidermis which were then infected with Pseudomonas aeruginosa. Reepithelialisation, the production of the pro- inflammatory cytokine TNF-α, and the presence of bacteria were monitored over time, showing healing in wounds without infection and those treated with antibiotics, and potential biofilm formation in infected wounds. The tissue-engineered skin developed here is suitable for use as an in vitro model of the infected chronic wound environment. Future work includes developing better methods for creating the physical wound and characterizing the bacterial biofilm in order to improve the reproducibility and clarity of results. Such a model will then be well-poised to begin testing potential chronic wound therapies in vitro. / Graduate / 2023-07-26