Cardiovascular disease is a leading cause of death worldwide. Changes in the cardiac extracellular matrix (ECM) are associated with cardiac pathologies such as cardiomyopathy and cardiac hypertrophy. The ECM is a dynamic scaffold of proteoglycans, fibrous proteins, and glycoproteins that sheathes and protects many organs and tissues, including the heart, by attenuating mechanical stress. Misregulation of ECM proteins triggers changes in matrix stiffness, which can lead to age-associated and congenital heart defects. ECM rigidity is also important to the migration of cells, such as hemocytes, the invertebrate blood cells. In the embryo, hemocytes also perform fibroblast functions, through the deposition of the ECM proteins Collagen and Laminin. Hemocytes are hypothesized to be critical for ECM assembly, and by extension, for heart development. The consequences of impaired hemocyte function in the embryo and during larval growth are unknown and are the focus of this research. Using Drosophila melanogaster as a model, I used genetic tools to manipulate hemocyte survival and motility to assess their role in ECM organization and structure around the heart. Concerted gene knockdown and confocal microscopy techniques were employed to evaluate the effects of altered hemocyte abundance and motility on hemocyte behaviour and resulting changes to the ECM. Here I provide evidence to support a role for hemocytes in the turnover of a vital ECM protein, the Type IV Collagen Viking. I also developed a novel protocol to photobleach and observe fluorescence recovery in intact, living larvae using confocal microscopy. Recovery of fluorescence of GFP tagged ECM is a measure of the rate of ECM protein turnover during development or growth. This novel technique has allowed for assessment of recovery of Viking-GFP after photobleaching in vivo, as a measue of Viking protein turnover at the cardiac ECM. This new technique can be employed to determine the turnover of other major ECM proteins. Combining hemocyte impairment with photobleaching provides the opportunity to observe innate protein turnover at the ECM in real time, both in normal and hemocyte-deprived matrices. Recovery of Viking-GFP fluorescence was also observed in hemocyte-deprived conditions. My findings reveal gradual recovery of Viking-GFP at the cardiac ECM in controls, and potentially slower recovery in hemocyte-impaired conditions. These observations suggest a role of hemocytes in ECM protein turnover. This work will help reveal the role of hemocytes in organizing the cardiac ECM and provides a novel technique for the in vivo assessment of ECM protein turnover. Ultimately, this research sheds light on how hemocyte function affects overall structure of the cardiac ECM and contributes to an enhanced understanding of how changes in this ECM influence predisposition to and progress of cardiac disease. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25017 |
| Date | January 2019 |
| Creators | MacDuff, Danielle |
| Contributors | Jacobs, Roger, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.002 seconds