| Description |
The Human Genome Project has revealed that the human body has about 25,000 genes. Of these genes, about 20,000 are known as protein-coding genes. If protein splicing and polymorphisms are taken into consideration, the number of proteins involved in human physiology is estimated to be in the billions. The importance of the interactions between these proteins with each other or with other biomolecules cannot be stressed enough in understanding how aberrant biomolecular interactions can cause diseases such as cancer. Thisdissertation seeks to develop and evaluate new technologies in detecting important breast cancer-related biomolecules and their interactions, such as HER2 and EGFR as the important biomolecules and the interactions of CD44 with HA, EGFR withEGF, and EGFR with HER2. By utilizing and further engineering already existing probes (e.g. fluorophore-conjugated HA and split-NanoLuc®), different methods can be used to understand the aforementioned biomolecular interaction dynamics. Chapter 2 explorespossible cellular binding mechanisms on differentiating HA molecular weights and how cell surface receptor CD44 non-discriminately binds to HA regardless of the HA chain length but has slight avidity. In Chapter 3, a single multifaceted tagsystem for protein production, purification, and functional assays is proposed that can be used in studying HER2 and EGFR dynamics. Chapter 4 introduces the concept of homogeneous immunoassay for the measurement of an important ivbiomolecule HER2 in both soluble form and as a cell surface receptor. Lastly, Chapter 5 discusses an immunoassay developed to monitor the change in EGFR-HER2 dimerization levels in a non-invasive way to the target proteins and its functionality is tested in adenocarcinoma breast cancer cells. Although this dissertation heavily focuses on examining biomarkers and biomolecular interactions in breast cancers, we expect that the same proof-of-concept can be used to analyze the interactions in other cancers or potentially other diseases. |
