Author: Mark Anthony Sellmyer
Publisher:
Published: 2010
Total Pages:
ISBN-13:
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A detailed understanding of living systems requires tools to examine and manipulate biological processes. Small molecules and optical imaging technologies are uniquely suited for this purpose. Small molecules enable the specific manipulation of biomolecular targets, and optical imaging permits the real-time observation of molecular and cellular processes in vivo. This dissertation describes a combination of chemical tool development and imaging strategies to address the following biological problems: 1) specific modification of the genome 2) exquisite control of protein function 3) observing cell-cell interactions in living animals. Chapter One describes a technology for targeted gene modification via the induction of double strand breaks in genomic DNA. The chapter begins with an overview of the field of gene targeting, and documents the design, synthesis, and testing of a novel method for high efficiency homologous recombination. The method relies on engineering two reagents, a small molecule DNA targeting element and a nuclease fusion protein. The targeting element, a peptide nucleic acid (PNA), was designed and synthesized to target DNA via Watson-Crick base pairing. The PNA also was covalently linked to the small molecule, trimethoprim, to recruit a DHFR nuclease fusion protein to a specific DNA site. Our studies show that the individual interactions of PNA/DNA and of PNA/nuclease can readily occur. Further, the ternary complex of PNA, DNA, and nuclease can form in solution. Chapters Two, Three, and Four describe the development and further characterization of a general method to perturb protein stability and function. Briefly, an unstable protein domain, termed a destabilizing domain (DD), can confer instability to a fused protein of interest and promote its rapid degradation. This instability can be rescued by the addition of a small molecule, Shield-1. Our work in Chapter Two describes the use of this system to regulate protein function in living mammals. In one example, we show that regulation of a secreted protein, the immunomodulatory cytokine IL-2, can control tumor burden in mouse models. Additionally, we used the DD to control the function of TNF-[Alpha] after systemic delivery to a tumor. Chapter Three expands on these in vivo efforts by employing the system to regulate secreted FGF2, an important modulator of bone formation, for skeletal tissue engineering. Shield-1 induction of FGF2 causes induction of bone formation in a calvarial-defect model. Chapter Four presents our observations on the behavior of the DD in various cellular environments --the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria- and in the presence of small molecules that modulate protein production, degradation, and local protein quality control machinery. These data indicate that the levels of the DD, in the presence and absence ligand, is dependent on its subcellular locale and protein homeostasis machinery. The fifth chapter of this dissertation reports the development of a method to assess spatiotemporal cell-cell relationships in real-time and in living animals. The method is based on small-molecule diffusion of an activatable substrate between two populations of cells, thus allowing assessment of cell-cell proximity in vivo via bioluminescence imaging. One cell population catalyzes the release of a caged luciferin. The free luciferin can diffuse to a nearby population of cells expressing luciferase capable of light-emitting catalysis. Thus the luciferase cells in closest proximity to the "pool" of free luciferin emit the most light. We demonstrate the utility of this system in vitro and in vivo and are currently investigating its use for the detection of cancer and early metastatic disease in mouse models.
