Dr. Amanda G. Paulovich

The focus of my laboratory is the study of human phenotypic variation. Sample projects include:

1. Development of high throughput, multiplexed technologies for targeted protein quantification in blood plasma and solid tissues. We use targeted multiple reaction monitoring mass spectrometry coupled to stable isotope dilution and anti-peptide antibody-based enrichment to measure the abundance of proteotypic peptides as surrogates for quantification of proteins of diagnostic interest. Initially, this work is being done in a highly controlled experimental system: inbred mouse strains genetically engineered to develop cancers. The use of mouse models allows us to minimize biological variation ("noise") and to generate as much sample as needed for technology development. Ultimately, we apply working technologies developed using the mouse model to measurement of candidate diagnostic markers in human patients.

2. Development of high throughput functional assays to determine human phenotypic variation in the cellular DNA damage response. The cellular response to DNA damage is clinically relevant in human cancer. For example, familial cancer syndromes mostly result from germline mutations that compromise the cellular DNA damage response. Second, somatic inactivation of the DNA damage response is ubiquitous in solid tumors and is associated with chromosomal instability. Third radiation and many chemotherapeutics used to treat cancers are DNA damaging agents. Little is known about naturally existing phenotypic variation in the DNA damage response amongst humans, aside from rare familial syndromes. To characterize phenotypic variation in the human population, we are developing high throughput, quantitative assays (e.g. ELISAs) to measure the kinetics of activation of the DNA damage response pathway following gamma-irradiation. Understanding human variation in this response may be clinically important for predicting risk for developing cancer as well as for predicting toxicity to cancer therapies. Also, because the cellular response to radiation is rapid, dose- dependent, time-dependent, and occurs at clinically relevant doses, these assays may also have utility for biodosimetry in the event of a nuclear disaster.

3. Elucidate the network of genes and pathways that buffer defects in the DNA damage response. The cellular DNA damage response shows robustness in that networks of multiple genes (from multiple cellular pathways) buffer the effects of defects in any one gene in the pathway. We use genetic studies in the model yeast Saccharomyces cerevisiae to discover interacting genes and pathways determining sensitivity to DNA damage, and we subsequently test for conservation of these interactions in human cells using RNA interference. The ultimate goals of these studies are to identify novel therapeutic targets, to discover novel tumor suppressor genes, and to understand the underlying molecular mechanisms of the cellular DNA damage response.