I am directly involved in the development of novel fluorescence tools for the discovery of cardiac calcium pump therapeutics. The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the calcium pump responsible for maintaining cellular calcium homeostasis. Diminished SERCA function has been directly linked to numerous degenerative disease states, such as heart failure. The pathological progression of heart failure is associated with an elevated level of cytosolic calcium, and impairs the function of the muscle contraction-cycle. The overarching goal of my research is to discover novel small-molecule effectors, capable of enhancing SERCA’s ability to pump and store calcium within the sarcoplasmic reticulum (SR). Drugs that increase the calcium pumping efficiency of SERCA will restore calcium homeostasis by reducing the calcium content in the cytosol, and enhance impaired cardiac function.
The process of drug discovery is a high-risk effort, and involves screening millions of small-molecules to fortuitously discover a lead compound with high-therapeutic potential. The precise placement of two fluorescent proteins at specific locations along SERCA’s cytosolic headpiece, allows for the detection of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorescent proteins. I have generated human cell lines that overexpress this fluorescent fusion protein and created a live-cell biosensor. The rate of energy transfer (FRET) is dependent on the distance between the fluorescent probes and linked to the enzymatic activity of SERCA. FRET tracks SERCA’s structural status, while it pumps calcium into the sarcoplasmic reticulum. These biosensors are grown in vast quantities, harvested, and utilized for high-throughput drug screening. The cells are dispensed into high-density microplates, where each well contains a different compound.
Through my research, I have demonstrated that FRET can be utilized for the purposes of high-throughput drug screening by using proprietary fluorescence technology, capable of recording the nanosecond fluorescence decay rate (lifetime) and the full emission spectrum. Both lifetime and spectral modes offer incredibly fast speeds, with high resolution and precision. High-throughput screening by lifetime mode offers the advantage of resolving the structural status of the FRET biosensor because the mole fraction of each structural state is assessed, and candidate compounds found during the screening process can be characterized by their structural effect on the biosensor. High-throughput screening by spectral mode increases assays precision by taking into account the shape of the fluorescence emission spectrum. The shapes of these spectra are decomposed into the contribution of known components by a novel spectral unmixing method, and further used to accurately evaluate FRET. When coupled with lifetime mode, spectral-based drug screening increases assay precision and removes artifacts from cellular autofluorescence and fluorescent compounds. The complementary advantages of coupling spectral and lifetime fluorescence measurements significantly reduce the rate of false-positives from high-throughput drug screens. The development of these technologies and FRET biosensor assays, drastically increases the probability of identifying a novel drug with great therapeutic potential.
I am currently interested in applying these novel technologies for the development of new FRET-biosensors targeting other promising therapeutic pathways. I have training and research experience in a broad range of fields (neuroscience, immunology, stem cells, molecular biology, structural biology, and optical fluorescence). My long-term career goal is to work in industry but still remain in close contact with academic research to hopefully make a lasting impact in both areas.
University of Minnesota, Minneapolis, MN Ph.D., Biochemistry, Molecular Biology, and Biophysics Advisor: David. D. Thomas, PhD
University of Minnesota, Minneapolis, MN BS,Neuroscience BS, Biochemistry
|2005-2007||University of Wisconsin River Falls, WI|
TEACHING & RESEARCH EXPERIENCE
2015 BIOC 4125, Laboratory in Molecular Biology and Biotechnology
2014 BIOC 4125, Laboratory in Molecular Biology and Biotechnology
2008-2009 Undergraduate Research Assistant, University of Minnesota (Mark Thomas, PhD)
2017 – Present Post-Doctoral Fellow, University of Minnesota, Minneapolis, MN
I am currently a post-doctoral fellow in the laboratory of Dr. David Thomas at the University of Minnesota. My major focus at this time is to finish preparing and writing manuscripts that I started as a graduate student. I am also involved in writing grants, tutorials, help documents, and training students. I am collaborating with multiple labs in the development of biosensors corresponding to other disease-related molecular targets.
2012-2017 Graduate Fellow, University of Minnesota, Minneapolis, MN
Tory Schaaf, Kurt Peterson, Benjamin Grant, David Thomas, and Greg Gillispie. 2017. High Precision FRET Assays. U.S. Patent Application 62450188. Filed on Jan. 25, 2017.
2011-2013 Research Associate, R&D Systems, Minneapolis MN.
• Performed tissue culture.
• Transfected expression constructs for stable clone development.
• Screened for recombinant protein expression utilizing western blot, fluorescence microscopy, and flow cytometry.
• Utilized DNA cloning techniques for gene-isolation and VersaClone cDNA vectors.
• Effectively planned, organized, and multi-tasked to complete assignments in an efficient manner.
• Interacted positively with flow cytometry, cell culture, protein purification, antibody, and assay development groups.
• Demonstrated ability to work independently and with teams to complete objectives and goals.
2009-2011 Junior Scientist, University of Minnesota. Minneapolis. MN.
• Performed gene therapy preclinical trials in the laboratory of Dr. Rita Perlingeiro.
• Expressed micro-utrophin in human or mouse induced pluripotent stem cells lacking the dystrophin gene, which were then differentiated into skeletal muscle myoblasts and transplanted into animals models of muscular dystrophy.
• Performed molecular biology to generate new cell lines with cre-recombinase, retroviral, lentiviral, and sleeping beauty transposon systems.
• Performed RNA and protein analyses utilizing qRT-PCR and western blotting.
• Problem solved and developed new protocols for lentiviral production and cell sorting using FACS.
• Presented data and results bi-weekly to large groups.
• Aided in animal husbandry for an SPF animal colony with over 1,500 animals.
2009 Junior Scientist, University of Minnesota. Minneapolis. MN.
• Performed preclinical trials in the laboratory of Dr. Keli Hippen involving the suppression of Graft-vs-host disease (GVHD) by regulatory T-cell transplantation.
• Performed flow cytometry and immunohistochemistry to document disease progression and treatment in xenogeneic model of GVHD.
• Performed large-scale cell culture experiments.
• Assessed and graded severity of disease using H+E staining and imaging software.
• Isolated regulatory T-cells from peripheral blood mononuclear cells.
• Optimized multiplexed FACS analysis using up to eight different antibodies.
FUNDING & AWARDS
2016 Ross A. Gortner Award
2014-2016 NIH Chemistry-Biology Interface Training Grant
2012-2016 3M Graduate Fellowship
2013-2017 Arnold H. Johnson Graduate Fellowship
2014 Charles Carr / William Peterson Award
2008-2009 Undergraduate Research Grant (UROP)
Tory Schaaf, Kurt Peterson, Benjamin Grant, David Thomas, and Greg Gillispie. 2017. High Precision FRET Assays. U.S. Patent Application 62450188. Filed on Jan. 25, 2017, Patent Pending.
5. Lo, CH, N. Vunnam, AK Lewis, TL Chiu, BE Brummel, TM Schaaf, BD Grant, P Bawaskar, DD Thomas, and JN Sachs. An innovative high-throughput screening approach for discovery of small molecules that inhibit TNF Receptors. SLAS Discovery, Published online May 22, 2017
4. Schaaf, TM, KC Peterson, BD Grant, DD Thomas, and GD Gillispie. 2017. Spectral unmixing plate reader: high-throughput, high-precision FRET assays in living cells. SLAS Discovery 22:250-261. PMC5323330.
3. Schaaf, TM, KC Peterson, BD Grant , P Bawaskar, S Yuen, J Li, JM Muretta, GD Gillispie, and DD Thomas. 2017. High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA. SLAS Discovery 22:262-273. PMC5323330
2. Gruber, SJ, RL Cornea, J Li, KC Peterson, TM Schaaf, GD Gillispie, R Dahl, KM Zsebo, SL Robia, and DD Thomas. 2014. Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells. J Biomol Screening 19:215-222 (cover article). PMC4013825.
1. Filareto, A, S Parker, R Darabi, L Borges, M Iacovino, T Schaaf, T Mayerhofer, J Chamberlain, J Ervasti, R McIvor, M Kyba, and R Perlingeiro. 2013. An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells. Nature Communications 4: 1549. PMC3595133
Tory M. Schaaf, KC Peterson, BD Grant, J Li, P. Bawaskar, S Yuen, GD Gillispie, and DD Thomas. 2017. Spectral unmixing plate reader: High-throughput FRET-based screening in living cells to identify small-molecule effectors of SERCA. Biophysical Society 61th Annual Meeting, February 14, New Orleans, Louisiana.
Tory M. Schaaf, Ji Li, Samantha L. Yuen, Prachi Bawaskar, Benjamin D. Grant, Kurt C. Peterson, David D. Thomas, Gregory D. Gillispie. 2016. SUPR: Spectral unmixing plate reader for live-cell FRET biosensor drug screening. Biophysical Society 60th Annual Meeting, March 2, Los Angeles, California.
Tory M. Schaaf, Ji Li, Rocio Foncea, Simon J Gruber, Kurt C Peterson, Karl J Petersen, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2015. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Chemistry-Biology Interface Career Development Workshop. August 10, Vanderbilt University, Tennessee.
Tory M. Schaaf, Ji Li, Rocio Foncea, Simon J Gruber, Kurt C Peterson, Karl J Petersen, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2015. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Biophysical Society 59th Annual Meeting, February 10, Baltimore, Maryland.
Tory M. Schaaf, Kurt C. Peterson, Benjamin D. Grant, David D. Thomas, and Gregory D. Gillispie 2016. Spectral unmixing plate reader for high-precision FRET assays in living cells. Society of Laboratory Automation and Screening 5th Annual Meeting, January 23rd-27th. San Diego, California.
Tory M. Schaaf, Simon J Gruber, Kurt C Peterson, Karl J Petersen, Rocio Foncea, David A Bernlohr, Gregory D. Gillispie, and David D. Thomas. 2014. A two-color non-muscle SERCA FRET sensor for diabetes drug discovery using fluorescence lifetime detection. Biophysical Society 58th Annual Meeting, February 15th-19th, San Francisco, California.