Christine Mature
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The long term goal of this applicant, is to establish a career in basic research in the Department of Internal Medicine at Washington University Medical School. Areas of research will include the molecular regulation of lymphocyte activation, particularly the mechanisms involved in cell-mediated cytotoxicity. This project will allow Dr. Pham to obtain a strong foundation in advanced methods and experimental approaches upon which she can build her research career. Washington University School of Medicine has a long record of outstanding and innovative research and Dr. Ley has an established commitment to the development of new investigators. His laboratory is an ideal environment to gain the intellectual foundation and technical skills needed to pursue a scientific career. The long term goals of this project are to define and characterize the role of dipeptidyl-peptidase I (DPPI) in granzyme processing and to elucidate the molecular mechanisms by which cytotoxic lymphocytes kill their targets. Cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, and lymphokine- activated killer(LAK) cells contain granules that are directly cytotoxic for target cells. These granules contain perforin and several serine proteases termed granzymes. The importance of perforin and granzymes for cell-mediated cytotoxicity and apoptosis has been firmly established recently in several loss-of-function models. Studies of mice carrying null mutations in perforin and granzyme B also revealed that granzymes other than B may potentially play an important role in the perforin-dependent mechanism of cytotoxicity. To further define the contribution of other granzymes to this pathway of cytotoxicity, we propose to create a mouse deficient in all functional granzymes by creating a null mutation in DPPI, the enzyme thought to be responsible for processing all granzymes to their mature active forms. To achieve this goal, we propose the following specific aims: 1. We will completely characterize the murine DPPI locus by cloning, sequencing, mapping and defining the chromosomal location of the gene. 2. We will analyze the regulation of murine DPPI by analyzing RNA expression, protein expression, and activity in the tissue of adult mice, and in activated lymphocytes. 3. We will study the role of DPPI in granzyme processing and activation by expressing mDPPI cDNA in E.coli, creating active recombinant enzyme to process progranzyme B. 4. We will create a null mutation in the DPPI gene using homologous recombination in embryonic stem cells and analyze the function of immune effector cells derived from these mice.
Sarcopenia is a condition that is most common in aged animals, and is characterized by the loss of skeletal muscle mass and integrity, and can lead to physical disability and poor quality of life. Since skeletal muscle protein synthesis can be limited by the availability of amino acids, supplementation of limiting amino acids to ameliorate the progression of sarcopenia has become a topic of interest in companion animal research. Although t