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Portfolio of Past Research

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A sampling of my past research findings as a Research Fellow at Harvard Medical School and as a Ph.D. candidate at the University of Toronto.

Published in: Health & Medicine
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Portfolio of Past Research

  1. 1. Portfolio of Past Work Nish Patel, Ph.D.
  2. 2. Postdoctoral Research Harvard Medical School Sept. 2006 – Dec. 2009
  3. 3. Multi-Drug Resistance <ul><li>~ 40% of human tumors develop resistance to chemotherapeutic drugs </li></ul>
  4. 4. <ul><li>Paclitaxel® (Taxol) </li></ul><ul><li>a frontline chemotherapeutic agent </li></ul><ul><li>binds to  -tubulin preventing microtubule dynamics </li></ul><ul><li>FDA approved in 1992 for treatment of metastatic breast cancer </li></ul><ul><li>Clinically approved for the treatment of breast and ovarian cancer, in clinical trial for prostate, lung and several other cancers </li></ul><ul><li>Major limitation is its susceptible to several mechanisms to drug resistance </li></ul><ul><ul><li>Expression of the MDR phenotype </li></ul></ul><ul><ul><li>Alterations of the microtubule system </li></ul></ul>
  5. 5. Rationale: Increased understanding of the molecules associated with drug resistance would undoubtedly be useful for the discovery of new targets for the detection, delay or reversal of resistance. Objective: Identify proteins associated with Paclitaxel ® resistance and to examine their potential use as targets for modulating the resistant phenotype
  6. 6. <ul><li>Prohibitin1 </li></ul><ul><li>molecular mass of 30-32 kDa </li></ul><ul><li>Prohibitin1 forms a high molecular weight complex with Prohibitin2 </li></ul><ul><li>primarily localized to the inner mitochondrial membrane but also found on the cell surface </li></ul><ul><li>Best characterized function of prohibitin is as a chaperone </li></ul><ul><li>Interacts with E2F, p53, and pRb in the nucleus </li></ul>Vol. 11 No. 4 April 2005 pp192-197
  7. 7. Prohibitin expression is elevated in Paclitaxel resistant cell lines Paclitaxel Resistance Paclitaxel Resistance - + - + - + - + Cell line 1 Cell line 2 Cell line 1 Cell line 2
  8. 8. Prohibitin1 localizes to the mitochondria in both drug-sensitive and resistant cell lines Paclitaxel-sensitive Paclitaxel-resistant
  9. 9. Prohibitin1 is localized to the cell surface in drug resistant cell lines Phb1 Antibody Rhodamine-PHB1-binding peptide Paclitaxel-sensitive Paclitaxel-resistant Paclitaxel-sensitive Paclitaxel-resistant
  10. 10. Prohibitin1-binding peptide preferentially localizes to Paclitaxel-resistant xenografts after intravenous injection Rhodamine-PHB1-binding peptide Rhodamine-PHB1-binding peptide DAPI DAPI Paclitaxel-sensitive Paclitaxel-resistant
  11. 11. Effect of Prohibitin1 siRNA on Taxol Sensitivity of Paclitaxel-resistant Cells
  12. 12. The generation of Paclitaxel-resistant cell lines with stable knockdown of Prohibitin (PHB1) or GST 
  13. 13. Paclitaxel sensitivity in stable cell line expressing PHB1 or GST  shRNA
  14. 14. Paclitaxel Sensitivity of A549TR xenografts expressing control shRNA
  15. 15. Paclitaxel Sensitivity of A549TR xenografts expressing PHB1 shRNA
  16. 16. Paclitaxel Sensitivity of A549TR xenografts expressing GST  shRNA
  17. 17. Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST  shRNA
  18. 18. Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST  shRNA
  19. 19. EMERGING HYPOTHESIS: The selective spatial localization of Prohibitin1 may be necessary for the onset and/or maintenance for paclitaxel-resistance in human cancers.
  20. 20. SIGNIFICANCE: Prohibitin1 on the surface of tumor cells may represent a novel marker for the detection of taxane resistance in cancer patients either before or during the course of chemotherapy. It may also act as a novel target for therapeutic strategies for the treatment of drug-resistant tumors.
  21. 21. Ph. D. Candidate Research University of Toronto Jan. 2001 – Mar. 2006
  22. 22. Insulin Stimulates Glucose Uptake Pancreas GLUT4 is the major insulin-responsive glucose transporter
  23. 23. Several stimuli stimulate GLUT4 translocation Stress
  24. 24. <ul><li>How is signal specificity achieved? </li></ul><ul><li>Activation of unique signalling molecules </li></ul><ul><li>Signal amplitude and time course </li></ul><ul><li>Unique subcellular loci </li></ul>
  25. 25. GLUT4 Glucose
  26. 26. Insulin induces GFP-actin remodelling and membrane ruffling in L6 skeletal muscle cells Confocal Fluorescence Microscopy Scanning Electron Microscopy
  27. 27. HYPOTHESIS: Insulin-induced actin remodelling is needed for signalling molecules to meet with GLUT4 in time and space
  28. 28. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify signalling molecules that regulate in insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
  29. 29. Insulin-mediated cellular redistribution of GFP-PH-GRP1 reflects its PI-3,4,5-P 3 binding capacity Patel et al., Mol. Cell Biol. , 2003 Insulin
  30. 30. Insulin-induced actin remodelling promotes the spatial localization of specific signalling molecules within the actin mesh
  31. 31. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify signalling molecules that regulate insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
  32. 32. GLUT4 (10 nm; arrowhead) and p110  (5 nm; arrow) colocalize within insulin-induced actin structures Basal Insulin
  33. 33. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify the signalling molecules that regulate insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
  34. 34. <ul><li>member of A ctin depolymerizing factor/ C ofilin (AC) family </li></ul><ul><li>21 kDa </li></ul><ul><li>ubiquitously expressed in varying cell types </li></ul><ul><li>binds F-actin and G-actin </li></ul>Cofilin: a regulator of actin dynamics <ul><li>Active State = unphosphorylated at Ser-3 </li></ul>Hypothesis: Cofilin is necessary for insulin-stimulated actin remodelling and GLUT4 translocation to the cell surface
  35. 35. Cofilin1 siRNA disrupts insulin-induced actin remodelling
  36. 36. Cofilin1 siRNA inhibits insulin-induced GLUT4myc translocation * p < 0.005, n = 6
  37. 37. EMERGING HYPOTHESIS: The selective spatial localization of insulin signalling molecules within the actin mesh may ultimately lead to GLUT4 arrival at the plasma membrane.
  38. 38. SIGNIFICANCE: A defect in insulin signalling leading to actin remodelling may contribute to insulin resistance in the skeletal muscle and to the onset of type 2 diabetes.

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