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Understanding Cellular Responses through Biochemical Analysis: A Comprehensive Guide

This course led by Dr. M.H. Ghahremani focuses on cellular responses and their biochemical analysis. Participants will explore various methods to measure changes in cells, including morphological studies, proliferation analyses, and biochemical assays. The curriculum covers transcription changes, mRNA and protein level assessments through techniques like RT-PCR and Western blotting. Emphasis is placed on understanding second messengers, secretory chemicals, and enzymatic activities. By the end, participants will gain insights into cellular interactions and the methods used for accurate measurement and analysis.

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Understanding Cellular Responses through Biochemical Analysis: A Comprehensive Guide

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  1. Cellular Response:Biochemical Analysis Dr. M. H. Ghahremani Reference: 1-Masters, J.R.W. (2000). Animal cell culture, 3rd Ed., Oxford. 2-Sambrook, J.; Russell, D.W. (2001). Molecular cloning: a laboratory manual,3rd Ed., CSH press. Dr. M.H.Ghahremani

  2. The goal of this course • How to get response from cell • How to measure changes in cells • What kind of assays one can use in cells • How biochemical assays will provide responses Dr. M.H.Ghahremani

  3. How can we use cell for responses? • Morphological studies • Proliferation studies • Cell Death studies • Biochemical changes • Cell to cell interactions Dr. M.H.Ghahremani

  4. Cellular changes • Transcription changes • mRNA levels • RT-PCR • Real time PCR • Northern blot • Translation changes • Protein levels • Immunohistochemistry • Western blot Dr. M.H.Ghahremani

  5. Biochemical changes In Cytoplasm • Change in second messengers • Change in secretory chemicals • Change in enzymatic activities • Change in protein interactions In nucleus • Change in nuclear enzymes • Change in transcription Dr. M.H.Ghahremani

  6. Second messengers • Cyclic nucleotides • cAMP, cGMP • Interacellular • extracellular • Radioimmunoassay • ELISA • Radioisotope Dr. M.H.Ghahremani

  7. Second messengers • Intracelluar Calcium • Single cell • Cell suspension • Chromophore • Fura2-AM • Radioisotope • Patch clamp Dr. M.H.Ghahremani

  8. Secretory chemicals • Hormones • Neurotransmitters • Signaling molecules • Radioimmunoassay • ELISA • Radioisotope Dr. M.H.Ghahremani

  9. Enzymatic activities • In the intact cell • Extracted form What to consider • Substrate • Reach to enzyme • Quantitative measurement • Low background • Chromophores • Radioisotope Dr. M.H.Ghahremani

  10. Enzymatic activities What to measure • Enzyme kinetic • Enzyme localization • Substrate specificity • Enzymes to measure • PKA, PKC, PLC • MAPK, JNK • COX, LOX • Caspases Dr. M.H.Ghahremani

  11. Phosphorylation • Activation of proteins (or Enz) • Inactivation of proteins (or Enz) How to measure? • Addition or elimination of Phosphate • Using 32P • Using Mass Spectroscopy • Using Specific Antibody • Localization • Substrate specificity • Protein Interactions Dr. M.H.Ghahremani

  12. Protein interactions • Measure physical interaction • Protein-protein interaction • Protein-chemical interaction • Immunopercipitation • Fusion protein • Yeast two hybrid Dr. M.H.Ghahremani

  13. Protein interactions • Measure biochemical interactions • Change in enzymatic activity • Substrate specificity • Effect on other biochemical pathway Dr. M.H.Ghahremani

  14. Protein interactions • Measure Protein-DNA interactions • Change in Interaction • DNA finger printing • Gel shift assay • Yeast One hybrid • Change in Transcription • Effect on other proteins Dr. M.H.Ghahremani

  15. Dr. M.H.Ghahremani

  16. 4.30 Dr. M.H.Ghahremani

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