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SC BIOL 425 - Chapter 4 Powerpoint(2)

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Slide 1Bei XIE et. alSlide 3DifferentiationHL-60 differentiationSignal TransductionSignal TransductionSlide 8Slide 9Discussion of the Xie PaperDiscussion of the Xie PaperSlide 12Signal Transduction: The Big Picture!Slide 14Slide 15Chapter 4: today’s labSlide 17Writing the introductionWriting the introductionWriting the introductionMaterials & MethodsResultsResultsResultsWriting the discussionChapter 4BEI XIE ET. AL“Fibronectin-mediated Cell Adhesion Is Required for Induction of 92-kDa Type IV Collagenase/Gelatinase (MMP-9) Gene Expression during Macrophage Differentiation”MutipotentTotipotent: able to form every type of cell in the bodyPluripotent: able to form most cell types, but not limited to a specific systemMultipotent: able to form multiple cell types, but restricted to a specific lineage/system= Pro-myeloid cell, ie HL-60 cellWhite Blood CellsRed Blood CellsNerveBoneOther TissuesHierarchy of Stem Cells TotipotentPluripotentBlood Stem CellsOther Stem CellsMuscleDifferentiation•The process by which a cell becomes more specialized in its function.•This is accompanied by many changes which may include:–Changes in morphology –Changes in gene expression –Changes in protein expression–Enter apoptosis•We know from the Collins paper that HL-60 cells can be induced to differentiate into various cells of the white blood lineage.HL-60 differentiationHL-60GranulocytesSignal Transduction•In HL-60 cells, differentiation takes place by a process known as signal transduction.•An extracellular signal binds to a receptor on the surface of a cell, and induces certain reactions to occur within the cell.•These reactions then result in the changes in morphology, gene expression, and protein expression associated with the cells gaining a specialized function.Signal Transductionhttp://www.scq.ubc.ca/wp-content/uploads/2006/07/transduction.gif Examples of Ligands:PMA and DMSOhttp://www.dnalc.org/resources/3d/13-transcription-advanced.htmlhttp://www.dnalc.org/resources/3d/16-translation-advanced.htmlGene expression: Central dogma of molecular biologyThe principle of directional flow of genetic information proceeds from:DNADNA replication•transcription (copying information without “language” change) from DNA to RNA•translation (language change from nucleotide sequence to amino acid sequence) from RNA to protein. ProteinRNAtranslationtranscriptionDiscussion of the Xie Paper: PMA-induced differentiation of HL-60 cells•HL-60 cells were induced to differentiate into macrophages/monocytes by treatment with a ligand, PMA.•PMA: phorbol 12- myristate 12-acetate–A potent tumor promoter that activates certain pathways in the cell (via protein kinase C)–Is a light colored powder which is soluble in DMSO–Induces HL-60 cell differentiation into monocytes•DMSO: dimethyl sulfoxide–A colorless liquid–The solvent used to make a PMA solution–Acts as a control for PMA treatment conditions–Induces HL-60 cell differentiation into granulocytesDiscussion of the Xie Paper•The authors showed that a certain protein, PKC- , was also important to MMP-9 expression and cell adhesion and spreading associated with macrophage differentiation.•HL-525 cells are deficient in PKC- , what does this mean for MMP-9 expression in the paper?•What did the inhibitors (H-7, HA-1004) do to MMP-9 expression in macrophage differentiated HL-60 cells and monocytes?•PKC- very important for PMA-induced macrophage differentiation!!Discussion of the Xie Paper•The authors showed that a certain gene was induced during PMA-induced HL-60 macrophage differentiation.•Scientists also tested for MMP-9 gene induction and protein expression in normal cellular monocytes with PMA and M-CSF.•What are MMPs?Discussion of the Xie paper: PMA induces macrophages/monocytes to express MMPs:•Matrix Metalloproteinases–Proteases capable of degrading the extra cellular matrix–Involved in cell invasion and migration (metastasis, angiogenesis)•Remember, these are immune response cells!•Matrix metalloproteinase 9 (MMP-9)–Proteinase, Gelatinase–Produced as a pro-enzyme (pro-MMP9) which is exported outside the cell, and is cleaved to form MMP-9, a now active enzyme which acts on collagen, gelatin, elastin, pro-TNF-α, and interleukins What else is different about PMA induced HL-60 cells? Xie paper: integrin composition at cell surfaceSignal Transduction: The Big Picture!http://www.scq.ubc.ca/wp-content/uploads/2006/07/transduction.gif Examples of Ligands:PMA and DMSOPMA Signal Transduction PathwayIn the presence of PMA, a signal is triggered via receptors in the cell membrane to a secondary messenger, PKC-β. This signal is then amplified within the cell and results in the activation of a transcription factor which translocates into the nucleus and activates the transcription of the MMP-9 gene. The MMP-9 mRNA is then translated into pro-MMP-9, which is exported outside the cell, cleaved to form MMP-9, which then degrades proteins in the extracellular matrix (ECM).Hypothetical representation of treatment effects on HL60 cellsChapter 4: today’s lab•You will examine HL-60 cells that have been treated with PMA, DMSO, or neither1) You will view changes in the cells’ viability – Done for you already!•once a cell differentiates, it is no longer immortal, it is capable of under going apoptosis2) You will view changes in the cells’ morphology (shape and structure)•What do you expect to happen to viability and morphology upon treatment of HL-60 cells with PMA and DMSO?Biology 302 Lab Report Rubric (Point value in parentheses)1. INTRODUCTION (10)Context (4)Accuracy and relevance (3) Hypothesis (3)2. MATERIALS & METHODS (5) Procedure, Controls & Replication 3. RESULTS (10)Data Selection (4)Data Presentation (6) 4. DISCUSSION (20)Conclusions based on the data selected (10)Alternative explanations / Limitations of data (5)Significance of research (5)5. WRITING QUALITY (5)Writing the introduction•Today in lab, we tested the changes that occur in HL-60 cells when they are induced to differentiate , specifically changes in viability & morphology.•Before you begin, write out your hypothesis for this lab report; What are you testing with the experiments you performed today? You must then aim to provide a context for your experiment – cell system, reagents etc. •Provide information that is relevant to the study at hand! – Do not talk about what Biology is, or what a cell is


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