Tour-de-Force
Tour-de-Force Tour-de-Force
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed CostsSalary (PhD student):Bench-fee:40 000 EUR/year (total 160 000 EURO in 4 years)10 000 EUR/year (total 40 000 EURO in 4 years)Experiments andMaterials Year 1 Year 2 Year 3 Year 4General MaterialsCell lines €2.150 - - -Medium €400 €600 €600 €400Nocodazole €100 €100 €100 €100BrdU + antibodies €400 €400 €400 -Histone H3phosphorylation €500 €500 €500 €500Question 1Self-referencing system €23.000 - - -JC-1 mitochondrialdetection kit€200 - - -Question 2ROS probes €1.500 €1.500 - -Antioxidant enzyme kits - €800 - -Question 3Materials ROS level - - €400 -manipulationReal Time RT-PCR - - €500 -IP kinase assay - - - €1.000Antibodies - - - €5.00035 S pulse-chaseexperiment- - - €1.000Total: €5.250 €3.900 €2.500 €8.000€19.650For all machinery used during the proposed experiments, we will use the university as our resource forperforming these measurements. A total of € 242.650 funding is needed for the research proposal.SCI 332 Advanced Molecular Cell Biology Research Proposal 32
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed TimelineFor the entire research, a four-year time span has been allotted. The estimated duration of thesubquestions in all cell lines and conditions is shown in this overview.Question 1: Are there cyclic fluctuations in mitochondrial metabolic activity that are coordinated with thecell cycle?The total time span of question 1, including data analysis, will be around six months. Measurement of oxygen consumption and BrdU-labelling:4 months Measurement of membrane potential and BrdU-labelling:2 monthsQuestion 2: Do ROS levels fluctuate throughout the cell cycle?Approximately 19 months will be allowed for question 2. Making and testing the Hyper and PF-1 probe: Measurement of cytosolic and mitochondrial superoxide and hydrogenperoxide levels: Measurement of peroxisomal hydrogen peroxide levels: Measurement of cytosolic superoxide and hydrogen peroxidewhile NADPH oxidase is inhibited: Control for laser-induced ROS production: Measurement of antioxidant activity: Data analysis:3 months5 months1 month2 months1 month3 months4 monthsQuestion 3: Is the G 2 /M transition controlled by redox-dependent regulation of cyclin-cdk complexes?The total time span of this question will probably cover eighteen months. Determination doses antioxidants, antimycin A and hydrogen peroxide: 4 months Cell cycle progression with FACS:2 months IP Kinase assay and Western blot:2 months Northern blotting and real time RT-PCR:3 months 35-S pulse-chase experiments:1 month Further co-immunoprecipitation:2 months Data analysis:4 monthsThis planning ensures that the last months of the four year time span are available for processing all thedata and combining it into a conclusive research. It also leaves some space for unforeseen events, suchas repetition of experiments.Question 1Question 2Question 3Total Data AnalysisYear 1 Year 2 Year 3 Year 4SCI 332 Advanced Molecular Cell Biology Research Proposal 33
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<strong>Tour</strong>-<strong>de</strong>-<strong>Force</strong>: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed CostsSalary (PhD stu<strong>de</strong>nt):Bench-fee:40 000 EUR/year (total 160 000 EURO in 4 years)10 000 EUR/year (total 40 000 EURO in 4 years)Experiments andMaterials Year 1 Year 2 Year 3 Year 4General MaterialsCell lines €2.150 - - -Medium €400 €600 €600 €400Nocodazole €100 €100 €100 €100BrdU + antibodies €400 €400 €400 -Histone H3phosphorylation €500 €500 €500 €500Question 1Self-referencing system €23.000 - - -JC-1 mitochondrial<strong>de</strong>tection kit€200 - - -Question 2ROS probes €1.500 €1.500 - -Antioxidant enzyme kits - €800 - -Question 3Materials ROS level - - €400 -manipulationReal Time RT-PCR - - €500 -IP kinase assay - - - €1.000Antibodies - - - €5.00035 S pulse-chaseexperiment- - - €1.000Total: €5.250 €3.900 €2.500 €8.000€19.650For all machinery used during the proposed experiments, we will use the university as our resource forperforming these measurements. A total of € 242.650 funding is nee<strong>de</strong>d for the research proposal.SCI 332 Advanced Molecular Cell Biology Research Proposal 32
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