Elaborate on this aim, experiments proposed, what data you expect to collect and how that data will answer your question

1) Specific Aims Introduction to your problem (breast cancer and cell cycle, make the audience think that what you want to study is important). However, very little is known about … (what new information will your study add to the current knowledge). Therefore, I propose to … The current proposal will test the hypothesis that … (be specific here) The hypothesis will be tested using … (brief sentence on methods and materials). The specific aims of this proposal are: AIM 1: Specifically state the aim of your proposal in one sentence. Elaborate on this aim, experiments proposed, what data you expect to collect and how that data will answer your question. AIM 2: Feel free to pose another aim if you like. Briefly describe the significance of these outcomes to the field. 2) Background and Significance Here you can go in depth about 1. Breast cancer 2. The cell cycle and cancer 3. The significance of your channel in #2. You can include data and/or pictures from your references to support your hypothesis. For Example: Cardiotoxicity is a serious side effect associated with a number of antiarrhythmic compounds11;28. More surprising is that a large number of non-cardiac drugs are similarly associated with increased cardiac risk, despite the fact that they were not developed with cardiac ion channels as the intended target. In general, drug-induced cardiotoxicity is characterized by acute or subacute changes in the electrocardiogram (ECG), including prolongation of the QT interval. Prolongation of the QT interval is associated with an increased risk for torsade de pointe arrhythmia (TdP), ventricular tachycardia (VT) or ventricular fibrillation (VF)20. Currently, acute ECG changes have been associated with over 70 non-cardiac compounds, most of which inhibit the cardiac potassium channel hERG/IKr thereby prolonging cardiac repolarization25. Non-cardiac drugs, hERG block and QT prolongation Block of hERG/IKr currents by a number of noncardiac drugs such as terfenadine and astemizole clearly established the link between druginduced LQTS and inherited LQTS caused by mutations in hERG, the gene responsible for IKr potassium currents20;29. Mutations in hERG produce loss of function with a corresponding reduction in repolarizing cardiac IKr potassium currents causing prolonged QT intervals. Acquired LQTS is produced by non-cardiac drugs, and in nearly all cases is due to block of hERG/IKr currents25. Similar to the situation in inherited LQTS, the reduction of IKr currents by channel blockade in acquired LQTS prolongs the QT interval and is associated with an increased risk for adverse 3) Research Design and Methods Aim 1: Title of aim 1 For example: be specific about doses and/or concentrations of drugs and how you will apply. Therefore, in the first series of experiments we will test the effects of a range of concentrations of As2O3 and SSG on Ca2+ current in freshly isolated cardiac myocytes. Next be specific about how you will culture your cells and the conditions under which you will record. For example: For these and other experiments involving overnight exposure to test compounds, ventricular myocytes will be isolated and cultured overnight in M199 medium with or without the test compound. Patch clamp recordings will be made on the following day using solutions of the following composition (mM): NaCl (137), CsCl (5.4), MgCl2 (1.8), CaCl2 (1.8), glucose (10), and HEPES (10), pH 7.4 for extracellular; and CsMeSO4 (130), TEA Cl (20), MgCl2 (1), EGTA (10), HEPES (10), MgATP (4), Tris-phosphocreatine (14), Tris-GTP (0.3), and 50 U/ml creatine phosphokinase, pH 7.2 for intracellular. Currents will be elicited using 300 ms step depolarizations from a holding potential of -40 mV (to inactivate Na+ and low-voltageactivated Ca2+ currents) over a range of -50 to +70 mV in 10 mV increments to record current-voltage relations (I-V). To determine current densities, currents will be normalized to membrane capacitance determined using the membrane test function in pClamp software (Axon Instruments). Normalized I-Vs will be compared for differences between control and treated cells in both magnitude and activation range. Current traces will also be analyzed and compared for activation and inactivation time constants. This analysis may also provide clues to the mechanism of action i.e., changes in phosphorylation, channel number or gating properties. In addition, we will correlate significant effects on Ca2+ current amplitudes with changes in APD (as shown in figure 2B) where appropriate. Anticipated Potential Problems: This should be self explanatory. 4) Literature Cited

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