Respiratory Control in Mitochondria — Data Analysis and Research Paper

Honor code policy

Please use the data that your team collected from the first four experiments. You may work together with others in your team in aquiring data from chart records, including slopes, qualitative information, etc. If you have difficulty interpreting the records you may consult with other teams and (with proper acknowledgement) use another team's data for your analysis. In that event you may seek the other team's assistance in clarifying information on the records, such as names of and volumes of reagents that were added, chamber volume, etc. Otherwise, you are not to receive outside assistance in acquiring data from the records.

You are encouraged to discuss the results and their interpretation during and outside of lab. Under no circumstances, however, are you to receive or give help in actually writing the paper. All text and tables in your paper are to be your original material.

Specific requirements and suggestions

Focus on the overall objective. You are evaluating isolated rat liver mitochondria for its suitability as a model for studying mitochondria function. Isolated mitochondria were prepared from rat liver and a polarographic system was employed to conduct key experiments. Observed results are to be compared with predicted results based upon established theory. Use your prior experience to decide what results to present and how to present them most effectively.

Materials and methods

Look back at the example I provided for the first paper, the comments that you received, and review the guidelines. Recall that Materials/Methods should be very concise and generic. If you find yourself repeating yourself then you are probably doing too much. Remember, there are two objectives here. One is to permit the reader to judge the scientific merit of the methodology that you employed. The other is to document the general methodology, including compositions of solutions and reagents and kinds of procedures that were conducted. A reader might want to use all or part of the methods for some other objective involving measurement of oxygen consumption. It is doubtful that a reader will repeat each of the experiments that you ran. Recall that for the last study you did not report how each individual sample was prepared. Apply the same principle this time.

Consider the following situation. Different people did different sets of experiments, depending on time constraints and on changes to the methodology as they conducted experiments. Yet we all used the same system for measuring oxygen consumption and the same set of reagents. The exact same materials and methods section could be used word for word in each individual paper, despite the differences in how specific experiments were designed. It's about the methodology, not about how the methodology was applied to specific experiments.

Results

When we write up a research paper we present results of successful experiments. To put it another way, we make sure that we have accomplished our objectives and are sure that our findings are valid before we attempt to publish them. That doesn't mean we wait until the outcome is as expected. We must be prepared to accept the conclusion that our hypothesis was invalid, if the findings tell us so. Some of our mitochondria experiments typically fail, sometimes due to contamination of media and/or reagents, sometimes because the source material is compromised, and sometimes because of errors we make.

You have choices of what results to report. Decide in advance if, for the discussion, you want to discuss your own results and what mitochondria properties may have been compromised, or if you would prefer to discuss results of experiments that gave us typical results. Do report the results of your first experiment. After that, you may continue to report your own experiments (facts only, of course). If you wish, you may extract data from the appropriate record posted here instead. These records were produced by valid experiments and are the property of each student in the class. The experiments that produced the posted records all gave predictable results.

On each record the chamber volume is marked (the experiments were from different investigators). The records appear as they would comiing at you from a chart recorder, with 100% saturation with oxygen to the right. Paper speed was 0.2 mm/sec. and each square represents 2 mm distance along the chart.

Remember that the results section is a write-up. One should be able to read your text and understand the context of the results, including all parts of the study. Recall, however (to parapharase the McMillan text, page 46) that it can be tedious and cumbersome to report a large amount of quantitative information in the text. For that reason, and also to provide the reader with ready access to important findings, we typically use a table for such data. The text and table should complement each other. That is, we don't repeat the numbers verbatim. An effective approach to the write-up might be to describe the changes you observed as you conducted the experiments in qualitative terms. Recall that we use figures only when it is important to show a pattern or trend in the data.

Recall that results must be converted to numbers with physically meaningful units. Is "percent" physically meaningful? Also think about how you might compare one experiment with another when you used different volumes of mitochondria suspension. Don't forget to round any uncertain quantities to reflect the actual level of precision with which we can report such information.

Chart records are raw data, not "graphs." Raw data, of course, is typically not included in a results section. They are analyzed and quantitative results presented in an appropriate manner. Thus, you should not incorporate the chart records into the paper itself.

Discussion

Thoroughly discuss the results from the preparation and study of isolated mitochondria. You may limit your discussion of the oxygraph studies to experiments one through four. If you can stay within the page limit you are welcome to discuss the optional studies, however they are difficult to interpret and results less predictable, especially when we do not have exceptionally good mitochondria preparations. It makes sense to discuss the experiments in sequence. They were designed to reveal properties of isolated mitochondria in a logical order.

To cover the material in sufficient depth you must provide a sound basis for your predictions and address any discrepancies between observed and predicted results. Such discrepancies are not necessarily fatal flaws in the experimental model, if there is an explanation based upon design of an experiment and/or limitations of the polarographic system or mitochondria in vitro. You must discuss the molecular mechanisms behind your predictions. For the sake of context you should also (briefly) explain the significance of mechanisms. For example, oxygen is indeed reduced to water by the ETS, but what is the real function of oxygen in the system – what purpose does it serve?

Regardless of the approach you take to the discussion, you must at some point demonstrate a good understanding of the concepts of respiratory control and of ADP-stimulated (State III) respiration.

Abstract

Very briefly provide a context for the study, then summarize all of the findings, both qualitative and quantitative. You should finish up with some conclusion that revisits the main objective of the study.

Introduction

Provide a context for the study. You should describe and defend the objective and provide reasons for choosing this model system. We don't put detailed methods in an introduction but you should describe the general experimental approach.