What is the purpose of the PhD Confirmation paper?

One thing I am still trying to reconcile in my mind is the GRS comments to me over the phone that this paper simply needs to demonstrate quality thinking, a good plan around the research that addresses the key research questions and that “they are not trying to test or trap me in this review process, it is simply to get a level of comfort that we each know what you are doing…” and how much further effort we need to put into this confirmation paper. 

Besides the GRS who else is going to read the confirmation paper and what depth of questions am I expected to answer ?

 

Right now I think I have a reasonable understanding of what we want to attempt and can participate in a discussion to explain the process at a high level. But in one of Nina’s comments recently she asked what are the steps I will follow in the blood collection, can it be stored, if so what temp and for how long before we have to use the sample. I would have thought for the confirmation paper all we would need to address is that we are aware of these questions and it is not the purpose of the confirmation paper to answer detailed questions but to give an overview of the research framework we are proposing and that it is the purpose of the PhD research to raise these detailed questions, drill down in the research journals and find the answers. 

I am not clear at this stage if this understanding is correct or should I expect the reviewers to really get stuck into the details of the research, ask tough questions and that I need to add in further evidence of what I am going to do to head off these potential problems. 

If this is the case then I will need to do further research on the various lab protocols etc. and we will not meet the time line even if it is extended to July 30^th, which would cause me some concern.

OXPHOX XFe-96 well plate design

The Seahorse XFe-96 well plate designs in the confirmation paper were based on comparing just PD samples across different age groups or gender.  In the design below comparisons with the healthy controls (HC) would have been made difficult because the HC sample were planned to be on different plates ie plates for the PD and plates for the HC.

In the updated well plate design we now have both the PD and HC samples for the same age category or gender in the same well plate. This design is better in that both the PD and HC samples are subject to the same treatments in the same XFe-96 test run rather than running two separate plates.

Under the new design, the samples for HC and PD will be prepared at the same time and submitted to the various inhibitors around the same time in the micro-chambers.

Applications of mass spectrometry-based proteomics in research

Today I did the Applications of mass spectrometry-based proteomics in research workshop. I did a similar workshop last year but at that stage I had no idea what proteomics was. At least this time round I had a better idea and most of the presentations on the new proteomic platform (Pierre Faou), types of proteomic tests available (Rohan Lowe) and LC-MS proteomic bioinformatics and visualisation (Harinda). I found the course very useful in getting some initial ideas beyond just comparing the protein abundance between PD and controls.

I have set up a Zoom meeting with Rohan Lowe for Wednesday 1st July at 10am to go over the following questions:

1. Research area: I think my main area is Post Translational Modification, id this correct?
   
   
2. Peptide Mapping:
• Can the mapping be done on a comparative basis?
• If not which technique would I use to compare the mitochondria in leukocytes between Parkinson's patients and healthy controls?
  
  
3. Abundance depletion: 
• With the new faster technology, would we still need to deplete the blood samples to lower the high abundance proteins such as albumin and globulin to make the low abundance proteins easier to detect?
• If we deplete the sample, will the remaining low abundance proteins need to be enriched? If so how would we do this with human blood samples?
  
  
  
  
4. Pierre mentioned you always run a sample test before the full proteomic analysis. What sort of sample should we think about?
   
   
5. Protocols:
• What is the process to identify the key experimental steps that enable the correct protocols to be identified?
  
  
6. Validation: How do we confirm the proteomic results are accurate - is there a validation process?
   
   
7. Chris Adda mentioned LTU research was subsidised - how does this work?
   
   
8. Bioinformatics: How is the relevant analysis derived? Does the proteomics team suggest the most appropriate analysis and does this as part of the proteomics package or do I do the analysis on line with software you give me access to?

 

 

Updated PhD Research Project Plan

In the earlier time line was it looked at doing all the experiments one after the other, then all the statistical analysis would follow and finally the thesis chapters would be written. The problem with this process is you will not know if some of the experimental runs have failed until much later down the track when you are doing the statistical analysis.

The changes made to the project plan now group each experiment as a self contained unit, doing the experimental run then the analysis for that run to find out how the results turned out. If the run was a success the analysis can be used to write up the chapter and journal paper for the thesis. In this way the thesis is progress as the experiments are done and hopefully each experiment results in both a published paper and completed thesis chapter.

The benefit of this is that I do not have to strain my memory at the end to think back to when I did the experiments to create the thesis chapters. As they are done as I progress the data, ideas and conclusions should be fresh and accurate.

Detailed PhD Research Plan (click to enlarge)

A summary version of the above detailed version will be used for the confirmation text.

Summary PhD Research Plan (click to enlarge)

PhD project plan time line

Started to work on an estimated project plan and based on my Honours experience with Seahorse analysis, we hydrated the well plate the day before, then prepared the sample and ran the test the following day. So each run essentially took 2 days. 

When each of the five studies are placed into a project time line it looks as if the experimental section could be completed by the December 2020. However this is based on the assumption that my well plate designs for the OXPHOS experiments will work and we can get most of the samples accomodated in a few XFe-96 plate runs, which I have reservation s about.

It will be necessary to discuss the OXPHOS runs with Dr Binger to get her guidance on setting up the well plate cartridge layouts. At this stage any material changes to the layout may significantly change the budget and time line, particularly if many more experiments are needed.

The initial draft time line which starts 1/7/2019 and completed on 12/9/2022 which is close to the 3.5 years the University now specifies for completion of a PhD.

Next step is to review the tasks and duration with Professor Crewther and Dr. Nina Riddell who are my supervisors.