Waking up the Lion ....

 BioTek Lionheart

Ideally the Seahorse XFe-96 should be coupled with a Citation 1 or 5 to enable cell counts, microscopy and to automatically pass the cell count data across to the Seahorse to normalise the data. At a cost of around $150,000 it is beyond our budget at present.

An alternative that we can explore is a BioTek Lionheart which LTU already owns and is located in the lab next to the Binger Lab where I will be working.

The long road for ethics approval

I have completed my ethics application and have referenced the Western Health ethics application number provided to me from WH, which covers us taking Parkinson's blood and Sheila running various cognitive tests. However Western Health have resubmitted their ethics to add my PD blood requirements, OXPHOS, Proteomic and Sheila’s cognitive tests as well as the COVID research, so that we are all on the same updated Western Health ethics application. Unfortunately the WH application may not be submitted until January or later.

I am wondering what the down side would be if we submitted our ethics application without a copy of the Western Health updated ethics approval. Ideally if we submit by Nov 29th, the committee would review it and pass back any comments and requests for further information which we could fix whilst we wait for Western Health's updated ethics approval in Jan or Feb. This could save us at least one month as it is unlikely our ethics application will be approved first time round.

LIMS and beyond ...

Completed the LTU COVID-19 risk assessment (8th November), as part of the LIMS safety requirements for access to LIMS and the Binger laboratory.

Both the risk assessment and a request my access to LIMS from Katrina were sent off on Tuesday 9th November with initial comments from the Biochemistry Head of School being supportive. Katrina expects approval by early next week 16th-20th November.

Choosing the Most Relevant Normalization Method

For some time I have been trying to work out how to normalise my OXPHOS sample prior to doing an assay. Finally found an Agilent paper "Methods and strategies for normalizing XF metabolic data to cellular parameters" which shows the following schematic solving one of my problems ie if I do not have access to a Cytation unit to automatically stain, count and photograph the cells, what method should I use?

Methods and strategies for normalizing XF metabolic data to cellular parameters

It would seem that unless we can find a Cytation to use, teh methods I can use to normalise my assay data are :

1.  Protein measurement,
2.  gDNA measurement, or
3.  Manual counting after cell dissociation

Locating healthy control blood

There are a few a few problems that I face with respect to the sourcing, collection, preparation and storage of the blood samples, namely:

1. We can get the Parkinson's blood from Sunshine hospital neurology clinic, but we are yet to work out where we can get age matched blood from healthy controls. One idea is to ask their partners, 
2. Once we have the blood, what are the laboratory protocols we should use for optimally preparing the blood ie centrifugation. Too slow and the whole blood does not seperate into the buffy coat, whilst too fast will damage the cells, and,
3. Once we have isolated the leukocytes, what is the best way and what temperature should we store the remaining blood samples and the prepared leukocytes.

I made contact with the Melbourne Blood Bank today and had a series of phone conversations which I will summarise below:

• The blood bank can provide us with a sample of blood for us to test and work out the best parameters to prepare, isolate the buffy coat and then store the remaining blood and isolated leukocytes – the cost of this sample blood is free to authorised research projects
• If you are an authorised project you need a permit to receive the blood. In a conversation with Francesca Hulme (Senior Transfusion Scientist), she is sending my details to Andrew Morrissey who is a senior scientist in the blood banks research supply chain, as she thinks he can provide me with the details around how we might qualify for research blood samples for free and be able to send us the paper work to get the process of approval and a permit organised.
• The blood bank research group have a project running under the leadership of Dr Lacey Johnson (Principal Research Fellow in the Product Development Research Group) looking at methods to extend the shelf life of blood products. Francesca will speak to Dr Lacey about our blood needs with respect to how we should store our whole blood and our isolated buffy coat and leukocytes.
• I also spoke with Francesca about preparation methods i.e. centrifugation, and she said that she spins down whole blood to get the buffy coat and then they filter out the leukocytes to leave just the platelets, where as we want to filter out the platelets. I asked if we could get a copy of their protocol and she was going to see if this could be sent to us. Francesca also said that Michael Wheeler at Monash medical centre had done a bit of work in separating out leukocytes and he may be willing to talk to me about the best way to isolate the leukocytes. She said she would pass my details onto Michael and ask him to ring me.
• Finally, since the blood bank can provide us a healthy blood sample, I explained to Francesca that whilst we had made initial arrangements to acquire some Parkinson’s blood, we were still thinking about how we could get age matched healthy blood for the Parkinson’s age groups and the young 30-40 yrs age group for the biomarker study. Francesca said that if we are eligible for the sample and get a permit they can also supply us with sufficient blood samples to match all our healthy controls for free.

I have my fingers crossed that we will be seen as eligible to get the blood and for free.

XF Real-Time ATP Rate Assay well plate set up

Cellular metabolic regulation allows cells to adjust for changes in ATP demand with subsequent changes in ATP production to maintain total intracellular ATP levels. By using the Agilent Seahorse XF Real-Time ATP Rate Assay we can measure total ATP production rates in living cells and to distinguish between the fractions of ATP that are produced from mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, the two main metabolic pathways responsible for ATP production in mammalian cells. 

The XF Real-Time ATP Rate Assay can also be used to calculate the ideal cell seeding density. By creating four different seeding density sources ie 0.5X, 1.0X, 2.0X and 4.0X we can determine which level for our particular cells is best. Optimal cell seeding numbers vary widely, though are typically between 5 × 10³ – 4 × 10⁴ cells per well and must be determined empirically. (For example, for 1 x 10⁴ cells per well, resuspend cells 1 x 10⁴ per 80 μL = 1.25 x 10⁵ cells per mL)

To simultaneously test the ideal seeding level for both Parkinson's and healthy control cells the following well plate design is proposed.

PhD Confirmation ....

Presented my Confirmation paper and presentation on Tuesday 8th September, and it was well received with unanimous agreement to confer me into the PhD program.

How many XFe-96 cartridges ?

In the updated XFe-96 cartridge well plate set-up below, we can compare between the age groups and genders between healthy controls and Parkinson's patients for study 1, as well as running a duplicate on the same plate.

This offers the advantage of both initial experiment and confirmation run being done under the same experimental conditions. 

One of the issues yet to be understood is the expectations for the number of runs required due to the variability or accuracy of the XFe-96 results, ie how many technical references are needed for this type of experiment - is one duplicate sufficient or do we need to run another plate to increase the testing from two cases to four cases ?

The good news is that if we need further plates the costs seem to be affordable if we assume we can buy the extra cartridges from the Binger laboratory rather than a bulk order from Agilent

Study 4 - Early Bio-marker for Parkinson’s Disease ... An update

Out of each of the studies I have planned, trying to utilise the data and information that I have learnt from the other studies together in such a way that we may highlight particular patterns that are linked with early cognitive or motor declines that can be potentially used as an early bio-marker is the hardest to draw to a conclusion that seems to make sense.

My current thinking is that we can't simply analyse the proteomic results, find particular protein aberrations, then analyse a younger sample ie 30 year olds, to see if the same proteins are showing any form of change or impairment. The problem is that the sample may be taken randomly from a younger person who may never go on to develop any form of neurodegenerative disease.

So how to overcome this dead end issue. We know from other studies that mitochondrial disease is definitely linked to other diseases such as coronary, vascular disease, diabetes, schizophrenia etc. which also have a higher correlation of being linked as comorbid diseases with Parkinson's disease.

By the time someone presents with Parkinson's they have already lost around 80% of their dopaminergic neurons, and research has indicated that this damage has been occuring over a period of upto 15 years. So if we can we identify a younger sample from individuals already showing signs of these other comorbid diseases and take our samples from this cohort, then this may increase the probability that some of the cohort will go on to develop Parkinson's in the future. An alternative tho this would be to run a cognitive battery of tests over the younger cohort and use samples that show cognitive changes compared to the test norm. 

"another day, another demise ..."

 Another day, another deluge of depressing emails on the damage Covid-19 is wreaking on the University. It looks like there will be another round of forced retrenchments and firings to get the OPEX down to minimize the well publicised $200 million budget deficit. 

"it's a much more dire situation that it was two months ago ..."

Sheila Crewther - 14th August 2020

In a Zoom meeting with Sheila and Nina, they are concerned that the research dollars are rapidly drying up and that I have to cut my planned research budget by over 50% before Sheila will let the confirmation paper go forward. The cut is possible, but will result in no room for making mistakes or repeating an experiment because of errors in sample preparation or failures in equipment. 

The really big assumption is that the Binger laboratory which has the XFe-96 is also well stocked with all the consumables I need because we are budgeting for only the consumables I need and not the requirement to buy the bulk packages ourselves.

 

Thought it was just me ...

Recently I came across this article in "The RED Alert" put out by the graduate research office at LaTrobe. 

"the burden of feeling like an imposter ..."

"Many of us in our group have experienced ‘imposter syndrome’. As with many other syndromes, graduate researcher imposter syndrome is a complex set of psychological feelings that in this case is hidden by the PhD cloak of feigned confidence. We pretend to know what we’re doing, what we’re saying, what we’re studying, while at the same time being fearful of difficult, wicked questions that our supervisors and progress committee may put to us. This uncertainty and insecurity is further compounded by a sense of ‘aloneness’, a feeling that we are travelling with others on parallel rather than intersecting paths. While our paths may appear to cross, there are very few people to talk to about our passion, our problems, hopes and anxieties. Instead, the anxiety continues to lurk, and each day we step onto another lonely path of head in PC in the hope that our individual journey will be worth it in the end and that in the meantime we are not found out ..."

And there I was, waiting for the Graduate Research Committee to quietly tap me on the shoulder and say. "Shane, I think it's time we had a little chat ..."

Study 4 - Early Bio-marker for Parkinson’s Disease ...

The final objective is to review the evidence from each of the five experimental studies to investigate the efficacy of using leukocytes to sense early deviations in bioenergetic levels and changes in metabolic stress associated with mitochondrial dysfunction. This capability would make leukocytes a potentially sensitive early biomarker for therapeutic and clinical intervention in neurodegenerative progression of PD (Braganza, Annarapu, & Shiva, 2020; Kramer, Ravi, Chacko, Johnson, & Darley-Usmar, 2014).

Statistical Analysis of Leukocyte Early Bio-marker

Correlation analysis will be used to review OCR and ECAR levels between PD and HC to establish high level relationships for any bioenergetic dysfunctions/impairment. Any changes detected will be tested for significance using between and within sample student t testing. Where patterns of significant change have been observed, multivariate analysis such as hierarchical and stepwise multiple regression will be used to factor out confounding patient data (i.e. BMI, weight etc.) and to investigate the veracity and accuracy of using age related mitochondrial changes in PD patient leukocytes as a predictor of predisposition for early development of PD or for increasing changes in the progression of existing PD symptomatology.

Study 3 - Proteomic Differential Pathway and Abundance Analysis ...

Changes in Protein Abundance by Age and Gender

Study 3 will support the signalling pathway analysis from study 2, by using a proteomic analysis of leukocytes across age and gender matched groups, to investigate differences in HC and PD participant protein abundance and gene expression along a specific signalling pathway.
Abnormal Protein Expression and Neurodegenerative Progression

A key manifestation of neurodegenerative progression in PD pathogenesis is the abnormal expression and aggregation of proteins through transcriptional and translational responses to biological perturbations, resulting in post-translational modifications (Wetie, Woods, & Darie, 2014), altered protein–protein interactions, protein degradation and functional changes to the proteome (Dixit, Mehta, & Singh, 2019; Li, Ganz, & Smit, 2019), in disease progression (Rubinsztein, 2006). Impaired protein expression has been consistently associated with cellular damage, mitochondrial bioenergetic dysfunction, metabolic impairment, increased oxidative stress, elevated proinflammatory responses and increased apoptosis linked to neurodegenerative progression in PD (Dixit et al., 2019; Jiang et al., 2019; Kitsou et al., 2008; Licker, Kovari, Hochstrasser, & Burkhard, 2009). Identification of specific protein changes along an impaired bioenergetic signalling pathway offers the possibility of developing targeted pharmacological and clinical solutions to slowing and treating PD.

Proposed Procedures

Identification of changes in protein expression and abundance will use liquid chromatography (LC) to fractionate blood samples into similar protein groups (Zhang et al., 2012) then mass spectrometry (MS) will fragment each of these long chain proteins into smaller peptides (Chin et al., 2008; Zhang et al., 2012). The molecular weight of the proteins present in the sample will then be calculated and compared against pre-established databases for identification. Microarray/RNA-Sequencing will be used to compare changes in specific leukocyte derived pathway mean differential gene expression levels between HC and PD participants (Ihnatova & Budinska, 2015). Microarray/RNA-Seq data will be analysed using an R statistic routine within Bioconductor v3.2 software.

Proteomic Analysis Workflow. The workflow diagram shows PD and HC blood samples are collected and initially lysed to release cellular content into the cytosol. The sample is then centrifuged with the resulting supernatant submitted to a liquid chromatograph where the proteins are ionised and fractionated into similar protein groups. These protein groups are then passed through a mass spectrometer which fragments the long chain proteins into smaller protein peptides to enable the calculation of the molecular weight of each peptide. Software then compares the calculated molecular weight to a predefined protein database to identify the proteins present in the sample. Adapted from Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies, by Philipp E Geyer. et.al., 2019. EMBO Molecular Medicine, 11(11), e10427. doi:10.15252/emmm.201910427.
Proteomic Differential Pathway Analysis

Differential protein comparisons between PD and HC participants by molecular weight (Zhang et al., 2012), and along mitochondria signalling pathways identified as impaired should reveal alterations in PD protein expression and abundance indicating the potential location of the mechanisms driving mitochondrial and protein impairment in PD. Understanding the changes in gene expression, protein alteration and the mitochondria mechanisms that are impaired will offer significant opportunities to target new clinical and pharmacological solutions in treating PD.


Proteomic Differential Pathway Analysis. Figure simulates an anticipated result for a proteomic differential pathway analysis comparing gene expressions between Parkinson disease participants and healthy controls along a specified pathway. (A) Differentially expressed OXPHOS protein heat map, (B) Protein-protein interaction analysis of the significantly differentially expressed proteins, and (c) Network map of impaired mitochondrial proteins. Adapted from A dose-dependent perturbation in cardiac energy metabolism is linked to radiation-induced ischemic heart disease in Mayak nuclear workers, by Azimzadeh, O., et.al. (2017), Oncotarget, 8(6), 9067-9078.

Understanding Membrane Permeabilization ...

My initial understanding of how permeabilization worked was that when the permeabilizing agent of recombinant cytolysin protein was injected to create large pores in the leukocytes outer membrane, we would see a normal OXPHOS blueprint from each of the working complexes and a flatline for the impaired complex.

However on reflection, once the leukocytes outer membrane was permeabilized and the cells contents leaked out into the cytosol, there would be no further substrates of any type left in the cell to fuel any of the enzyme complexes CI-CIV.

What I now expect to see is that once the leukocytes membrane is permeabilized, and a specific substrate is injected, the only enzyme complex that will be activated to process the substrate and produce ATP, will be the complex that can oxidise the substrate injected into the cell.

As an example, by selectively injecting succinate we can test complex II’s ability to produce ATP.  Any increase or change in OCR will indicate production of ATP indicating that Complex II is working normally. However if there is no OCR consumption, then Complex II has been identified as impaired suggesting that CII, may be the cause of the PD mitochondrial bioenergetic impairment. This then gives us a target for more specific clinical and pharmaceutical investigation.

Study 2 - Impaired Bioenergetic Signalling Pathways ...

Having identified bioenergetic impairment in PD patients, study 2 will analyse how the mitochondria signalling pathways involved in energy production are dysfunctional. Different substrates are oxidised by different enzyme complexes along metabolic pathways to generate ATP. 

Permeabilizing the leukocyte plasma membrane causing large pores on the outer membrane surface, allows the cell contents to leak out into the cytoplasm. It is then possible to selectively control the type, amount and timing of particular substrates delivered into the cell for oxidation by the mitochondria during OXPHOS and ATP production. 

Study 1 - Bioenergetic changes ...

The first study will look at analysing changes in bioenergetic levels between Parkinson disease participants (PD)and healthy controls (HC) across differing age groups and by gender.

Initially in this study the levels of mitochondrial oxidative respiration will be measure using a Seahorse XFe-96 to simultaneously measure the oxygen consumption rate (OCR) and changes in glycosylation via the Extracellular Acidification Rate (ECAR).

Both oxygen and glucose are necessary in the production of ATP, so by measuring the OCR & ECAR we can estimate the ATP level of production. By measuring OCR & ECAR for healthy individual over the two age groups (40-50 & 60-70 yrs.), we can set up a benchmark to identify how mitochondrial bioenergetic levels change as individuals age.

These levels can be then used as a benchmark for comparison to the same measures in Parkinson disease participants as they age and also between gender.

Research Studies slowly coming together ...

Overall the focus of my PhD research will be on changes in mitochondrial bioenergetics and how the lack of available energy in the form of adenosine triophosphate (ATP) is linked to a wide variety of disease pathologies. 

Specific Studies : 

1. Impact of mitochondrial bioenergetic dysfunction/failure on aged Parkinson’s Disease patients
2. Identify impaired bioenergetic signalling pathways in PD 
3. Proteomic analysis of specific pathway changes in protein abundance
4. Investigate the viability of Leukocytes as a bio-marker of Parkinson’s Disease

Confirmation Presentation ...

Beginning work on my presentation. The talk will need to be around 25 minutes and cover most aspects of my research methods, expectations, potential issues and overall project timing. Like every talk you have to put together, you end up with more information than you are allowed to present. I am only up to study 1 and already I have 25 slides.

I think the way to proceed is to get all the information I want for each study and then at the end trim the number back by deleting and combining information. 

Laura Bassi Scholarship

Drafting up an application for the Laura Bassi Scholarship which was established by Editing Press in 2018 with the aim of providing editorial assistance to postgraduates and junior academics whose research focuses on neglected topics of study, broadly construed, within their disciplines. 

The scholarships are open to every discipline and are awarded three times per year: December, April, and August. The value of the scholarships are remitted through editorial assistance for a PhD at $2,500.

I do not think there is any cash involved, just the equivalent dollar value in the editing press assistance with getting my PhD thesis professionally edited.

Update - 11th July: Turns out both Sheila & Philippe had misread the application, thinking it was for $24,000 pa paid out as $8,000 three times a year. When I contacted them to discuss, the decision was made to not apply at this time as we cannot see any value in getting Editing Press involved in reviewing each of my articles that will constitute each of the key PhD chapters.

How many patients are significant ?

In order to calculate an effective sample size that will yield a significant result I have set up Gpower using the Linear Multiple Regression: Fixed model single regression co-efficient with the following parameters:

1. Confidence interval of 95%
2. Critical value of 0.05%
3. Power of the test at 80%, and 
4. Large Effect size of 0.80
5. Number of predictor variables set at 1

The analysis results in a proposed sample size of 10 which seems close to the 10 sample we are expecting to get access to from the hospital.

Proteomic Protein Targeting ....

Yesterday (1st July 2020), I had a meeting with Rohan Lowe over in Proteomics to outline the planned OXPHOS experiments and to discuss how study 1 looks to confirm mitochondrial impairment and study 2 attempts to tracks each of the ETC complexes to identify the signalling pathway associated with the source of the impairment. From this initial outline we then talked about the practicalities of using leukocytes, setting up protocols and the types of proteomic methods that might be appropriate ie label free, protein mapping, depletion etc.

Whilst it was a short high level discussion three interesting concepts that came out of the discussion:

1. Rohan commented that our Seahorse experiments and data will generate a mass of information to help them with the design of our experiments and that if all goes according to plan the inter-related nature of studies 1 & 2 will be a great source of meta data that will be very useful in defining the proteomic experiments.
   
   
2. When we discussed protein mapping, what seem to offer the greatest opportunity (at least at this early stage), was if we know from study 2 which pathway is impaired i.e. the use of pyruvate reveals poor OXPHOS indicating the TCA cycle and Complex I are the potential source of the impairment, then we can look to identify specific proteins involved in that process and investigate their abundance. In a similar way, if we look at another neurodegenerative disease such as Alzheimer’s or Huntington’s and find proteomic analysis has identified a specific protein as a problem, then an alternative for us is to bring the study 2 pathway analysis together with the other neurodegenerative proteomic analysis and ask if that problem protein in Alzheimer’s is also a problem in PD and is it on our identified impaired pathway.
   
   
3. We discussed how a sample run might work and he suggested we meet with them early ie when we are lining up the samples and before we begin the Seahorse experiments to ensure we can define a collection protocol that meets the quality criteria for preparing the OXPHOX and proteomic samples at exactly the same time so the meta data will be comparable.
   

I think I need to begin looking at proteomic research on neurodegenerative disease where specific proteins have been identified as a problem i.e. missing, low abundance, mutated etc. so that we can see if asking the question “ if it is a problem in that disease, is it also a problem in PD ?”

How are we going so far ....

I was reflection on how we are going as a PhD "team" and whilst I am enjoying the research and writing the papers, I cannot help lamenting the impact Corona is taking on all those casual conversations I am missing, where parts of ideas are exchanged and discussed to form even better leads to follow up. 

Sheila has such an enormous wealth of knowledge, that I know it may seem selfish, but sometimes I feel I am really missing out on learning something new and unexpected. So I am looking forward to the end of Corona and getting back to normal so we can get those discussion and ideas flowing.

Designing and researching the confirmation paper was interesting but I feel it would have been a significantly more enriching experience if there had of been lots of conversation along the way.

 Just a thought.

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.