Thesis The Chronic Fatigue Syndrome, mitochondrial dysfunction and monocyte differentiation, 2025, Brinksma

[Dolphin]

The Chronic Fatigue Syndrome, Mitochondrial Dysfunction and Monocyte Differentiation

thesis.unipd.it

https://thesis.unipd.it/retrieve/10e77794-1684-4d87-b51a-5a4f4d547f9c/Brinksma_Esther.pdf.pdf

UNIVERSITÀ DEGLI STUDI DI PADOVA
DEPARTMENT OF BIOMEDICAL SCIENCE
Interuniversity Bachelor’s degree programme in Biology of Human and Environmental Health Final dissertation

The Chronic Fatigue Syndrome, Mitochondrial Dysfunction and Monocyte Differentiation

Supervisor Professor Carlo Viscomi Candidate: Esther Lilian Brinksma Student ID number: 2091238

Abstract

Abstract​

The Chronic Fatigue Syndrome (CFS), also known as Myalgic Encephalomyelitis(ME), is a complex, multisystem disorder characterized by persistent fatigue and a wide range of symptoms as well as post-exertional malaise(PEM).

CFS is observed in an estimated 50% of Long COVID patients.

While the cause of CFS remains unclear, it is hypothesized to develop after viral infections such as EBV.

Mitochondrial dysfunction and immune dysregulation are strongly implicated. In this study, we investigated mitochondrial dysfunction and monocyte differentiation in CFS.

For the mitochondrial research we used cultured HUVECs treated with patient-derived serum, healthy serum, and untreated media controls, with and without the addition of K21, all cultured in triplicate.

Mitochondrial morphology was assessed via fluorescence microscopy using GFP staining to visualize the mitochondria, while protein expression (p53, MFN1, DRP1 and LONP1) was analyzed by Western blots. Microscopy revealed mitochondrial fragmentation in CFS-treated cultures.

Western blot analysis showed significant differences in mitochondrial protein expression comparing CFS treated samples to healthy controls, however not all the data is consistent with visual observations.

This suggests protein concentrations and therefore mitochondrial func tioning are affected but more research is required.

To assess immune dysfunction, we cultured monocytes, treated with patient and healthy serum and controls with untreated media.

Monocyte differentiation was evaluated using ELISA to detect markers of M1 (IL-12) and M2 (IL-10) macrophages.

Results showed high intra-patient variability, suggesting the need for additional replicates and patient samples to reach statistical significance.

These findings support the hypothesis of mitochondrial impairment in CFS and provide a basis for future references into monocyte polarization as a contributing factor to chronic inflammation in this condition.

 

[DMissa]

Seems like this was a project as part of a bachelor's degree but we will need better evidence of serum effects on mitochondrial morphology than individual images. Are Audrey and Charlie still doing the replication attempt on this? (Prusty's work or whatever it was) @chillier ?

My gut is that I am not convinced the mitochondria are the interesting thing in terms of potential serum factors. If there is an effect of some mysterious thing in serum any effects on mitochondria can come from any number of signalling pathways that change much of how the cell operates. Plus Audrey and Charlie's myoblast work shows that there is not a universal mechanism at play relating to effects of serum specifically on mitochondria.

We really need to move towards thinking about specific sequences of events involving known or plausible signalling relationships in discrete cells / tissues of interest.

 

[Hutan]

Daniel, were you able to access the pdf? I'm not managing to yet, not sure if the problem is me.

Audrey and Charlie @chillier did some Seahorse studies of mitochondrial function after exposure to ME/CFS serum but didn't look at mitochondrial morphology. I don't think they were planning to do more studies on 'something in the blood', but perhaps they were.

Was there blinding and/or automation in the analysis of mitochondrial morphology in this study?

What is K21 (something that was added to some samples)?

I don't think we have seen good evidence yet for mitochondrial fragmentation in cells direct from people with ME/CFS.

 

[DMissa]

Daniel, were you able to access the pdf?

yep

Audrey and Charlie @chillier did some Seahorse studies of mitochondrial function after exposure to ME/CFS serum but didn't look at mitochondrial morphology. I don't think they were planning to do more studies on 'something in the blood', but perhaps they were.

I remember reading an article saying they planned to look at morphology as well

Was there blinding and/or automation in the analysis of mitochondrial morphology in this study?

I didn't see anything about blinding but the only results were a few images, nothing quantitative. Small sample, clearly done in not a lot of time by an undergraduate student (understandably, probably with no budget and severe time constraints and little technical experience). Nothing to run away with.

What is K21 (something that was added to some samples)?

broad spectrum drug that also induces mitophagy used as a sort of control condition

I don't think we have seen good evidence yet for mitochondrial fragmentation in cells direct from people with ME/CFS.

Yeah I'm not aware of any. I mentioned the Prusty paper because I vaguely remember something about it in there and that in the article I'm remembering that Audrey and Charlie were going to try to replicate it, but I've never been able to completely understand or remember Prusty's papers so yes I am not aware of any clear evidence in favour.