- Circadian rhythms refer to biological cycles that occur over approximately 24 hours and regulate functions like sleep and cell metabolism. This circadian clock is present even without light and involves phosphorylation of proteins. - Phosphorylation is a post-translational modification that can activate, deactivate, or modify protein function by adding a phosphate group. It plays a key role in the circadian clock mechanism. - The core circadian clock mechanism involves a transcription-translation feedback loop between a positive arm comprising WC-1/WC-2 proteins and a negative arm comprising FRQ/FRH proteins that is conserved between fungi and animals.

2. https://www.rd.com/wp-content/uploads/2016/03/01-9-ways-to-become-a-morning-person-stretch.jpg

3. https://bento.cdn.pbs.org/hostedbento-prod/blog/20170323_201718_558049_angry-bat-1248391.jpeg

4. • Circadian rhythms refer to cycles that revolve
around our days (~24 hours)
• This circadian clock regulates many biological
functions – from sleep to cellular metabolism
• This circadian clock is present even in the
absence of light, showing the importance in
regulation of cell function in our days
Artwork by Emma Darvick - https://giphy.com/gifs/illustration-sunset-sunrise-3o6ZsWSWIkZYHLHTyM

5. • Phosphorylation is a reversible, post-
translational method of altering proteins
• Phosphorylation occurs from the binding of a
phosphate group to an amino acid
• These modifications can cause proteins to be
activated, deactivated, or even modify their
function in the cell
Gla086 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]
Serine
Phosphorylated Serine

6. • The biological makeup of the ‘core clock’ is
conserved between fungi and animals
• There is a transcription-translation negative
feedback loop between a heterodimer complex
that can be defined as a positive arm and a
negative arm
• The positive arm influences genes unrelated to
this core clock mechanism – these genes are
dubbed clock controlled genes (ccgs)
• The positive arm is comprised of WC-1/WC-2
proteins
• The negative arm is comprised of FRQ/FRH
proteins

8. • The many small sources of variation from non-
biological sources, e.g. different reagents and
handlers, are known as batch effects
• Batch effects likely influence all experiments
but are of greater effect in high-throughput
analyses like proteomics
• LIMBR, a python package designed for
circadian proteomics data, was used to
control for batch effects

9. • A positive value of γ indicates damping, or
decreasing amplitude over time
• A negative value of γ indicates forcing or
increasing amplitude over time
• When γ = 0, the data is considered in truly
harmonic oscillation
Pattern Legend
A = damped
B = forced
C = harmonic
D = overexpressed
E = repressed Hannah De los Santos et. al, ECHO: an Application for Detection and Analysis of Oscillators Identifies Metabolic Regulation
on Genome-Wide Circadian Output, Bioinformatics, , btz617, https://doi.org/10.1093/bioinformatics/btz617

10. Phosphopeptide Expression over 48 Hours
– All ECHO data
• Expressions are sorted from top to bottom by
phase and normalized to be within the range
[-1,1]
• Each column represents a 2-hour interval
• Our oscillatory data can be classified into
three groups by their amplitude change
coefficient (γ)
• Total ECHO expressions: 255

11. Phosphopeptide Expression over 48 Hours
– Harmonic
• Harmonic classified data (−0.03 ≤ γ ≤0.03)
ebbs and flows consistently over time
• Total Harmonic expressions: 70

12. Phosphopeptide Expression over 48 Hours
– Forced
• Forced classified data (−0.15 ≤ γ ≤ −0.03)
increases in amplitude over time
• Total Forced expressions: 79

13. Phosphopeptide Expression over 48 Hours
– Damped
• Damped classified data (0.15 ≤ γ ≤ 0.03)
increases in amplitude over time
• Total Damped expressions: 55

14. • Investigate the ECHO data for
regulatory proteins and any relations
to the core clock function
• FRQ protein was found in the ECHO
data and classified as overexpressed
• Identify specific phosphorylated
amino acids that may be key in
circadian regulation
- FRQ