|dc.description.abstract||The Earth’s oblique rotation results in changes in light and temperature across
the day and time of year. Living organisms evolved rhythmic behaviours to
anticipate these changes and execute appropriate responses at particular
times. The current paradigm for the biological clocks in several branches of life
is an underlying biochemical oscillator mainly composed by a network of
repressive transcription factors. The slow decay in their activity is fundamental
for generating anticipatory dynamics. Interestingly, these dynamics can be well
appreciated when the biological system is left under constant environmental
conditions, where oscillation of several physiological readouts persists with a
period close to 24 hours, hence the term “circadian clocks”, circa=around
In plants the model species Arabidopsis thaliana has served as an invaluable
tool for analysing the genetics, biochemical, developmental, and physiological
effects of the oscillator. Many of these experimental results have been
integrated in mechanistic and mathematical theories for the circadian
oscillator. These models predict the timing of gene expression and protein
presence in several genetic backgrounds and photoperiodic conditions.
The aim of this work is the introduction of a correct mass scale for both the
RNA transcript and protein variables of the clock models. The new mass scale
is first introduced using published RNA data in absolute units, from qRT-PCR.
This required reinterpreting several assumptions of an established clock model
(P2011), resulting in an updated version named U2017. I evaluate the
performance of the U2017 model in using data in absolute mass units, for the
first time for this clock system.
Introducing absolute units for the protein variables takes place by generating
hypothetical protein data from the existing qRT-PCR data and comparing a
data-driven model with western blot data from the literature. I explore the
consequences of these predicted protein numbers for the model’s dynamics.
The process required a meta-analysis of plant parameter values and genomic
information, to interpret the biological relevance of the updated protein
parameters. The predicted protein amounts justify, for example, the revised
treatment of the Evening Complex in the U2017 model, compared to P2011.
The difficulties of introducing absolute units for the protein components are
discussed and components for experimental quantification are proposed.
Validating the protein predictions required a new methodology for absolute
quantification. The methodology is based on translational fusions with a
luciferase reporter than has been little used in plants, NanoLUC. Firstly, the
characterisation of NanoLUC as a new circadian reporter was explored using the clock gene BOA. The results show that this new system is a robust,
sensitive and automatable approach for addressing quantitative biology
I selected five clock proteins CCA1, LHY, PRR7, TOC1 and LUX for absolute
quantification using the new NanoLUC methodology. Functionality of
translation fusions with NanoLUC was assessed by complementation
experiments. The closest complementing line for each gene was selected to
generate protein time series data. Absolute protein quantities were determined
by generation of calibration curves using a recombinant NanoLUC standard.
The developed methodology allows absolute quantification comparable to the
calibrated qRT-PCR data. These experimental results test the predicted
protein amounts and represent a technical resource to understand protein
dynamics of Arabidopsis’ circadian oscillator quantitatively.
The new experimental, meta-analysis and modelling results in absolute units
allows future researchers to incorporate further, quantitative biochemical data.||en