| dc.description.abstract | Eukaryotic cell growth, maintenance and differentiation relies on the dynamic structure of nuclear chromatin, the macromolecular complex consisting primarily of DNA and histones. Changes to chromatin structure and chemistry may lead to alterations in gene expression, resulting in functional and developmental processes in cells. Additionally, biomechanical properties of the nucleus, which play a role in mechanical signalling pathways, are also affected by chromatin conformation. The regulation and effects of chromatin dynamics in cellular processes have yet to be fully elucidated. Therefore, novel techniques for assessing chemical and mechanical signatures of cells undergoing chromatin changes during cell differentiation at the single cell level have great potential for 1) phenotypic characterisation of single cells for research and clinical purposes and 2) further unravelling the complex coordination of intracellular changes that occur during cell developmental steps and triggering of disease. In this thesis, I have studied chromatin remodelling in immune cells using vibrational spectroscopy and microfluidics. Single cell measurements were conducted through optimisations of experimental and data analysis parameters. Vibrational spectroscopy methods included FTIR spectroscopic imaging and Raman microscopy, both label-free techniques that measure the interaction of light with chemical properties of a sample by interrogating its molecular vibrations. Microfluidics is a technique for manipulating fluids at a submillimetre scale. It was utilised here to enable live cell Raman mapping, as well as for deformability cytometry for assessing mechanical properties of the cell nuclei. To initiate an immune activation, B cells were incubated with a cytokine (CIT) cocktail. The biomechanical property, nuclear auxeticity, was investigated in B cells using deformability cytometry. This property has previously been shown in transitioning embryonic stem (ES) cells, and chromatin decondensation has been determined to hold a regulatory role. Chromatin decondensation was therefore induced in B cells through immune activation (CIT treatment) or Trichostatin A (TSA) treatment. These cells were compared to untreated control cells. A subset of cells for both the CIT and TSA treatment, had auxetic nuclei. No control cells had auxetic nuclei. These results showed nuclear auxeticity in B cells for the first time, and linked it to chromatin decondensation in agreement with previous ES cell data. Using FTIR spectroscopic imaging and Raman microscopy, spectral features associated with chromatin and DNA changes during immune B cell activation were identified. Peak ratios for distinguishing between non-activated and activated immune cells were determined - for FTIR imaging: a DNA-to-protein peak ratio, and for Raman mapping: a peak ratio between two neighbouring peaks, both associated with nucleic acid. Both peak ratios measured the relative change in a peak associated with νs(PO2-), which was therefore shown to be a potential spectral marker for label-free characterisation of immune cells pre- and post activation. The biological origin of the FTIR spectral features was further investigated using additional cell treatments. Chromatin decondensation, intiated through CIT or TSA treatment, gave rise to similar change in the DNA-to-protein peak ratio. This supported the hypothesis that the νs(PO2-) spectral changes can be used to monitor structural changes occurring in chromatin and DNA itself. Finally, the key biological pathways influencing the whole range of Raman spectral differences between non-activated and activated B cells were investigated. Partial least squares (PLS) regression was performed on Raman maps and transcriptomic data. It was determined that a linear correlation exists between the two data types. Transcripts of high importance for this correlation were identified. These included the B cell receptor genes and a number of transcripts of regulatory proteins with known roles in immune activation. Transcripts not previously linked to immune activation were also identified. In summary, novel techniques for phenotypic characterisation of single cells were explored using both chemical and mechanical measurements of B cells undergoing immune activation. Previously unidentified biochemical and biomechanical factors influencing B cell activation were identified. These have added new layers to our understanding of this process and thus revealed potential new research directions. Furthermore, chromatin decondensation and transcriptional changes are key responses during all cell differentiation processes and disease development. Therefore, these experimental approaches have great potential for investigating other cell types and cellular processes. | en_GB |
