On the functional organisation of the genome in mammalian nuclei

Imperial Users onl

Development and application of fluorescence lifetime imaging and super-resolution microscopy

This PhD thesis reports the development and application of fluorescence imaging technologies for studying biological processes on spatial scales below the diffraction limit. Two strategies were addressed: firstly fluorescence lifetime imaging (FLIM) to study molecular processes, e.g. using Förster resonance energy transfer (FRET) to read out protein interactions, and secondly direct imaging of nanostructure using super-resolution microscopy (SRM). For quantitative FRET readouts, the development and characterisation of an automated multiwell plate FLIM microscope for high content analysis (HCA) is described. Open source software was developed for the data acquisition and analysis, and approaches to improve the performance of time-gated imaging for FLIM were evaluated including different methods to despeckle the laser illumination and testing of an enhanced detector. This instrument was evaluated using standard fluorescent dye samples and cells expressing fluorescent protein-based FRET constructs. It was applied to an assay of live cells expressing a FRET biosensor and to FRET readouts of aggregation of a membrane receptor (DDR1) in fixed cells. A novel instrument, combining structured illumination microscopy (SIM) with FLIM, was developed to explore the combination of SRM and FLIM-FRET readouts. This enabled the simultaneous mapping of molecular readouts with FLIM and super-resolved imaging. The SIM+FLIM system was applied to image collagen-stimulated DDR1 aggregation in cells, to image DNA structures during the cell cycle and to explore interactions between cell organelles. A novel SRM approach based on a stimulated emission of depletion (STED) microscope incorporating a spatial light modulator (SLM) was developed to provide straightforward robust alignment with collinear excitation/depletion beams, aberration correction, an extended field of view and multiple beam scanning for faster STED image acquisition. The performance of easySLM-STED was evaluated by imaging bead samples, labelled vimentin in Vero cells and the synaptonemal complex in homologs of C. elegans germlines.Open Acces

Disentangling the 4D Nucleome

The dynamical relationship between 3D genome structure, genome function, and cellular phenotype is referred to as the 4D Nucleome (4DN). 4DN analysis remains difficult, since multiple data modalities must be integrated and comprehensively studied in order to obtain new insights. In my dissertation work, I present a computational toolbox which offers both novel and established methods to integrate and analyze time series genome structure and function data. I also provide an extension of the 4DN that captures the contributions of the maternal and paternal genomes. I uncover differences between the two genomes’ structural and functional features across the cell cycle, and reveal an allele-specific relationship between local genome structures and gene expression. In addition, I present a computational framework for analyzing multi-way genomic interactions which allow us to identify transcription clusters in the human genome. Finally, I introduce a computational method to characterize the differences between memory and plasma B cells in the adaptive immune system, which guide us to develop an immune system inspired learning system.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169908/1/lindsly_1.pd

Physical Mechanisms of DNA repair: A single molecule perspective

Wuite, G.J.L. [Promotor]Peterman, E.J.G. [Promotor

Maintenance of metaphase chromosome architecture by condensin I

"Faithful segregation of the genome into two daughter cells is one of the most fundamental events for every living organism. In each round of the cell cycle, cells need to orchestrate a sequence of complex steps to replicate their genetic material, pack it neatly into mitotic chromosomes and perform their precise separation when all the prerequisites are met. One of the most fascinating questions in biology is to understand the internal organization of mitotic chromosomes. Even though mitotic chromosomes were first described around 140 years ago, how exactly interphase DNA molecules are packed to become mitotic chromosomes is still a mystery. Despite the lack of precise details about chromosome condensation mechanisms, it is believed that in the heart of this process lies a group of protein complexes called condensins. The mechanism by which condensins are able to enforce or guide the condensation process is yet unknown. In this thesis, we will present our advances in understanding condensin’s function in maintaining mitotic chromosome compaction and internal architecture.(...)