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Abstract:

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.Comment: No Comment

Abstract:

Diseases caused by viruses represent a serious global health concern. In this thesis, I present novel methods for labelling, imaging and analysing virus particles. I also describe mathematical and computational models that can be used to describe biological structures, such as viruses. In studies of viral structure, pleomorphism, and articularly filamentous morphologies, are frequently overlooked.To characterise and better understand viral filaments, I developed and optimised an imaging protocol for individual virus particles that captured the heterogeneity of viral shape. I also produced analysis pipelines that took this image data to study the structure of viruses at the individual virion level in order to find an overall picture of the populations of virions that exist. These pipelines investigated virus size, patterning of surface proteins and the location of viral genome. I also demonstrated a novel virus labelling strategy that specifically labels virus that will aid in the future study of viral assembly and infection. I also used mathematical modelling to further understand the role of viral morphologies. I modelled virus particles as a pressure vessel to study engineering stress in relation to virus, bacteria, and cellular morphology. Together these advancements will further our ability to study viruses and our knowledge of virus structure

Abstract:

Bacterial transcription by RNA polymerase (RNAP) is spatially organized. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich medium, with several studies linking RNAP clustering and transcription of rRNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons (Δrrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting five or six of the seven chromosomal rrn operons. Spatial analysis in Δrrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at cell endcaps, where the remaining rrn operons reside. These results support a model where RNAPs in Δrrn strains relocate to copies of the remaining rrn operons. In rich medium, Δrrn strains redistribute RNAP to minimize growth defects due to rrn deletions, with very high RNAP densities on rrn genes leading to genomic instability. Our study links RNAP clusters and rrn transcription, and offers insight into how bacteria maintain growth in the presence of only 1–2 rrn operons.