The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.

The self-assembly of structured particles into monodisperse clusters is a challenge on the nano-, micro- and even macro-scale. While biological systems are able to self-assemble with comparative ease, many aspects of this self-assembly are not fully understood. In this thesis, we look at the strategies and rules that can be applied to encourage the formation of monodisperse clusters. Though much of the inspiration is biological in nature, the simulations use a simple minimal patchy particle model and are thus applicable to a wide range of systems. The topics that this thesis addresses include: Encapsulation: We show how clusters can be used to encapsulate objects and demonstrate that such `templates' can be used to control the assembly mechanisms and enhance the formation of more complex objects. Hierarchical self-assembly: We investigate the use of hierarchical mechanisms in enhancing the formation of clusters. We find that, while we are able to extend the ranges where we see successful assembly by using a hierarchical assembly pathway, it does not straightforwardly provide a route to enhance the complexity of structures that can be formed. Pore formation: We use our simple model to investigate a particular biological example, namely the self-assembly and formation of heptameric alpha-haemolysin pores, and show that pore insertion is key to rationalising experimental results on this system. Phase re-entrance: We look at the computation of equilibrium phase diagrams for self-assembling systems, particularly focusing on the possible presence of an unusual liquid-vapour phase re-entrance that has been suggested by dynamical simulations, using a variety of techniques.

Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.

GATA1 and KLF1 are transcription factors that regulate genes which are important for the development of erythroid cells. The GATA1 transcriptional co-factor FOG1 has been shown to be essential in a wide range of GATA1 dependent cellular functions. Here we tried to understand the diverse mechanisms by which GATA1 and KLF1 recognize their binding sites, how the GATA1 recognition mechanisms are affected by complexation with either FOG1 or KLF1 and how the GATA1 recognition mechanisms affect the transcriptional regulation of the erythroid differentiation. We profiled the DNA binding specificities/affinities of a GATA1 fragment (mGATA1NC), that contains only the two DNA binding domains (N-terminal and C-terminal Zn finger), and the DNA binding specificities/affinities of a KLF1 fragment (mKLF1257-358), that contains the three DNA binding domains, using a novel methodology that combines EMSA with high throughput sequencing (EMSA-seq (Wong et al., 2011a)). We also profiled the DNA binding specificities of the C-terminal Zn finger of GATA1 alone (mGATA1C), the wt-mGATA1, the wt-mGATA1/wt-mFOG1 complex and the mGATA1NC/mKLF1257-358 complex. At first, we confirmed that the N-terminal Zn finger of GATA1 has a strong preference for the “GATC” motif, whereas the C-terminal Zn finger of GATA1 has a strong preference for the “GATA” motif. Next, we found that in the mGATA1NC, both DNA binding domains can bind simultaneously a wide range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. The wt-mGATA1 did not show the ability to bind in the same co-occurring motifs implying an effect of the non-DNA binding domains of the protein in the regulation of its DNA binding specificities. On the contrary, complexation of wt-mGATA1 with the wt-mFOG1 partially restored its ability to bind in a now limited range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. Similar observations were made for other pairs of GATA1 N-terminal and C-terminal Zn finger specific motifs. We then projected the GATA1 DNA binding specificities/affinities in vivo and we classified the GATA1 ChIP-seq peaks in low, medium or high affinity based on the number of the GATA1 motifs. We noticed that high affinity GATA1 ChIP-seq peaks tend to appear in regions with low nucleosome occupancy. We also noticed that GATA1 ChIP-seq peaks in the enhancer regions are usually high affinity whereas GATA1 ChIP-seq peaks in the proximal promoter regions are usually low affinity. Additionally, we observed that high affinity GATA1 ChIP-seq peaks are usually found in regions with increased levels of H3K4me2 and are associated with a higher decrease in the H3K4me3 levels on the TSS of the nearby genes. None of these GATA1 related in vivo observations were found for the KLF1 ChIP-seq positions. These findings significantly advance our understanding of the DNA binding properties of GATA1, KLF1 and their complexes and give an insight on the importance of the GATA1 DNA binding affinities in the regulation of the erythroid transcriptional program. Overall the work establishes an experimental and analytical framework to investigate how transcriptional co-factors can change the DNA binding specificities of specific transcription factors and how integration of the transcription factor DNA binding affinities with in vivo data can give novel insights into the transcriptional regulation.

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

In addition to a central circadian clock in the suprachiasmatic nucleus (SCN), zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These peripheral tissues express a complement of clock genes that can be synchronized with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only opsin that has been proposed as a candidate to mediate this cellular photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals. Phylogenetic analysis demonstrated that this gene family consists of three main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two paralogues in the zebrafish genome. Their predicted amino acid sequences contain most of the characteristic features for the function of a photopigment opsin, as well as seven transmembrane segments indicative of a G protein coupled receptor (GPCR) superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR) reveals that the tmt opsin genes in zebrafish are both temporally and spatially regulated. To investigate if these tmt photopigments mediate light-activated currents in cells, each opsin was expressed in vitro and the responses characterised by calcium imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric analysis, and bioluminescence reporter assay. Collectively, these data suggest that some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly, the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10, the potential spectral tuning sites involved in UV detection were tested. As part of this study, tmt opsin cDNAs were isolated from three populations of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed for a direct comparison between the tmt opsins present in the dark adapted species (cavefish) versus those of the light adapted species (zebrafish). It is hoped that the findings from this project will contribute to our understanding of non-visual light detection in fish and the evolution of their non-image forming photoreception.

Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.

Norrin is a secreted cystine-knot growth factor that plays critical roles in vascular development in the brain, retina, and cochlea, as well as the uterus. Although Norrin is unrelated to the lipid-modified morphogens Wnts, Norrin activates the canonical Wnt/β-catenin pathway by binding to receptor Frizzled4 cysteine-rich domain (Fz4-CRD) and co-receptors of low density lipoprotein receptor related protein 5/6 ectodomain (Lrp5/6-ECD) in conjunction with Tetraspanin-12 (Tspan-12). Like Wnts, Norrin has limited extracellular diffusion properties as a result of associating with heparan sulfate proteoglycans (HSPGs). Mutations lead to inherited disordered retinal vascularization diseases such as Norrie disease, familial exudative vitreoretinopathy and coats' disease. However, the molecular mechanism of how Norrin initiates signalling by engagement with Fz4, Lrp5/6, and HSPGs has remained unresolved. Here, novel strategies for protein production of recombinant human Norrin and Fz4-CRD as well as the complex are developed. The crystal structures of Norrin and its complex with Fz4-CRD, plus complex bound with the heparin mimic sucrose octasulphate, and unliganded structures of Fz4-CRD are presented. These structural data together with biophysical and cellular assays not only reveal the Fz4 and Lrp5/6 binding sites on distinct patches of the Norrin surface, but also indicate the HSPGs binding site on Norrin and Fz4-CRD as well as providing a framework to explain numerous disease-related mutations. Structural comparison with Xenopus Wnt8 in complex with mouse Fz8-CRD provides molecular insights for our understanding of ligand-receptor binding specificity and promiscuity, which has important implications for developing therapeutic strategies against Norrin dependent retinal disorders, and cancers caused by abnormal Wnt signaling.

Magnetic fields influence the rate and/or yield of chemical reactions that proceed via spin correlated radical pair intermediates. The field of spin chemistry centres around the study of such magnetic field effects (MFEs). This thesis is particularly concerned with the effects of the weak magnetic fields B₀ ~ 1mT relevant in the ongoing debates on the mechanism by which animals sense the geomagnetic field and on the putative health effects of environmental electromagnetic fields. Relatively few previous studies have dealt with such weak magnetic fields. This thesis presents several new theoretical tools and applies them to interpret experimental measurements. Chapter 1 surveys the development and theory of spin chemistry. Chapter 2 introduces the use of Tikhonov and Maximum Entropy Regularisation methods as a new means of analysing MARY field effect data. These are applied to recover details of the diffusive motion of reacting pyrene and N,N-dimethylaniline radicals. Chapter 3 gives a fresh derivation and appraisal of an approximate, semiclassical approach to MFEs. Monte Carlo calculations allow the elucidation of several "rules of thumb" for interpreting MFE data. Chapter 4 discusses recent optically-detected zero-field EPR measurements, adapting the gamma-COMPUTE algorithm from solid state NMR for their interpretation. Chapter 5 explores the role of RF polarisation in producing MFEs. The breakdown in weak fields of the familiar rotating frame approximation is analysed. Chapter 6 reviews current knowledge and landmark experiments in the area of animal magnetoreception. The origins of the sensitivity of European robins Erithacus rubecula to the Earth’s magnetic field are given particular attention. In Chapter 7, Schulten and Ritz’s hypothesis that avian magnetoreception is founded on a radical pair mechanism (RPM) reaction is appraised through calculations in model systems. Chapter 8 introduces quantitative methods of analysing anisotropic magnetic field effects using spherical harmonics. Chapter 9 considers recent observations that European robins may sometimes be disoriented by minuscule RF fields. These are shown to be consistent with magnetoreception via a radical pair with no (effective) magnetic nuclei in one of the radicals.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.