In human monocyte-derived macrophages (MDM), the human immunodeficiency virus type 1 (HIV-1) assembles on deeply invaginated domains of the plasma membrane, or Intracellular Plasma Membrane-Connected Compartments (IPMC). Some of the membranes forming the IPMCs are decorated with thick, electron-dense, cytoplasmic coats. A range of proteins typically found in focal adhesion complexes (FAC) cluster at these coat structures. I show that the FAC protein CD18 co-localises with IPMCs in MDMs and primary alveolar macrophages. Upregulation of CD18 during macrophage differentiation coincides with the appearance of IPMCs, and downregulation of CD18 by RNAi perturbs the integrity of IPMCs in uninfected and HIV-infected MDMs. These observations suggest that CD18 and FACs are necessary to form, or maintain, IPMCs in MDMs. Bst-2/Tetherin inhibits the release of HIV by physically linking budding virions to the plasma membrane of host cells. The HIV-1 protein Vpu counteracts Tetherin, thus permitting HIV propagation. I show that type 1 interferons induce Tetherin expression in primary MDMs by around one order of magnitude. Tetherin localises to the cell surface, the trans-Golgi network, and IPMCs. Vpu downregulates Tetherin from the plasma membrane, and, in the absence of Vpu, HIV is retained in IPMCs. My data indicate that so-called virological synapses play a major role in the direct cell-cell transmission of HIV from macrophages to CD4+ T cells, and that Tetherin inhibits this intercellular spread. Thus, I show that Tetherin restricts HIV propagation from primary human macrophages regardless of the mode of transmission. Overall this study identifies structural components crucial for the integrity of the HIV assembly compartment, and characterises the Tetherin-mediated restriction of HIV in macrophages.

The circadian clock is known to regulate a wide range of physiological and cellular processes, yet remarkably little is known about its role during embryo development. Zebrafish offer a unique opportunity to explore this issue, not only because a great deal is known about key developmental events, but also because of the highly decentralised nature of its circadian system. Moreover, specific stages of the cell cycle are clock-controlled in zebrafish cell lines and during embryo development, making zebrafish an ideal model to explore the interactions between these two cellular oscillators. This thesis presents: i. Identification of an evolutionarily conserved cell cycle regulator, cyclin-dependent kinase (CDK) inhibitor 1d or p20, which is closely related to the CDK inhibitor p21. Both p20 and p21 are rhythmically expressed, directly clock-controlled, and regulators of the G1/S transition of the cell cycle. However, their expression patterns differ both temporally and spatially, leading to a unique mechanism whereby S-phase is set to different times of the day in a tissue-specific manner, depending on the balance of these two inhibitors. ii. Description of a wide range of day lengths to which the zebrafish circadian clock can be entrained (from at least 16 to 32 hr days). Clock entrainability is demonstrated by rhythmicity of both clock-related gene expression and cell cycle timing. Furthermore, this remarkable wide range of entrainment for a vertebrate is observed both in vitro (zebrafish cell lines) and in vivo (zebrafish larvae). iii. Identification of numerous rhythmic genes during zebrafish development, including neuroD and additional transcription factors whose rhythmic expression is restricted to the developing retina. Rather surprisingly, however, these circadian expression rhythms are only observed in differentiated retinal photoreceptors (both in larvae and adults), suggesting a new and important role in regulating the rhythmic expression of phototransduction components.

Inherited diseases and cancer are often characterized by single DNA base mutations that can result in altered gene expression, altered mRNA splicing, or changes to the protein structure. The effects of the latter category on protein function and how this is related to disease is the easiest of these to understand. Pathogenic deviations (PDs) are utations reported to be disease-causing, while true single nucleotide polymorphisms (SNPs) are understood to have a negligible effect on phenotype. With recent developments in biotechnology, the most relevant being the increased reliability and speed of sequencing, a wealth of information regarding SNPs and PDs has been acquired. Quite apart from the analytical challenge of analysing this information with a view to identifying novel therapies and targets for disease, the challenge of simply storing, mapping, and processing these data is significant in itself. This thesis builds on earlier work in the Martin group in which a database (SAAPdb) was developed to map mutation data to protein structure and allow the likely local protein structural effects of a mutation to be evaluated. In this thesis, a general introduction to the relevant biology (Chapter 1) and bioinformatics tools and resources (Chapter 2) is provided. In Chapter 3, the Single Amino Acid Polymorphism database (SAAPdb) is described and the work done to fix bugs and update the data is outlined. Despite this work, owing to continuous maintenance problems identified when updating the program, the Martin group has now switched to using a ‘pipeline’ version that no longer relies on any pre-calculated data stored in a database. Earlier work performed during a Masters project showed that some of the analyses were extremely sensitive to tructural details. These analyses have been updated and extended, confirming earlier results. Consequently, some of he analyses were updated to replace Boolean True/False (Good/Bad) assignments with energy or pseudo-energy alues. A pseudo-energy potential was developed for evaluating the effects of mutations to-proline or from-glycine (Chapter 4) and a new full-energy method for assessing the effects of side-chain clashes was evaluated (Chapter 5). method using the structural analyses data together with random forests to predict whether a mutation will be amaging was then developed (Chapter 6). This method was demonstrated to be better than all competing individual methods. A variation of this approach was used to distinguish between two phenotypes (hypertrophic ardiomyopathy – HCM, and dilated cardiomyopathy – DCM ) caused by mutations in the cardiac beta-myosin gene (MYH7, Chapter). The thesis finishes with a general discussion and conclusions (Chapter 8).

Cytochrome c oxidase (CcO) reduces O2 to water with four electrons from cytochromes c2+ and four matrix protons. The energy released is conserved in the protonmotive force by translocation of four additional protons into the intermembrane space. Electrons are transferred via CuA, haem a, to the binuclear centre; haem a3 and CuB, where O2 is reduced. Four major aspects of its structure/function have been investigated in this study. Mid-infrared (IR) spectroscopy has been used to probe redox-induced structural changes. By using electrochemically-poised samples of cyanide- and carbon monoxide-ligated bovine CcO these redox-linked IR changes were shown to be linked primarily with CuA and haem a metal centre transitions with fewer changes associated with transitions in haem a3 and CuB. CcO contains a cross-linked Tyr-His which is believed to form a Tyr radical in the PM intermediate. In this work, electrochemical conditions to induce Tyr-His model compound radicals have been combined with IR spectroscopy to record IR reference spectra. This has aided tentative assignment of IR bands at 1572 cm-1 or 1555 cm-1 to v8a(C-C), 1519 cm-1 to v7a(C-O.) to the phenoxyl radical and at 1336 cm-1 to a His ring stretch of the cross-linked structure in the PM state of bovine CcO. There is strong evidence from mutagenesis studies in bacterial CcOs that the well-conserved D channel is the proton translocation pathway. However, mutagenesis studies in a human/bovine hybrid CcO of an extensive hydrophilic H channel suggest that it fulfils this function, at least in mammalian CcOs. A structural model of Saccharomyces cerevisiae CcO produced by homology modelling indicates that it also contains an H channel (Maréchal, A., Meunier, B., Lee, D., Orengo, C. and Rich, P. R. (2012) Biochim. Biophys. Acta. 1817, 620-628). However, measurements of the H+/e- stoichiometry of a yeast H channel mutant (Q411L/Q413L/S458A/S455A) suggest it is not critical for proton translocation in yeast CcO. The nuclear-encoded subunit 5 of yeast CcO has two isoforms, 5A and 5B. COX5A is expressed aerobically and COX5B below 1 µM O2. They are reported to alter core catalytic activity; however comparisons were not strictly controlled. Here mutants were constructed where COX5B expression was controlled by the COX5A promoter yielding wild type levels of aerobically expressed 5B isozyme. Interestingly, this 5B isozyme exhibits the same catalytic activity and oxygen affinity as the 5A isozyme and the previously observed elevated activity must arise from a secondary effect.

The morphogenetic processes underlying gastrulation are driven through tightly localised cell rearrangements that occur in both ectodermal and mesodermal cells. While some of the cellular movement involved in the involution of the mesodermal cells have been studied in detail, and the movement of ectodermal cells have also been identified, we know very little about what regulates the movement of ectodermal cells during gastrulation. During gastrulation, the ectoderm suffers a dramatic expansion where an initial multilayered tissue becomes single-layered through a process called epiboly. During epiboly of the ectoderm, the superficial layer remains intact, whereas deep cells intercalate between each other, which accounts for an increase in the area of the tissue and the expansion of the superficial layer. However, the mechanism underlying radial intercalation of deep cells during epiboly remains to be unveiled. In zebrafish, radial intercalation of deep cells is thought to partially control the process of epiboly through which the embryo thins and wraps the underlying yolk. Mutants that impair epiboly have been known for some time and resulted from mutations of half-baked (which encodes for Ecadhrein). However, the role of Cdh1 in zebrafish epiboly has remained controversial and no detailed characterisation of the role Cdh1 in deep ectodermal cells of Xenopus gastrulation has been demonstrated. In this project, we study Xenopus epiboly in pursuing of a mechanism that could help us to understand how cell rearrangements are controlled throughout gastrulation. We have found that at in Xenopus gastrulae, the complement component C3a is expressed in the superficial layer superficial layer of the ectoderm, whereas its receptor (C3aR) is distinctly expressed in the deep layer. Complement factors are well characterized components of the immune response, and C3a has been described with chemoattractant activities by binding to it cognate receptor C3aR. We also found that in an in vitro assays deep layer cells move towards the superficial layer, showing for the first time chemotaxis of deep cells towards superficial ectoderm. Given the important role of C3a as a chemoattractant, we hypothesised that sensing cells from the deep layer of the ectoderm would be attracted to the C3a-expressing cells, superficiallayer, and that chemotactic movement contributes to radial intercalation of ectodermal cells. Indeed, in this thesis we demonstrate for the first time that cells from the deep layer of the ectoderm move towards the superficial layer in a process controlled by the anaphylotoxin complement component C3a. We also prove that C3a signalling through C3aR is indispensable for proper gastrulation as embryos lacking C3/C3aR in the ectoderm showed impaired epiboly and thicker ectoderm.

In primary human monocyte-derived macrophages (MDM), HIV assembles on complex intracellular plasma membrane-connected compartments (IPMCs). Currently, it is unclear whether, in addition to assembly at IPMCs, HIV uses the cell surface of MDM, as viruses that bud at the cell surface may dissociate and be lost from the samples. As the endosomal sorting complex required for transport (ESCRT) machinery is required for the final scission events that release assembled virus from the plasma membrane, this question was addressed by depleting the ESCRT components Tsg101 and ALIX, or generating mutant HIV-1 strains defective in recruiting the ESCRT machinery, to arrest HIV budding and visualise all budding events. Using siRNA, ALIX and Tsg101 were depleted efficiently in MDM. Although the effects on virus release were minimal, Tsg101 had a greater effect than ALIX for HIV release in MDM. In the second approach, I generated mutants (HIV-1 PTAP−, YP−, PTAP−YP−, Δp6) defective in recruiting the ESCRT machinery, by mutating the sequences that bind Tsg101 and ALIX. The mutants were characterised on HEK 293T cells, and the release of PTAP−, PTAP−YP− and Δp6 was inhibited. Analysis by electron microscopy (EM) showed that the mutants indeed produced arrested viruses. As the mutants are defective in release, I developed a method to rescue HIV-1 PTAP− and PTAP−YP− viruses for infecting MDM. The cells were infected with the rescued mutants, and analysed using immunofluorescence and EM. By confocal microscopy, 77% of the cells had viruses in the IPMCs. Using EM, immature viruses were found predominantly (97%) in the IPMCs. Estimates of membrane area revealed enrichment of HIV in the IPMCs. This study provides the first conclusive evidence that HIV is targeted to IPMCs in MDM. This may shield the virus from immune surveillance during virus assembly, with potential impact on cell-to-cell transmission and disease progression.

Complement activation of C3 via the alternative pathway involves the generation of C3u and C3b. The inactivation of C3u and C3b by factors H and I results in the inactive fragments C3c and C3d. Several key details of the factors controlling C3b activity are poorly understood. Many pathogeneses are associated with defective complement regulation. Analyses of 317 mutations in four proteins revealed hotspots in the C-terminal domains of factor H and the C3d region of C3, highlighting their importance in dysfunctional protein-protein interactions. Crystal structures of C3b showed the C3c and C3d regions to be in contact with each other. By combining ultracentrifugation and scattering data, this compact crystal structure was confirmed in 50 mM NaCl buffer. In physiological 137 mM NaCl, the C3c and C3d regions in C3b had separated. The compact structures were well explained by the E1032-R102 salt-bridge between C3d and C3c. Its importance was tested by mutagenesis of C3d (A1032), whereupon the interaction was no longer observed. This result explained the different properties between the major C3S and C3F polymorphic forms of C3. The interactions between the different regions of C3 in 50 mM and 137 mM NaCl were explored. C3, C3u and C3b underwent dimerization and the dimerization site was located at their C3d domain. C3b and C3u dimerized the strongest, followed by C3, and these were explained by extended or compact C3d conformations in C3b and C3u. The interactions between C3d and SCR-19/20 of factor H is central for complement regulation. The stoichiometry of this interaction was controversial, being either 2:1 or 1:1. The combination of multiple biophysical techniques showed unequivocally that the correct stoichiometry is 2:1. This outcome clarified a key aspect of how factor H regulates C3b at C3d-decorated host cell surfaces.

The oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV) is the aetiological agent of Kaposi’s sarcoma (KS). The majority of KSHV infections result in latency, which allows for a persistent host cell infection. During latency, only a limited number of viral genes and at least 12 KSHV microRNAs (miRNAs), ten of which form a cluster, are co-expressed from the KSHV Latency Associated Region. The aim of this project was to investigate the molecular mechanisms and consequences of KSHV miRNA-mediated regulation of cellular signaling. To identify signaling pathways that are altered by KSHV miRNA cluster expression, a Collaborative Enzyme Enhanced Reactive ImmunoAssay (CEER) was performed in lymphatic endothelial cells (LECs) expressing the KSHV miRNA cluster versus control. In addition, a PepChip Kinomics Array in KSHV miRNA cluster expressing LECs was analysed to find signaling pathways that were affected by KSHV miRNA cluster expression. Data from both screens together suggested the regulation of the ERK-TAL1 signaling pathway by the KSHV miRNA cluster. Subsequent experimental validations in LECs and 293T cells showed that the ERK signaling pathway is indeed negatively regulated by the KSHV miRNA cluster through increasing RAF1 S43 phosphorylation. Moreover, KSHV miRNA cluster expression decreases TAL1 S122 phosphorylation and increases total TAL1 protein levels in LECs. 293T cells that exogenously express TAL1 also show an increase in TAL1 protein levels upon KSHV miRNA cluster induction. Interestingly, the RTA promoter was shown to contain a putative binding site for TAL1, which could suggest a potential role for TAL1 in the regulation of the RTA, as well as in the maintenance of KSHV latency. Taken together, the outcome of this PhD project identified the ERK-TAL1 signaling pathway as a novel pathway targeted by the KSHV miRNA cluster and added insights to the role of TAL1 in KS.

The de novo folding of newly synthesised polypeptide chains, exposed in a vectorial manner to the crowded cellular environment, often requires the assistance of molecular chaperones. The ribosome-associated molecular chaperone, trigger factor (TF), facilitates the early folding events of such nascent chains at the ribosomal exit tunnel. The investigation of the structural and dynamic properties of this interaction, at high resolution, presents a challenge due to the dynamic nature of the interaction and the intrinsic conformational heterogeneity of nascent chains. Additional complexity comes from the competition between TF self-association into a dimeric state, the interaction with ribosome-nascent chain complexes (RNCs) and the interaction with isolated protein substrates independent of the ribosome. The sensitivity of NMR spectroscopy to both structural and dynamic changes make this technique uniquely suited to the investigation of the trigger factor chaperone. This thesis presents the application of selective isotopic labelling strategies and advanced TROSY optimised NMR experiments to the study of the TF dimerisation equilibrium, providing a detailed understanding of its kinetics and thermodynamics and insights into the solution structure of the TF dimer. The improved understanding of the mechanism of TF dimerisation forms the basis for the interpretation of observations made in the presence of substrate proteins. Preliminary results on the interaction of TF with two disordered model ribosome- nascent chain complexes reveal that ribosome-associated nascent chain substrates are significantly perturbed by the presence of TF, even in the absence of predicted TF binding motifs. The study of TF and the corresponding isolated polypeptides reveals that limited interaction occurs in the absence of the ribosome. This early NMR investigation of the TF chaperone provides the first residue-specific details of the highly dynamic interaction of TF with nascent polypeptides and begins to elucidate the role of this chaperone in the process of co-translational folding.

This thesis characterises the head direction system in pre-weanling rats around the time of eye-opening. Head direction (HD) cells are neurons in a distributed network of brain areas that fire action potentials as a function of the animal’s directional orientation relative to its environment. Environmental sensory information and self-motion cues are used to update the directional signal, with visual landmarks being particularly salient in controlling the preferred firing directions of HD cells. HD cells have previously been shown to be adult-like as early as postnatal day 16 (P16) in the rat pup (Wills et al 2010, Langston et al 2010), just after its eyelids come unfused and coinciding with the first spontaneous exploration of its environment. To determine if the HD cell circuit can organise before the onset of patterned vision, I recorded from the postsubiculum (PoS) before eye-opening in pre-weanling rats. The earliest instance of HD cells is found at P12, three days before eye opening. This early HD signal carries low directional information content and lacks stability both within and across trials. However, once the eyes of the rats are open, the HD system matures rapidly, with a dramatic increase in the number of directional cells and the quality and reliability of their directional signal. A prominent visual landmark is also able to exert control over HD responses within 24 hr of eye-opening. The data suggest that the directional circuit can be organised in the absence of visual spatial information, while patterned vision is rapidly integrated once it becomes available, for accurate and reliable orientation in space.