Theses - Biochemistry
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Item Embargo Engineering and deploying FRET-based biosensors to illuminate cellular phytohormone dynamics coordinating environmental stress responsesTang, BijunSynthesised in plants in small quantities, phytohormones are naturally occurring chemical messengers that play critical roles in regulating plant growth and development as well as triggering responses to external stimuli. The precise regulation of phytohormone biosynthesis, catabolism and transport is crucial to maintain these messengers’ concentration, allowing different parts of the plant to communicate and coordinate responses to changing environmental conditions. Understanding the biology of phytohormones has therefore developed to be an important field of study. In this thesis, I focused on two specific hormones, Gibberellin (GA) and Salicylic acid (SA). I improved and characterised the next-generation Gibberellin Perception Sensors (GPS) based on the GPS1 in Chapter 3 and successfully designed and engineered a novel FRET-based biosensor for SA, Salicylic acid Sensor 1 (SalicS1) in Chapter 5. Using these biosensors, I examined the relationship between repatterning of the corresponding phytohormones and plant reprogramming under different stress conditions in Chapter 4 (GA) and Chapter 6 (SA). Aims of this thesis are summarised in the Figure below. These biosensors allowed the monitoring of changes in phytohormone levels with high spatial and temporal resolution and provided valuable insights into the complex interplay between phytohormones and environmental stimuli at the cellular level. While nuclear-localised GPS1 (nlsGPS1) has been found to bind bioactive GA4 with high affinity and good signal-to-noise ratio, other GPS1 biosensor properties remained to be optimised and diversified. By modulating the interaction interface between the sensory domains AtGID1C and the truncated DELLA domain of AtGAI, we have successfully increased the *in vitro* reversibility of GPS1, and GA hypersensitivity phenotypes were reduced, resulting in GPS2. In my project, GPS2 was fully characterised. I further attempted to expand the range of GPS biosensor affinities through mutagenesis and by deploying higher affinity GID1 variants from other plant species. By altering the linkers between fluorescent proteins (FPs) and binding domain, I created GPS3 with a much-improved signal to noise ratio *in vitro* which allows accurate detection of smaller changes in GA levels particularly at low concentrations, although such properties were not observed *in planta*. I used nlsGPS1 to study the relationship between GA's redistribution and abiotic stresses, including nutrient deficiency, salinity stress, high sugar stress and osmotic stress. I then focused on SA which is best known as a plant defence hormone and is also involved in increasing plant tolerance to several abiotic stresses. In this arm of my project, I developed a novel FRET based biosensor, SalicS1, to directly detect SA levels in live plants with unprecedented resolution. I screened SA receptors and their interaction partners from multiple species as ligand sensory domains. Combinations of various cyan-yellow FPs as FRET pairs and a set of linker variants connecting these four moieties generated single biosensor fusion proteins that were evaluated for the optimal SalicS1. SalicS1 response to SA was tested first *in vitro* after purification from yeast and then *in planta* in stable transgenic *Arabidopsis* lines, both in a dose dependent manner. Using more low pH tolerant FPs will allow biosensors to be more widely subcellularly targeted, particularly in the acidic environments of the vacuole and apoplasm. My preliminary evidence indicate that some FRET pairs could lead to successful low pH-tolerant biosensors. Further engineering is needed to develop high signal-to-noise ratio low pH-tolerant biosensors (SalicSLowpH and GPSLowpH) to elucidate subcellular phytohormone distribution. Nuclear localised SalicS1 (nlsSalicS1) were further used in studying the redistribution of SA levels under abiotic stresses. It revealed that SA were reduced in *Arabidopsis* seedlings roots. Other abiotic stresses, including low temperatures and salinity stress, were found to affect cellular SA levels, depending on the duration of exposure. In conclusion, I fully characterised the GPS2 and created GPS3. I also engineered a novel FRET based SA sensor, SalicS1, to allow SA levels to be monitored in cellular scale *in vivo*. I attempted to design diversified sensor variants to be targeted to acidic subcellular environments. Taken together, my thesis has engineered and applied biosensors to advance our understanding of phytohormone redistribution at a high spatiotemporal resolution under stress conditions.Item Open Access The crystal structure of human Navβ3-Ig domain and its implicationsNamadurai, SivakumarThe mammalian Voltage-gated sodium (Nav) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Navβ subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Navα subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Navβ-subunit isoforms, Navβ1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD). This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, *in vitro*), Proximity Ligation Assay (full-length Navβ3, *in vivo*), Atomic Force Microscopy (isolated full-length Navβ3, *in vitro*) and Photo-activated Localisation Microscopic experiments (full-length Navβ3, *in situ*) support the view that the Navβ3 subunit can form trimers when expressed in cells. The biological significance of Navβ3 subunit trimerization is discussed. Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced.Item Open Access Studying Microglia-Mediated Neurodegeneration Using CoculturesBirkle, TimothyMicroglia are resident innate immune cells of the central nervous system with potent phagocytic and inflammatory capabilities. These cells are crucial in both health and disease, including neurodegenerative diseases. Genetics studies have linked Alzheimer’s disease and other diseases to genes that affect microglial functions, and evidence indicates that microglial phagocytosis and inflammation control neuronal function and survival. Some microglial activity may be beneficial during neurodegeneration, but excessive release of pro-inflammatory cytokines and reactive oxygen/nitrogen species is a hallmark of neurodegenerative disease and can promote neurodegeneration. Additionally, microglial phagocytosis of the protein aggregates driving proteinopathic diseases may be beneficial, but excessive microglial phagocytosis of synapses or neurons may contribute to neurodegeneration. Overall, study of microglia-mediated neurodegeneration is essential when working towards microglia-targeted therapies for dementias, such as Alzheimer’s disease, and other conditions. In order to study these interactions between microglia and neurons, *in vitro* model systems are required that include both cell types. Use of these models has often been limited to low-throughput experiments due to practical challenges and the need for careful manual analysis of individual cell types. In this work, I used a neuron-glia coculture model in which inflammatory activation of microglia with lipopolysaccharide (LPS) or other stimuli results in neuronal loss, addressing the above limitations by building an imaging and analysis workflow using recent methods and machine learning tools. This enabled accurate, automated analysis of images from primary cocultures. Early tests of these higher-throughput assays identified potential roles for urokinase (uPA) and spleen tyrosine kinase (SYK) in microglia-mediated neurodegeneration. uPA is an extracellular protease that may also regulate migration, inflammation and proliferation in association with its receptor uPAR. SYK signals downstream of other microglial cell surface receptors that have been linked to brain diseases, including Alzheimer’s, such as TREM2, CR3 and CSF1R. In this work, uPA and SYK were investigated further using assays for microglial survival, inflammation, and phagocytosis. Here, I found that uPA may influence inflammatory neurodegeneration, as well as microglial proliferation and phagocytosis, but it remains unclear which of uPA’s many signalling mechanisms drive this. A broad uPA inhibitor affecting both proteolysis and receptor binding prevented LPS-induced, microglia-dependent neuronal loss in cocultures, potentially by depleting microglia and affecting their morphological and phagocytic response to LPS. However, more specific inhibitors of either proteolysis or receptor binding produced only weak effects, if any. Interestingly, exogenous uPA caused proliferation of microglia, suggesting a further role for uPA signalling in these cells. Meanwhile, inhibitor studies found that SYK also regulates neurodegeneration while affecting microglial survival, inflammation, and phagocytosis, which fits with existing knowledge on SYK and its upstream receptors. Finally, a high-content screen for drugs and targets that control microglia-mediated neurodegeneration was developed, using the primary neuron-glia cocultures and new image analysis methods. This novel proof-of-concept validated the use of neuron-glia cocultures in high-content assays when combined with the image analysis developed here. The data identified contributions from steroid hormones, adrenergic receptors, and MAPK signalling (amongst other pathways). Overall, this work has used updated image analysis methods to investigate the roles of uPA and SYK in microglial biology and microglia-mediated neurodegeneration, as well as showing proof-of-concept for using neuron-glia cocultures in screens for drugs and targets influencing neurodegenerative disease. This adds to the increasing literature on targeting microglia for therapies against neurodegeneration, while validating new assays to study neuron-glia interactions for both target discovery and investigation of the complex mechanisms controlling microglial function.Item Open Access Investigations of the assembly and function of the Toxoplasma microporeMercado Saavedra, Brandon*Toxoplasma gondii* is an apicomplexan parasite that can invade all warm-blooded animals. One of the characteristic features of this parasite is its pellicle, a complex three-layer membrane that includes flattened vesicles that run the entire parasite. *Toxoplasma*’s pellicle has conferred the parasite protection, cell shape, and motility, which is important for the parasite’s host invasion and survival. However, this pellicle has created a barrier to material exchange processes such as endocytosis. This has raised the question of how *Toxoplasma* and other members of the same group perform endocytosis with this extra set of membranes. *Toxoplasma*’s endocytosis is not fully understood. The first insights about this process started with the electron-microscopy images of pore-like plasma membrane invaginations with electro-dense material around it, called micropores. Therefore, it was suggested that *Toxoplasma* would have clathrin-dependent endocytosis through this pore. Later studies proved that *Toxoplasma* ingests material from its environment in intra and extracellular stages. Nevertheless, these studies did not necessarily provide evidence that this material uptake was via endocytosis, nor that it happened through the micropores. This thesis has studied the *Toxoplasma* micropore and its relation with endocytosis. The *Toxoplasma* micropore is a fixed structure present in 2-3 dedicated pits in each cell. It is located closer to the anterior end of the parasite, probably due to its early synthesis during daughter cell assembly. Its molecular composition included known endocytosis proteins such as the AP2 adaptor complex, EPS15, and a dynamin-related protein (DrpC). It also included other proteins implicated in endocytosis in other apicomplexan parasites, such as UBP1 and K13. The micropore is an important part of the parasite’s pellicle since the recruitment and assembly of its components start during early budding, and the depletion of some of these components leads to pellicle disruptions. The molecular composition and endocytosis assays developed by collaborators of the Waller lab, conclude that the micropore is the site of endocytosis in *Toxoplasma*. *Toxoplasma*’s endocytosis occurs through a fixed structure that is part of the parasite’s pellicle. Growth plaque assays proved that endocytosis is essential for the parasite’s survival, and depletion of some of its components (K13 and ISAP1) leads to pellicle ruptures. Replication assays showed that endocytosis is not essential for the parasite’s nutrition. Nevertheless, the inhibition of endocytosis led to failure in egress. Phenotypic observations of endocytosis-inhibited parasites provided evidence about the loss of the rosette-like intracellular organisation and plasma membrane-bound cytosolic extensions. These observations suggest that the main use of endocytosis in *Toxoplasma* is plasma membrane homeostasis. Overall, this thesis has provided evidence about the *Toxoplasma* micropore, its assembly, and the importance of endocytosis in this apicomplexan parasite.Item Embargo Novel Structures of RAD51 Reveal Mechanisms in DNA Damage Repair and Genomic StabilityAppleby, RobertThe RAD51 protein contributes to the maintenance of genomic stability by promoting the repair of DNA double-strand breaks and the protection of DNA replication forks. RAD51 functions alongside the tumour suppressor protein BRCA2 to catalyse DNA strand-exchange reactions which form an integral part of Homology-Directed Repair. RAD51 has been the subject of decades of research which has helped to elucidate many mechanisms underpinning its function, however numerous key questions still remain. In this thesis I present three-dimensional structures of RAD51 nucleoprotein filaments together with biochemical and biophysical data that reveal new insights into how RAD51 contributes to maintaining the stability of our genome. High-resolution structures of RAD51 filaments on single- (ss-) and double-stranded (ds-) DNA revealed the presence of a second metal cation at the ATP-binding site, which forms the basis for a mechanism of ATP-hydrolysis dependent filament disassembly, confirmed by the structure of a RAD51 filament in the presence of ADP. Here I also describe two structures of RAD51 bound to the C-terminus of BRCA2, which show how BRCA2 binds to and stabilises RAD51 filaments during DNA replication and repair. A low- resolution structure of a RAD51 synaptic filament is also presented here, which suggests that the mechanism of recombinase-catalysed strand exchange is conserved throughout the three domains of life. Furthermore, I demonstrated that RAD51 can bind to DNA damaged by base hydrolysis, based on the structure of RAD51 nucleoprotein filaments that reveal specific recognition of abasic sites. Finally, I show that RAD51 can bind RNA substrates, as established by RAD51 filament structures bound to ssRNA and a DNA : RNA hybrid. Collectively these structures and supporting biochemical experiments highlight new mechanisms of RAD51 function in both DNA repair and DNA replication.Item Open Access Studies on voltage-gated sodium channel β3 subunit structure and cancer-related functions using single-chain variable fragment antibodies and bioinformaticsLiu, HengruiVoltage-gated sodium channels (VGSC), embedded in the plasma membrane of cells, play a pivotal role in generating sodium currents and action potentials. The mammalian VGSCs consist of a large pseudo-tetrameric pore-forming α subunit, the channel protein, which associates with one or more β-subunits. In humans, nine types of VGSC Nav1 channel α subunit isoforms (Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) and four types of β subunit isoforms (β1, β2, β3, and β4) (gene names: *SCN1-11A* and *SCN1-4B*) have been identified in various tissues. This thesis describes the use of single-chain variable fragment (scFv) antibodies to study the binding site of the β3 subunit on the pain-sensing channel, Nav1.7, and investigate the role of the VGSC β3 subunit in cancer guided by bioinformatic analysis. *In silico* docking was used to construct a binding model between the Nav1.7 α subunit and β3 subunit, and molecular dynamic simulations were employed to assess the binding stability of this model. These computational structural analyses provide an *in silico* model that was validated in subsequent scFv mapping experiments. Using scFvs that specifically recognized distinct regions of Nav1.7, the binding site of the β3 subunit on the Nav1.7 α subunit was identified. The results substantiate that the β3 subunit binds to the Nav1.7 α subunit in a manner akin to the β1 subunit. This study offers a valuable strategy for studying the extracellular domain of plasma membrane complexes under cellular conditions, complementing cryo-electron microscopy (cryo-EM) and X-ray crystallography approaches. In the context of cancer research, the systematic literature review and bioinformatics analysis indicated that the expression of the VGSC β3 subunit gene (*SCN3B*) is associated with reduced glioma severity and regulated glioma immunity and migration. Subsequently, the effect of the β3 subunit on glioma cell migration was experimentally investigated. The results reveal that the β3 subunit inhibits glioma cell motility via the β3 subunit immunoglobulin (β3 Ig) domain and this function is not reliant on chemo-sensing. Instead, the β3 Ig domain governs actin-based cell protrusion, reducing network actin (lamellipodia and membrane ruffle) while augmenting bundle actin (filopodia) in glioma cells. Finally, through immunoprecipitation and mass spectrometry, several pathways underlying the regulatory function of the β3 subunit in glioma cell actin organization were identified. In conclusion, this study advances our understanding of the structure and significance of the VGSC β3 subunit in cancer biology.Item Open Access Microglial activation and regulation by secreted chaperonesReid, KyleMicroglia are brain-resident macrophages and play pivotal roles in central nervous system (CNS) development, homeostasis and pathology. Calreticulin and LRPAP-1 are ubiquitously expressed protein chaperones that aid in protein folding and processing within the endoplasmic reticulum (ER). Both proteins might also be released into the extracellular space, but, if so, it is unclear whether and how they affect microglial functions when present extracellularly. Microglia are the primary innate cells of the CNS and one of the first cell types to respond to signs of injury or inflammation. However, the mechanisms that mediate and regulate this early immune response are unclear. In this work, I show stressed microglia (inflamed, ER-stressed or apoptotic) and neurons (crushed) release calreticulin into the extracellular culture media, where it reaches nanomolar levels. Applications of nanomolar calreticulin activated microglia in culture to release pro-inflammatory cytokines and chemokines, and inhibited microglial proliferation. Nanomolar calreticulin also upregulated surface MHC-II and upregulated the expression and release of APOE, but did not change the expression of 11 other genes associated with disease-associated microglia. Microglia also migrated towards media containing extracellular calreticulin. Overall, this suggests that calreticulin can be released from stressed brain cells, and this released calreticulin can act as an alarmin to recruit and activate microglia. Calreticulin apparently activated microglia by stimulating toll-like receptor 4 (TLR4) signalling, as nanomolar calreticulin could not activate microglia or a TLR4 reporter line when i) intracellular TLR4 signalling was blocked, ii) binding to TLR4 was blocked with function blocking antibodies, or iii) the hydrophobic binding pocket formed between TLR4 and its co-receptor MD2 was blocked. Microglial activation was also inhibited when calreticulin was pretreated with sugars, so TLR4 activation may require calreticulin’s carbohydrate-binding domain. Calreticulin partially oligomerised under the same conditions used to activate microglia, so oligomeric calreticulin might contribute to activation, but this remains unclear. Thus, calreticulin is a microglial alarmin, and activates microglia by activating TLR4. LRP-1 and related LDL family receptors mediate many cell functions, and these receptors are inhibitable by extracellular LRPAP-1. However, it is not known whether extracellular LRPAP-1 is a physiological (or pathological) regulator of these receptors, because it is not known whether LRPAP-1 is released extracellularly in physiological conditions and concentrations sufficient to inhibit these receptors. In this work, I found that microglia activated with LPS or ER-stressed with tunicamycin released nanomolar levels of LRPAP-1. Released LRPAP-1 was detected on the surface of microglia, and anti-LRPAP-1 antibodies induced internalisation to peri-nuclear compartments, consistent with LRPAP-1 being bound to endocytic LDL family receptors. Extracellular LRPAP-1, applied at levels released by stressed microglia, did not activate microglia, nor did it prevent LPS neurotoxicity in mixed neuronal-glial cultures. However, extracellular LRPAP-1 did inhibit microglial phagocytosis of dead cells and isolated synapses. Amyloid beta (Ab) is implicated in Alzheimer’s disease (AD), and LRPAP-1 can bind and regulate Ab uptake in a variety of cells. I show extracellular LRPAP-1 regulates microglial uptake of Ab in a serum- and concentration dependent manner. Extracellular LRPAP-1 inhibited Ab fibrillization. In mixed neuronal-glial cultures, extracellular LRPAP-1 increased Ab bound and/or internalised by neurons but reduced Ab neurotoxicity. Thus. LRPAP-1 can be released by stressed microglia to inhibit microglial phagocytosis, inhibit Ab fibrillization and inhibit Ab neurotoxicity. More generally, this work supports the novel concept that released LRPAP-1 may be an extracellular regulator of LRP-1 and related LDL family receptors and their multiple functions. Taken together, these findings indicate that calreticulin and LRPAP-1 are secretable regulators of microglial function and are extracellular chaperones.Item Restricted Item Embargo An omics study into the molecular impact of autosomal dominant APP and MAPT mutations on the cerebral cortexHnátová, SilviaFamilial Alzheimer’s disease (AD) and frontotemporal dementia (FTD) are characterised by an age of onset typically between 30 and 50 years of age and are often linked to autosomal dominant mutations. This dissertation focuses on APP and MAPT mutations causal to early onset AD or FTD, which affect the dosage or isoform ratio balance of the key proteins underlying AD/FTD: amyloid beta and tau. Using a combination of in vitro iPSC-derived neuronal cultures carrying MAPT E10+16 and APP duplication mutations and post-mortem human brain tissue from patients carrying MAPT E10+16, APP V717L and APP duplication mutations, I describe the generation of transcriptomics and proteomics datasets with the goal of uncovering transcriptional and translational pathways driving AD linked to APP and MAPT mutations. The first half of this thesis focuses on the differentiation of patient-derived iPSCs carrying MAPT E10+16 and APP duplication mutations and isogenic control iPSCs into neuronal cultures using the directed differentiation protocol. I first describe the CRISPR/Cas9 strategy to correct APP copy number in iPSCs derived from patients carrying APP duplication mutation. Using MiSeq sequencing, I confirm a successful monoallelic APP knockout in iPSCs carrying APP duplication mutation, thus generating two isogenic APP duplication iPSC lines (APPDUPiso). Next, I characterise the neuronal cultures derived from iPSCs carrying MAPT E10+16 and APP duplication mutations and isogenic control iPSCs differentiated using the directed differentiation protocol. Last, using a single-cell RNA sequencing dataset of mature cultures derived from each iPSC line, I identify an upregulation of cholesterol biosynthesis genes in excitatory and inhibitory neurons and astrocytes carrying MAPT E10+16 mutation compared to the isogenic control cells. I corroborate cholesterol synthesis upregulation in VGLUT1+ excitatory neurons carrying MAPT E10+16 mutation using an independent organoid-derived single-cell RNA sequencing dataset (Bertucci et al., 2023). Small RNA sequencing from bulk cultures carrying MAPT E10+16 mutation points out miRNA-mediated repression of cholesterol efflux, suggesting an overall prioritisation of cholesterol internalisation in the cultures carrying MAPT E10+16 mutation. The second half of this dissertation focuses on the generation of spatial transcriptomic and bulk proteomic datasets using frontal lobe human brain post-mortem tissue from patients carrying patients carrying MAPT E10+16, APPV717I, APP V717L and APP duplication mutations. Using spatial transcriptomics on FFPE slides from three patients carrying MAPT E10+16 mutation and five control patients, I identify dysregulation of transcripts potentially relevant to the MAPT E10+16 tauopathy in the prototype spatial transcriptomic dataset. Using bulk TMT proteomics design from a bigger cohort of patients, including patients carrying APP V717L and APP duplication mutations, I corroborate the results from the spatial transcriptomics dataset. In addition, I identify differential protein abundance in samples from patients carrying MAPT E10+16 mutation, including a potentially novel marker of MAPT E10+16 tauopathy: RNA-binding protein Nop58. Preliminary results from immunohistochemistry experiments staining for Nop58 in the FFPE slides from patients carrying MAPT E10+16 mutation suggests formation of Nop58+ inclusions in the grey matter of these patients, potentially related to stress granule formation. The role of Nop58 in the brain or in tauopathies has not yet been described. The results from my dissertation provide a platform for future examination of cholesterol biosynthesis and Nop58 in tauopathy underlined by MAPT E10+16 mutation. Whilst these results may be specific to the experimental models deployed in this dissertation, cholesterol synthesis upregulation was previously described as potentially related to pathological hyperexcitability in neurons carrying MAPT mutations, and stress granule formation remains an intensive area of research in neurodegenerative diseases.Item Embargo The role of the microglial P2Y₆ receptor in ageing, development, and tauopathyDundee, JacobPrior research has shown that activation of the microglial P2Y₆ receptor (P2Y₆R) with uridine diphosphate (UDP) induces microglial phagocytosis. Also, young mice with knockout of the P2Y₆R gene (*P2ry6⁻´⁻*) were found to perform worse in memory tests compared to *P2ry6⁺´⁺* mice, but *P2ry6⁻´⁻* mice were protected from ageing-associated memory loss. Finally, in a chronic model of tauopathy, *P2ry6⁻´⁻* mice were protected from both TAU-induced memory loss and tauopathy. The goal of this thesis was to investigate microglial-dependent mechanisms might protect *P2ry6⁻´⁻* mice from ageing- and tauopathy-induced memory loss, as well as possible roles for P2Y₆R in brain development. However, as P2Y₆R is found on numerous peripheral cells throughout the body, it cannot be dismissed that the cause of the effects observed by P2Y₆R deficiency in mice may not solely be microglial-dependent. I found that microglia from *P2ry6⁻´⁻* mice had no significant changes in microglial migration, cytokine release, or phagocytosis of beads. However, P2Y₆R deficiency substantially reduced the phagocytosis of synaptosomes. 4-month-old (young) and 17-month-old (old) mice were investigated to study ageing-associated effects. There was also a large increase of synaptic material within microglial lysosomes with age of *P2ry6⁺´⁺* mice, but this apparent ageing-associated phagocytosis of synapses was not present in aged *P2ry6⁻´⁻* mice. Similarly, there was a loss of synapses with age in hippocampal CA1 stratum radiatum and the somatosensory cortex in *P2ry6⁺´⁺* mice, but no such synaptic loss was observed in aged *P2ry6⁻´⁻* mice. Together, these findings indicate that P2Y₆R mediates microglial phagocytosis of synapses with age. Postnatal day 15 (P15) and P30 mice were investigated as active microglial pruning is occurring at this timepoint. Synaptic protein levels were observed to be dysregulated in P15 *P2ry6⁻´⁻* mouse brain homogenates compared to *P2ry6⁺´⁺* mice. P30 *P2ry6⁻´⁻* mice, but not P15 *P2ry6⁻´⁻* mice, showed higher levels of synapses compared to *P2ry6⁺´⁺* in multiple regions associated with memory. This increase in synapses was coupled with reduced internalisation of synaptic material within CD68+ lysosomes in Iba1+ microglia in P30 *P2ry6⁻´⁻* mice. This indicates that P2Y₆R-dependent phagocytosis of synapses contributes to synaptic loss late in development. *In vitro*, P2Y₆R deficiency reduced microglial phagocytosis of myelin debris. *In vivo*, P15 *P2ry6⁻´⁻* mice, but not P30 *P2ry6⁻´⁻* mice, were observed to have reduced cortical myelination compared to *P2ry6⁺´⁺*, with no change in the internalisation of a myelin-associated protein within CD68+ lysosomes in Iba1+ microglia. 4-month-old *P2ry6⁻´⁻* were observed to have reduced cortical myelination compared to 4-month-old *P2ry6⁺´⁺* mice. This was coupled with increased internalisation of a myelin-associated protein within CD68+ lysosomes in Iba1+ microglia with 4-month-old *P2ry6⁻´⁻* mice. This suggests that lack of P2Y₆R-dependent phagocytosis of myelin early in development results in a myelination defect. Mice expressing P301S TAU have TAU hyperphosphorylation and reduced memory, and memory is rescued when crossed with *P2ry6⁻´⁻* mice. However, there was no difference in synaptic density in multiple regions associated with memory, and no change in the internalisation of hyperphosphorylated TAU within microglial lysosomes in *P2ry6⁻´⁻* mice with P301S TAU. Activation or inhibition of P2Y₆R did not affect lysosomal exocytosis by microglia in culture. P2Y₆R did not regulate microglial phagocytosis of TAU fibrils or cells with TAU fibrils in ways that could explain why P2Y₆R deficiency reduced tauopathy in mice. Thus, why P2Y₆R deficiency reduces tauopathy and the associated memory loss in mice remains unclear. Overall, the research described in this thesis shows that the microglial phagocytic receptor P2Y₆R mediates microglial phagocytosis of synapses during development and ageing, and additionally affects myelination during development, summarised in the graphical abstract below. Together these changes may explain why P2Y₆R deficiency affects memory, and suggests that, after development, P2Y₆R may be a good target to prevent excessive synaptic loss in pathology and ageing.Item Open Access Investigating the effects of calreticulin and beta-galactosidase on microglial functions and neuronal lossKitchener, Emily Jessica AnnMicroglia are the primary immune cells of the central nervous system (CNS); they have dynamic roles in brain development, maintaining homeostasis, and responding to insult and injury, thereby contributing to CNS health. However, increasing evidence highlights a role for microglial dysfunction in many brain pathologies, like Alzheimer’s disease (AD) and Parkinson’s disease (PD). Understanding how and when microglia are beneficial, detrimental or both, is an active area of research and, elucidating the underlying mechanisms involved in neurodegeneration could uncover novel therapeutic avenues. In this work, I investigated how microglial functions and neuronal loss were affected by calreticulin and β-galactosidase, two proteins associated with ageing, chronic inflammation, and neurodegeneration. The aggregation of amyloid-β to form oligomers and insoluble amyloid plaques in the brain is a hallmark of AD. Amyloid-β can be directly neurotoxic and induce pro-inflammatory activation of microglia, which may contribute to neurodegeneration. Molecular chaperone proteins are commonly found intracellularly, where they interact with proteins to prevent their aggregation and facilitate proper folding. The endoplasmic reticulum-resident chaperone protein, calreticulin, can be released from microglia and has been found to bind amyloid-β. I investigated whether exogenous calreticulin affects amyloid-β aggregation and amyloid-β induced neurotoxicity. *In vitro* assays revealed inhibition of amyloid-β fibrillisation by calreticulin, and transmission electron microscopy showed that calreticulin promoted formation of larger amyloid-β oligomers. Furthermore, exogenous calreticulin was protective in the context of amyloid-β-induced neuronal loss in primary mixed neuronal-glial cultures. Together this data suggests that calreticulin might act as an extracellular chaperone for amyloid-β and be neuroprotective, hence treatments increasing extracellular calreticulin in the brain might be beneficial for AD. Glycohydrolase enzymes, including neuraminidase 1 (Neu1) and β-galactosidase, play a fundamental role in the degradation of glycoproteins and glycolipids, to maintain cellular constituent turnover and glycosylation homeostasis. Neu1 hydrolyses terminal sialic acid residues to expose galactose residues, which can then be hydrolysed by β-galactosidase. Previously it was thought that these enzymes were exclusively located in lysosomes, but recent evidence has found Neu1 activity also associated with the external surface of the plasma membrane. As Neu1 can be structurally and functionally coupled to β-galactosidase in a lysosomal multienzyme complex, I investigated whether BV-2 microglia and primary rat microglia have increased extracellular β-galactosidase activity when activated by a variety of inflammatory stimuli, including lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Inflammatory activation of microglia increased β-galactosidase activity at the cell surface and increased β-galactosidase protein levels extracellularly. Extracellular β-galactosidase might remove galactose residues from the surface of microglia and neurons, potentially disrupting homeostasis. I found that addition of β-galactosidase to primary mixed neuronal-glial cultures caused a significant loss of neurons and promoted microglial activation. Whereas inhibition of β-galactosidase in LPS-stimulated cultures reduced LPS-induced neuronal loss and microglial activation, suggesting that β-galactosidase may activate microglia in a way that promotes neuronal loss. Together, this work elucidates novel effects of calreticulin and β-galactosidase on microglial function and neuronal loss, which may contribute to understanding the roles of these proteins in neurodegeneration and disease.Item Open Access Structure & Function of Bacterial Transport Machines in their Cellular ContextKirykowicz, Angela MaryMembranes confer cells with individual identity and capacity to regulate their response to their environment. A critical aspect of having a membranous partition is the ability to transport substances into and out of cells as part of life-sustaining functions. In pathogenic bacteria, transporters aid infection and survival in the host. Two such transporters in Gram-negative bacterial species are the MacA-MacB-TolC (MacAB-TolC) antibiotic efflux pump and the Type I Secretion System (T1SS), responsible respectively for antibiotic resistance and export of protein virulence factors. To pass the Gram-negative envelope in a one-step translocation process, both machines use a tripartite system, consisting of outer membrane protein TolC, a periplasmic adapter protein (MacA or haemolysin D (HlyD) in the T1SS), and an inner membrane protein (MacB or haemolysin B (HlyB) in the T1SS). Both use the power of ATP-hydrolysis to export their substrates. Here, I utilise computational and experimental approaches to elucidate the mechanism of function for both machines. I conduct molecular dynamics (MD) simulations of membrane embedded HlyB component of the T1SS with and without its haemolysin A (HlyA) substrate as *in silico* experiments. I also conduct MD simulations with and without substrate for a related peptidase. I show that substrate recognition is via conserved charge-charge interactions. I also show that HlyB has an asymmetric preferential interaction with cardiolipin when its substrate is present, which is not seen in the peptidase simulations. I propose that this preference is part of the mechanism of transport, with cardiolipin providing energy via the proton-motive force. I test this hypothesis through flow cytometry detection of labelled substrate trapped T1SS in a mixed population of cells, by comparing parental MG1655 *Escherichia coli* with a cardiolipin deficient MG1655 strain. I found that the cardiolipin deficient strain has reduced T1SS levels compared to its parent. To aid structural studies, I optimise the expression of the T1SS using a flow cytometry based sequential design strategy where conditions are iteratively tested via detection of substrate trapped T1SS and updated until no more improvement can be made. I also test purification strategies for single-particle cryo-electron microscopy studies. Finally, I apply further bioinformatic approaches and synthesise my computational and experimental results to propose a mechanism of transport and suggest future experimental tests. I conduct MD simulations of MacB in membrane with and without a trapped lipid. I show that this trapped lipid locks MacB into an open state, allowing for substrate entry into the pump. I contextualise the results by comparing MD simulations to MacB-like structures and propose a revised mechanism of transport as a function of its free-energy landscape. Lastly, I explore the use of cryo-electron tomography (cryo-ET) as a method to obtain *in vivo* structural insights. I show that the use of “ghost” partially lysed *E. coli* can produce high-contrast specimens for tomography. I collect a tomographic dataset of “ghost” MacAB-TolC containing cells and apply subtomogram averaging. Preliminary results suggest that MacAB-TolC forms an array in cells, and that MacB is structurally flexible, likely in its nucleotide-binding domain. Together, these studies of the MacAB-TolC efflux pump and the T1SS shed light on their function and suggest new avenues of research to explore in order to fulfil the goal of finding novel inhibitors.Item Embargo Electrochemical and genetic tools for analysing and engineering bioenergetic processes in cyanobacteriaLawrence, Joshua; Lawrence, Joshua [0000-0002-9250-8690]Oxygenic photosynthesis provides energy for the majority of Earth’s ecosystems. It is catalysed by photosynthetic electron transport chains: a collection of free and proteinbound redox cofactors found within the specialised thylakoid membranes of photosynthetic organisms. Cyanobacteria, to our knowledge, are the first organisms to have evolved oxygenic photosynthesis, and contribute approximately 25% of the Earth’s primary photosynthetic productivity. Cyanobacteria are also essential for maintaining important biogeochemical processes, such as the nitrogen cycle. Additionally, researchers have demonstrated how the electron transport chains of cyanobacteria can be ‘rewired’ for the sustainable production of electricity, fuels, pharmaceuticals, plastics, and high-value chemicals. However, research on cyanobacteria has been limited by the complex nature of their thylakoid membrane electron transport, which differs from that of plants and algae, not least because it includes a respiratory electron transport chain. Furthermore, the available tools for analysing and engineering cyanobacterial electron transport are scarce compared to those for model plant and green algal species. This thesis addresses this research challenge by developing a range of electrochemical and synthetic biology tools to analyse and engineer cyanobacterial electron transport. Firstly, a reproducible method of extracting cyanobacterial thylakoid membranes and wiring them to highly-structured electrodes was developed. By conducting electrochemistry experiments, electron transport pathways within these modified electrodes were determined, thereby establishing the technique as an analytical platform for studying cyanobacterial thylakoid membrane electron transport. The technique was used to answer biological questions inaccessible to other techniques, such as measuring plastoquinone reduction in different conditions. The technique was also utilised to engineer bio-photoelectrochemical systems for solar-powered electricity generation. An additional electrochemical platform was created and used alongside a series of analytical chemistry methods to study the role of outer membrane vesicles in cyanobacterial iron transport. The electron transport chains of cyanobacteria utilise numerous iron-containing redox cofactors, making iron availability essential for their assembly, function, and maintenance. This research revealed that outer membrane vesicles selectively uptake Fe3+ ions, answering a longstanding question on cyanobacterial physiology. Finally, in addition to these electrochemical platforms, a series of synthetic biology tools were created to aid the genetic manipulation of electron transport in cyanobacteria. These include improvements to an existing DNA assembly technique, and the creation of plasmids, selectable and counter-selectable markers, and CRISPR systems for use in different cyanobacterial species. This research provides crucial tools for advancing the understanding and engineering of bioenergetic processes in cyanobacteria.Item Embargo Glucuronic Acid Patterning on Xylan in Arabidopsis thalianaDavis, KatharinePlants represent the largest living source of biomass on Earth. Most of this biomass comes from the polysaccharide-rich secondary cell wall. Industrially, polysaccharides have a number of potential key uses. For these to be possible, it is important to understand how polysaccharides are synthesised and arranged to confer structural properties to the cell wall. The hemicellulose xylan is the second-most abundant secondary cell wall polysaccharide after cellulose. For angiosperms, the xylan backbone consists of repeating xylosyl residues and is decorated along its length by acetate and glucuronic acid (GlcA). It is likely that the position of these decorations, created by Trichome-Birefringence-Like (TBL) and Glucuronic acid substitution of Xylan (GUX) enzymes respectively, are key for the xylan molecule to perform a hypothesised linking function between cellulose and lignin, contributing to the wall’s recalcitrance to enzymatic degradation. While GUX1 is responsible for evenly spaced GlcA decoration, which covers the majority of the xylan backbone, GUX2 positions GlcA in clusters covering a smaller area. This work investigated the source of this activity difference and the impact of GlcA patterning on secondary cell wall structure and phenotype. The use of site directed mutagenesis in this work allowed investigation of the source of well-documented patterning differences between GUX2 from angiosperms, specifically AtGUX2, and gymnosperms, focusing on PtGUX2, and identified an SRF amino acid motif in PtGUX2, absent in angiosperms, crucial for creating consecutive GlcA decorations on the xylan backbone. Taken together with results from mutagenesis of the predicted active site of AtGUX1 and AtGUX2, this work suggested that the amino acid composition in the GUX catalytic C-terminus, including a GT8 domain, plays a key role in activity and patterning. However, creation of GUX chimeras showed that addition of the AtGUX1 N-terminal amino acid sequence to the C-terminus of AtGUX2 led to a significant increase in activity compared to the full length AtGUX2 enzyme. The chimera also produced an unusual patterning of unevenly spaced GlcA additions, extended along the xylan backbone. Interestingly, this uneven patterning did not lead to an increase in the proportion of xylan adopting a three-fold screw conformation in the plant cell wall. Combined with results from previous literature, this finding suggests that an even acetylation pattern may have more relevance for xylan conformation than GlcA patterning. Analysis of the activity of chimeras with swapped GUX and TBL29 N and C-terminal domains suggested that GUX and TBL29 activity exist in a fine balance, crucial for xylan patterning. Mass spectrometry results indicated that GlcA decorations by AtGUX2 were generally positioned closer to acetate than decorations by AtGUX1. Intriguingly, GlcA decorations by GUX1-2 were surrounded by less acetylation than GlcA by AtGUX2, and vice versa for the GUX2-1 chimera. Altogether, these results heavily suggest that the GUX N-terminus also plays an important, although perhaps indirect, role in determining activity.Item Controlled Access An Intermediate Heme Threshold is Necessary for the Activation of the Stress Response and Redox Homeostasis in YeastHe, YiyiRecent work in our lab and others has revealed that a certain threshold of mitochondrial respiration (40% of wild-type capacity) is necessary to promote chronological lifespan (CLS) extension via its dual roles in metabolic reprogramming to accumulate storage carbohydrates (trehalose and glycogen) and in the activation of the stress response to maintain homeostasis. The activation of the stress response includes the expression of molecular heat shock proteins (HSPs) and the antioxidant defence system mediated by the transcription factors Msn2/4, Gis1 and Hsf1. Here, we reveal that an intermediate threshold of heme biosynthesis is sufficient to activate the stress response and maintain mitochondrial redox homeostasis but not mitochondrial respiration. In the first result Chapter (Chapter 3), using the hem1∆ mutants supplemented with different concentrations of 5-ALA, multiple thresholds of heme biosynthesis were shown to be necessary for fermentative cell growth, starvation-induced HSP gene expression and redox homeostasis, and mitochondrial biogenesis and respiratory growth. The essentiality of heme synthesis was probably due to its roles in synthesis of unsaturated lipids. An intermediate threshold (~55% of the WT level) was required for the full activation of the HSP gene expression, restoration of mitochondrial superoxide and fermentative cell growth to WT levels. Higher heme levels than the intermediate threshold were required for inducing respiratory growth and mitochondrial biogenesis. In the second result Chapter (Chapter 4), transcriptome studies were conducted to reveal why the intermediate heme threshold was sufficient to activate the stress response and mitochondrial redox homeostasis. It has been found that the intermediate heme threshold is necessary to promote the transcription of ribosome biogenesis to support fermentative growth, while higher heme levels than the threshold are required to coordinate ribosome biogenesis and metabolic reprogramming in response to glucose starvation to promote respiratory growth. The intermediate heme threshold was sufficient to activate the expression of the HSP genes mediated by Msn2/4 and Gis1, and the Hap4-dependnet genes involved in oxidative phosphorylation. However, biochemical assays indicated that the intermediate heme threshold was not sufficient for the full activation of the antioxidant defence system. These data suggest that mitochondrial redox homeostasis is maintained at the intermediate threshold possibly due to limited respiration to generate ROS and the moderate activation of the anti-oxidant defence system to remove ROS, both of which are dependent on heme levels. In the final result Chapter (Chapter 5), genetic and biochemical assays were conducted to find why heme deficiency leads to defective stress response and redox imbalance. Firstly, heme deficiency below the intermediate threshold leads to hyperpolarization of mitochondria and the accumulation of active Ras on mitochondria during glucose starvation. Removal of Ras2 from heme-deficient cells enhanced catalase and mitochondrial superoxide dismutase activities, restored hydrogen peroxide to WT levels, but had little impact on the levels of labile heme or mitochondrial superoxide. These data suggest that heme deficiency below the intermediate threshold leads to redox imbalance due to excessive generation of mitochondrial superoxide and compromised antioxidant defence system. Secondly, unlike the cox mutants, the ER-resident NADPH oxidase Yno1 was not responsible for ROS accumulation in the heme-deficient mutants. Finally, exogenous hemin was shown to rescue the redox imbalance in mitochondrial respiratory mutants independently of the SOD and catalase activities or the heme oxygenase Hmx1. Put together, the above findings suggest that the intermediate heme threshold is necessary to maintain redox homeostasis through at least two distinct mechanisms: preventing aberrant Ras2 signalling to activate the Msn2/4- and Gis1-depedent stress response, and activating Hap4-dependent gene expression involved in oxidative phosphorylation to prevent excessive generation of mitochondrial superoxide. Labile heme may also participate in ROS removal independently of the antioxidant defence system. Given the role of heme in mitochondrial function and redox homeostasis, the intermediate heme threshold may provide a new perspective for understanding mitochondrial dysfunction and high levels of ROS in age-related diseases and exploring their potential therapies.Item Embargo Development of a reliable screening platform for PET hydrolysing enzymes in an ultra-high throughput setupSchaefer, ElenaThe increasing production of Polyethylene terephthalate (PET) combined with inadequate waste management and a lack of effective recycling technologies has led to the accumulation of PET material and significant environmental pollution. Bio-catalysis is a promising solution to address the recycling or upcycling of PET in an environmentally friendly and cost-effective manner. However, currently the low efficiency of known PET hydrolysing enzyme (PHE)s and the limited scope to scale up their production get in the way of using them at an industrial scale. Efforts have been made to identify novel PHEs and to improve the catalytic efficiency of known PHEs by using rational design approaches, applying homology searches, and/or machine learning. However, the capability of assessing the efficiency of PHEs is limited to low-throughput functional assays. Since PHEs stem from different enzyme families general findings on one PHE cannot easily be transferred to other known PHEs. Thus functional assays that are compatible with high throughput screens would enable far larger explorations of protein sequence space and thus increase the probability to identify highly active and industrially relevant PHEs. The present PhD thesis is aiming to develop a sensitive and reliable screening platform compatible with both low and ultra-high-throughput assay formats to support the directed evolution of known PHEs. Such a screening platform would facilitate the acquisition of large and high quality data sets of structure-function relationships of single residues within a PHE. These data-sets can thus be used to support computationally aided rational design approaches for enzyme improvement as well as to uncover a deeper understanding sequence landscape and function of PHEs which can in turn be applied to identify novel PHEs from metagenome data-sets. The core of screening platform is thereby formed by a platform organism, which contains both the gene encoding a PHE variant, as well as a transcription factor based synthetic gene circuit that senses and reports on the accumulation of the PET hydrolysis product terephthalic acid (TPA). Thus, the platform organism allows the use of PET as a substrate and enables a direct link of PET hydrolysis activity to a detectable signal, enabling genotype-phenotype coupling while facilitating the downstream screening process (Fig. 1). To screen for PHE activity, single platform organisms that carry the PHE library are compartmentalised (in e.g. microtiter plates or droplet microfluidics) to allow the extracellular PET hydrolysis reaction to feedback to the host organism and trigger the synthetic circuit while avoiding cross activity between two reactions and PHE variants. Upon completion of the reaction, the platform organism which now contains information on both genotype and phenotype of the PHE variant, can be pooled with the other host organisms and subjected to FACS for high-throughput screening of the library. In the course of this PhD project the host organisms *E. coli*, *P. putida* and *S. cerevisiae* have been identified as suitable platform organisms that have been validated to secrete a range of PHEs. Furthermore, functional TPA sensors have been developed for each of the determined host organisms. The functionality of both the PHE expression as well as the TPA sensor unit have successfully been tested individually and in concert but are yet to be tested when hosted within one and the same organism. This work offers a promising approach to improve the efficiency and stability of PET hydrolases by enabling the screen of large variant libraries, and thus to contribute to a sustainable PET waste management and a circular PET economy.Item Open Access Investigation of the interactions between Trypanosoma congolense invariant surface glycoproteins and components of the mammalian immune systemMcDowell, AliceAfrican trypanosomes are single-celled eukaryotic pathogens of humans and animals. As they are extracellular in vertebrate hosts, their cell surface forms the primary host-pathogen interface. Invariant surface glycoproteins (ISGs) are a large family of cell surface proteins with recent expansion and diversification in *Trypanosoma congolense*. *Trypanosoma brucei* ISG65 is known to be a receptor for complement C3, and *T. brucei* ISG75 has been implicated in trypanocidal drug uptake mechanisms. The function of other ISGs including all ISGs in *T. congolense* is unknown. The aim of the work here was to identify ligands for ISGs in *T. congolense*. First, the *T. congolense* ISG gene repertoire was investigated as a whole. Analyses of the sequences and synteny of ISGs in the available genome sequences were used to define a list of *T. congolense* ISGs. A phylogenetic analysis of *T. congolense*, *T. brucei* and *T. vivax* ISGs indicated that diversification of ISGs occurred before and after speciation events in the African trypanosome clade, and gave estimations of which ISGs are most closely related. *T. congolense* ISGs were found to be generally upregulated in the mammalian life stage (bloodstream form), supporting the hypothesis that these proteins may function as receptors for ligands in the mammalian host. Selected *T. congolense* ISGs were produced as recombinant proteins using bacterial and mammalian expression systems. Pulldown assays and mass spectrometry were used to identify ISG binding partners in goat serum. Putative ligands for two ISGs were identified. Protein-protein interactions were confirmed by surface plasmon resonance with purified ligands. ISG 64/65C was identified as a putative receptor for complement C3. ISG LBA was identified as a putative receptor for complement C4. The binding of ISG LBA to C4 did not disrupt the interaction between C4 and C4b binding protein *in vitro*. The putative C4 receptor, ISG LBA, was found to be non-essential in bloodstream form cells in culture, as demonstrated by successful production of a cell line in which both alleles were deleted by targeted homologous recombination. A polyclonal rabbit antiserum raised against recombinant ISG LBA was used to confirm presence of the putative receptor in wild type *T. congolense* protein lysates, and absence in lysates from the ISG LBA -/- cell line. Immunofluorescence assays were not conclusive, but were suggestive of surface localisation of ISG LBA in wild type *T. congolense*. This work represents the first characterisation of large cell surface protein receptors of bloodstream form *T. congolense*, and the first characterisation of a putative C4 receptor in an African trypanosome. This represents a major advance in our understanding of the interactions between trypanosomes and the mammalian immune system. This thesis provides a useful tool for the further study of ISGs, and is a significant step towards understanding the role of this protein family in African trypanosomes.Item Open Access Structural and functional elucidation of four putative PQS-binding proteins in Pseudomonas aeruginosaGrimm, Larson*Pseudomonas aeruginosa* is a multi-drug resistant, human opportunistic pathogen. If left untreated, *P. aeruginosa* can cause severe to life-threatening infections in people with burns, cystic fibrosis, and in immunocompromised patients. During chronic infections, *P. aeruginosa* primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing (QS). There are three key QS systems in *P. aeruginosa* responsible for driving global changes in virulence gene expression: the *las*, *rhl*, and *pqs* systems. Each of the *las*, *rhl*, and *pqs* systems rely on a receptor-autoinducer relationship: these receptor-autoinducer complexes are LasR-OdDHL, RhlR-BHL, and PqsR-PQS, respectively. When the receptors (LasR, RhlR, PqsR) bind with their cognate autoinducer (OdDHL, BHL, PQS, respectively), they act as transcription factors that ultimately stimulate the expression of hundreds of virulence-associated genes. The influence these QS systems have on the expression of virulence determinants has led to decades of scientific research focusing on the characterisation of these regulators. Although LasR and PqsR have been structurally elucidated, the RhlR crystal structure has long eluded characterisation and has been highly sought after due to its obvious potential as a therapeutic target. In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the *pqs* autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed *in vitro* using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” *pfpI* deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the *pfpI* deletion mutant and the *pfpI* transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the *pfpI* transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the *pfpI* deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism. Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of *pfpI* mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in *P. aeruginosa*. Lastly, proteomic analysis of a *pfpI* deficient mutant revealed global dysregulation of key biological processes.Item Embargo Quantitative characterisation of single-cell circadian rhythms in cyanobacteria Synechococcus elongatusEremina, AleksandraLife on Earth is affected by periodic fluctuations in light caused by our planet’s rotation. Circadian (24-h) rhythms enable living systems to adapt to environmental cycles and anticipate the changes in their surroundings. To perform their functions, the clocks must be sensitive to the entraining cues and simultaneously resilient to perturbations in their environments. It is unclear how clocks have evolved to find this balance in any species. Here, we used the cyanobacterium *Synechococcus elongatus* to investigate the clock dynamics in individual cells otherwise masked in bulk studies. To do this, we developed a microfluidic setup, allowing us to carefully control cellular environments and quantify circadian gene expression and clock-modulated cell growth and division. Using quantitative time-lapse microscopy, we measured the robustness of cyanobacterial circadian rhythms with previously inaccessible precision. We observed extremely robust timekeeping under various deterministic light environments. Using clock mutants, we revealed that the rhythm robustness under constant light is maintained even without individual regulators of the clock. In contrast, perturbations to the core clock genes affected free-running rhythms and clock entrainment in individual cells. Exposed to variable light periods, the clock buffered the noise by shifting its phase only by a fraction of the perturbation. The clock also made cell cycle duration less variable under such conditions. However, the clock timing was sensitive to the meteorological fluctuations in light amplitude. Comparing wild-type and clock-less cells, we observed clock-induced growth acceleration during the day. This growth advantage was consistent in the absence of light amplitude fluctuations and remains to be understood under more natural conditions. Overall, we provided a novel experimental framework for studying the robustness of individual clocks and revealed the principles of clock-environment interactions useful for informing strategies for altering natural and synthetic clocks for biotechnological applications.Item Open Access Cell-type-specific responses to DNA damage from acute and chronic radiationLowe, DonnaIt is well understood that radiation causes damage to DNA, which triggers a DNA-damage response often centred on the signalling pathways of ATM, with the subsequent involvement of proteins such as p53, p21 and H2AX. Many experiments have been performed with cell lines derived from different origins and donors which, while invaluable for studying specific effects, restrict direct assessment of response variability between different cells, tissues and individuals. This thesis investigates normal tissue responses to DNA damage from acute and chronic radiation, including exploration of cell-type and individual-specific differences in DNA-damage response. Due to the difficulty of obtaining different primary cell types of isogenic background (necessary to prevent confounding results by inter-individual variation), there are still unanswered questions regarding the extent and details of cell-type-specific responses to ionising radiation. I have developed a method for isolation of primary cells from human tissue and compared cellular outcomes and molecular signalling in response to an X-ray dose. Using these primary cells, I show that cell type causes far more variability in radiation response, with keratinocytes, which have highest p21 protein levels, being most susceptible to cell cycle arrest and senescence, while donor variability is limited. Another issue that has received insufficient attention is the biological effects of chronic radiation. Both experimental and epidemiological evidence highlight the damaging and disease-causing effects of high doses of radiation. As there are limited facilities worldwide to perform the experiments, understanding of the cellular response to chronic radiation is not well known. To address this, I focused on characterising the response of primary cells isolated from neonatal foreskin donors and exposed to chronic radiation. Not surprisingly, results show that chronic radiation elicited many classic DNA-damage response outcomes such as cell cycle arrest, apoptosis and senescence. However, unlike acute radiation, the chronically irradiated cells demonstrated a reduction in histone levels accompanied by significant senescence induction and increased global transcription, indicating deregulation of gene expression. These characteristics are associated with age-related pathologies. Therefore, I extensively tested epigenetic age in these cells, which uses consistent changes in DNA methylation with age to accurately predict age. This demonstrated that epigenetic age was not altered by chronic radiation, but since this only captures certain aspects of ageing, the results suggest that chronic radiation increases wear-and-tear aspects of ageing. Overall, the work described in this thesis contributes to understanding the extent of normal radiation response variability in donors and cell types. It also reveals histone reductions associated with cellular senescence as an effect of continuous exposure to low doses of ionising radiation that may have wider implications for ageing and the incidence of specific pathologies.