This project investigated the role of the OmpA linker in Escherichia coli mechanical integrity. Three mutant constructs were generated: α-helix linker, intrinsically disordered (IDP) linker, and double-length linker. Western blotting confirmed proper expression and folding of the IDP and α-helix linkers; the double linker appeared as a smear, indicating multiple conformations. Functional assays including efficiency of plating (EOP) under ampicillin and EDTA stress, and hypo-osmotic shock of ∆ompA ∆lpp strains, showed that the IDP linker maintained mechanical integrity comparable to wild-type, while the α-helix linker was less protective. The double linker performed similarly to wild-type in hypo-osmotic shock but had heterogeneous expression. Results indicate that linker flexibility is important for OmpA-mediated cell envelope strength.

This project aimed to expand the multiplexing capacity of the Vibrio cholerae Type I-F CRISPR–Cas system by designing diverse gRNA scaffold variants. Six scaffold variants were generated computationally using NoRDe to introduce sequence diversity while preserving secondary structure. Synthetic DNA fragments were cloned into two plasmid backbones (pSPIN v3 for single-guide and pDonor for 12-guide arrays), sequence-verified, and transformed into Escherichia coli strains. Gel electrophoresis and colony PCR confirmed successful integration. Among single-guide arrays, only the 4.6 scaffold variant consistently integrated reliably, suggesting scaffold diversification affects RNA processing. The 12-guide arrays were assembled successfully, but their activity was inconsistent in E. coli, supporting the idea that structural changes may interfere with guide maturation. Scaffold diversification improved synthesis feasibility and reduced assembly issues, demonstrating practical advantages despite functional variability.

This project examined how growth factor signals influence how pancreatic cancer cells interact with and respond to their surrounding environment. Using PANC-1 pancreatic cancer cells, the study focused on the effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on key signalling pathways and cell–matrix adhesion sites. Cells were stimulated with either growth factor and analysed over time using protein assays and fluorescence microscopy. The results showed that EGF triggered a rapid activation of ERK signalling followed by sustained Akt activity, while HGF produced weaker ERK activation but a strong, shorter-lived Akt response. These signalling differences were linked to changes in focal adhesions, with EGF associated with early adhesion turnover and HGF promoting greater redistribution of adhesion proteins linked to cell movement. Overall, the findings highlight how different external signals can shape cancer cell behaviour by coordinating biochemical signalling with physical interactions at adhesion sites, improving understanding of mechanisms that may contribute to pancreatic cancer progression.

This project investigated how EF-hand motifs in Marchantia polymorpha calcineurin B-like (CBL) proteins contribute to calcium sensing during plant salt-stress signalling. As soil salinisation threatens global food security, understanding calcium-based signalling pathways is essential. Plasmids expressing wildtype and EF-hand mutant CBL-C proteins were generated in Escherichia coli, followed by protein purification and calcium-binding assays. Initial Ni-NTA purification successfully isolated a ~26 kDa CBL-C protein, enabling calcium-shift assays that showed optimal mobility detection using SDS-free gels, SDS-free loading buffer and no heat denaturation. Later purifications were unsuccessful, limiting further biochemical analysis. Golden Gate cloning was used to produce several EF-hand mutants; sequencing confirmed correct assembly only for mutants 1+2 and 1. Despite technical barriers, the project established key methods for expressing, purifying and functionally assessing CBL proteins, contributing to understanding how early-diverging land plants decode calcium signals during salinity stress.

This project investigated the determinants of HTLV-1 retroviral integration into the human genome, aiming to identify host factors influencing site selection and potential disease progression. Computational analyses compared integration sites from in vitro infections, in vivo patient samples, and matched random controls, focusing on proximity to histone modifications and replication timing of chromosomes. ChIP-seq data from IMR90 cells were used to assess enrichment near 22 histone marks, validated against primary CD4⁺ T-cell data. HTLV-1 showed enrichment near transcriptionally active marks, particularly H3K4me3 and H3K9ac, while H3K79me1, H3K79me2, and H4K20me1 showed counterselection in vivo, suggesting possible host repression mechanisms. Integration was modestly biased toward early-replicating chromosomes in vitro and late-replicating chromosomes in vivo, with acrocentric chromosomes preferentially targeted. These results highlight the role of chromatin accessibility in integration and suggest potential interactions with PP2A-B56 and epigenetic regulators. Future work will validate findings in CD4⁺ T cells and identify host partners to better understand HTLV-1 integration and its link to disease risk.

This project investigated the potential of lipid transporters (LTs) to enhance terpenoid production in engineered Escherichia coli by alleviating intracellular toxicity. Four LTs were cloned individually and in combination into plasmids and co-expressed with terpenoid biosynthetic pathways producing amorphadiene, linalool, or cineole. Cloning success was confirmed via colony PCR and whole-plasmid sequencing, and transformed cells were cultured under controlled induction conditions. Terpenoid titres were quantified using GC-MS. Results showed that expression of the tested LTs, individually or combined, did not significantly increase terpenoid production or tolerance under the conditions tested, indicating these LTs likely do not bind the selected terpenoids. Despite negative results, this work provided valuable experimental data to refine computational models predicting LT-ligand specificity. Shadowing bioreactor runs further enhanced understanding of growth optimisation and large-scale fermentation.

This project investigated the relationship between the neuronally expressed intramembrane protease RHBDL3 and a potential interactor, protein X. Using immunoprecipitation in primary rat cortical neurons, RHBDL3 was shown to pull down ~10% of protein X, confirming a direct interaction. Five shRNA constructs targeting protein X were cloned into a pXAG vector, packaged into lentivirus, and used to deplete protein X in neurons. Knockdown was successful, but depletion of protein X did not alter RHBDL3 protein levels, indicating it does not regulate RHBDL3 stability. Alkaline phosphatase assays with artificial rhomboid substrates further demonstrated that RHBDL3 does not cleave protein X, nor is RHBDL3 activity affected by protein X overexpression. These results suggest that while protein X is a bona fide interactor of RHBDL3, it is neither a substrate nor a regulatory co-factor. This work provides tools and insight into RHBDL3’s neuronal interactions, relevant to neurodegenerative disease and ageing.

This project investigated how DNAJB6, a molecular chaperone, influences the ATPase activity of HSP70, a key protein involved in preventing misfolding and aggregation. DNAJB6 contains a J-domain that stimulates HSP70 activity, connected via a flexible GF-rich linker and a short Helix 5, which may autoinhibit the interaction. The study compared three DNAJB6 constructs: the J-domain alone (JD), JD with the GF-linker (JD-GF), and JD with the GF-linker plus Helix 5 (JD-GF-α5). ATP hydrolysis by HSP70 was measured using NADH-coupled assays and ³¹P NMR. Results showed that JD strongly stimulated HSP70 at low concentrations, while JD-GF-α5 had minimal effect at low concentrations but matched JD at higher concentrations, indicating concentration-dependent release of autoinhibition. These findings support a model where the GF-linker may enhance activity despite reduced binding affinity. The project provides functional validation of previous structural data and informs how autoinhibition and linker interactions regulate HSP70 activity.

This project aimed to generate GFP-fusion constructs of the MATE1 transporter in Dictyostelium discoideum to visualise its subcellular localisation and study its role in metabolite and xenobiotic efflux. Primers for the DdMATE1 coding sequence were designed in silico using Benchling, and PCR conditions were optimised to produce a single, high-quality amplicon. The PCR product was cloned into the pDT29 plasmid using BamHI and XhoI restriction sites to create an in-frame MATE1-GFP fusion. Ligation products were transformed into Escherichia coli, screened via colony PCR and restriction digestion, and successful clones were confirmed by Sanger sequencing. Four clones showed correct in-frame insertion and removal of the stop codon, producing functional MATE1-GFP constructs. Future work will involve transforming these constructs into D. discoideum for confocal microscopy, potentially complemented by MATE2-GFP fusions, to study transporter localisation and function across the organism’s life cycle.

This project investigated how the microtubule-associated protein EB1 from Plasmodium falciparum (PfEB1) interacts with microtubules compared to human EB1 (HsEB1). EB1 proteins regulate microtubule dynamics, which are crucial for cell division and parasite development. The CH domains of both PfEB1 and HsEB1 were expressed and purified, showing that the proteins were mostly monomeric and stable when snap-frozen. Co-pelleting and co-polymerisation assays indicated that both proteins specifically bind to taxol-stabilised and GMPCPP microtubules, with stronger binding to GMPCPP microtubules, suggesting preference for the GTP-like lattice. Binding also stabilised microtubules over time. Total internal reflection fluorescence microscopy gave mixed results, with HsEB1 showing binding to GMPCPP microtubules but PfEB1 showing minimal binding, possibly due to experimental artefacts. Preliminary cryo-EM data were collected but not fully processed. These findings establish methods to study PfEB1–microtubule interactions and provide a foundation for future structural and functional characterisation, potentially revealing parasite-specific mechanisms that could be therapeutic targets.

This project investigated the potential iron-dependent regulation of Neuropilin-1 (NRP1), a protein implicated in endothelial cell stability and protection against iron accumulation, which may play a role in diseases such as Alzheimer’s. Human embryonic kidney cells were first transfected with a control plasmid containing a canonical iron response element (IRE) fused to GFP, or GFP alone, and treated with increasing concentrations of iron citrate. Cells expressing IRE-GFP showed increased fluorescence with rising iron levels, whereas GFP controls showed no change, validating the system. In parallel, human vascular endothelial cells (HUVECs) were transfected with NRP1 or GFP constructs and analysed via immunostaining and RT-qPCR to assess NRP1 expression and interaction with VE-cadherin. These preliminary results establish a platform to test whether NRP1 mRNA contains a functional IRE, enabling iron-responsive translation. Understanding this mechanism may clarify NRP1’s cytoprotective role and provide insight into iron dysregulation in Alzheimer’s and other vascular diseases.

This project aimed to develop robust synthetic methodologies for archaeal lipid analogues to enable systematic biophysical studies and investigate membrane evolution. Archaeal lipids differ from bacterial lipids by containing isoprenoid ether tails, opposite glycerol chirality, and increased stability, yet their biophysical properties remain poorly characterised. Racemic ether phosphatidic acid (PA) with 12-carbon tails was synthesised from solketal, achieving 24% yield, while improved routes were developed for enantiopure ether PA from D-mannitol using sodium periodate cleavage and optimized reduction. Preliminary liposome formation studies indicated that Na⁺ salts have a lower critical vesicle concentration than NH₄⁺ salts, though aggregation required gentle rehydration. Phosphorylation under prebiotic conditions using urea was successful for mono- and di-alkyl ether glycerols. This work lays the foundation for future studies comparing homochiral and heterochiral membranes, investigating diverse headgroups and isoprenoid tails, and assessing advanced properties such as lipid packing, charge, and functional relevance.

This project investigated the RNA-binding ability of TRIM-NHL proteins. Full-length human TRIM-NHL protein was expressed in Escherichia coli and purified via HisTrap and size-exclusion chromatography, yielding 30 mg of >95% pure protein from 13 L culture. The NHL domain was crystallized using the sitting-drop method, though no crystals formed; pre-crystalline material was observed. RNA binding was assessed by EMSA using Cy5-labelled tRNA, showing decreased RNA mobility at higher protein concentrations, indicating binding. The observed interaction is consistent with TRIM-NHL recognition of RNA motifs, although the tRNA used showed lower affinity compared to sequences in the literature. The project successfully produced pure protein and demonstrated RNA-binding activity of TRIM-NHL.

This project explored enhancing CO₂ assimilation in Vibrio natriegens via the reductive glycine pathway (rGlyP) for electromicrobial production (EMP). Heterologous formate dehydrogenase (FDH) genes from Shewanella oneidensis, Cupriavidus necator, and Bacillus subtilis, including associated sulfurtransferases, were cloned into expression plasmids and sequence-verified. These FDHs were chosen for their reported catalytic efficiency, oxygen tolerance, and potential role in CO₂ reduction. Concurrently, knockout mutants for three glycine cleavage system (GCS) genes (gcvP, gcvH, 10795) were attempted to assess their impact on rGlyP function. Colony PCR results were inconclusive, indicating low editing efficiency and requiring further optimisation, such as redesigning tDNAs or extending homology arms. The FDH constructs are ready for functional validation via protein expression and activity assays, preceding isotope-labeling studies for CO₂ assimilation.

This project focused on using degron technology to study two synaptonemal complex proteins, SYCP1 and TEX12, which are normally involved in chromosome pairing during meiosis but are also expressed in some cancers. The aim was to generate plasmids that allow rapid degradation of these proteins in cells, enabling researchers to study their immediate role in cancer cell function. The project involved cloning donor plasmids containing the degron tag and Cas9/gRNA plasmids to insert the tag into the target genes using CRISPR. PCR optimisation, ligation, transformation, and test digests were performed to verify successful cloning. Positive results were obtained for the right homology arms, which were sent for sequencing, but challenges with the left arms and Cas9 plasmids remained unresolved within the project timeframe. Despite these setbacks, the work established a foundation for future experiments to rapidly deplete SYCP1 and TEX12.

This project established an anaerobic workflow for time-resolved serial X-ray crystallography at the T-REXX endstation to study enzyme dynamics under oxygen-free conditions. Microcrystals of Aerococcus viridans L-lactate oxidase (LOX) were produced using the hanging-drop method, with optimum crystallisation at pH 4.36–4.56 and 45% ethylene glycol, yielding high-quality diffraction. Initial aerobic crystallography confirmed the LOX space group (I4) and provided a 1.59 Å structure, including a refined model of a disordered active-site lid. To enable anaerobic experiments, a glovebox was purged to 5 ppm oxygen, and an airtight transfer chamber was designed to maintain anaerobicity when moving crystals to T-REXX, where the sample chamber achieved 30–170 ppm oxygen depending on configuration. Although time-resolved anaerobic data could not be collected due to protein delays, the project successfully developed protocols, established crystallisation conditions, and built infrastructure for future anaerobic studies of LOX and other oxygen-sensitive enzymes.

This project aimed to synthesise a trametinib-based PHOSTAC to target the RAF-MEK-ERK signalling pathway in cancer cells. PHOSTACs are bifunctional compounds that link Halo-tagged protein phosphatase 1 (Halo-PP1) to a target protein, promoting dephosphorylation and durable inhibition of signalling pathways. The trametinib-based design was intended to improve potency over previous mirdametinib-based PHOSTACs by simultaneously inhibiting MEK’s upstream and downstream interactions. A four-step synthetic route was planned, optimised using literature references, and executed experimentally. Initial steps, including deacetylation of trametinib and attachment of a chloroalkane to 6-PEG, were successful. Later novel steps required iterative optimisation, with the final amide coupling achieved using DMTMM and heat. The final PHOSTAC was confirmed by NMR and mass spectrometry, yielding 2.4 mg (26.7%). Preliminary assays indicate binding to both MEK and PP1, though not yet forming the desired ternary complex.

This project evaluated the effect of the antioxidant MitoQ on mitochondrial function in A549 cybrid cells carrying the m.3243A>G MELAS mutation. Cells were treated with MitoQ or DMSO controls, and mitochondrial membrane potential was measured using TMRM staining, while oxygen consumption rates were measured with the Seahorse XFe96 Analyzer. Mutant cells displayed significantly lower mitochondrial membrane potential compared to wild-type controls. Two-way ANOVA revealed significant effects of genotype, treatment, and their interaction on TMRM intensity. Seahorse analysis indicated that MitoQ treatment did not improve oxygen consumption or bioenergetic parameters in mutant or wild-type cells. The results confirm that MELAS cybrids have impaired mitochondrial function and show that MitoQ had no measurable beneficial effect under the tested conditions.

This project examined the effect of phosphorylation at T104A on LASP1 localisation in HeLa cells. LASP1 expression was confirmed by western blotting. Knockdown of LASP1 using shRNA constructs demonstrated successful reduction of endogenous protein. Transfection of wild-type and T104A LASP1 constructs showed similar expression levels, but immunofluorescence revealed altered localisation in the mutant, suggesting that phosphorylation at T104 influences LASP1 cellular distribution. These findings provide insight into the molecular regulation of LASP1 in HPV-positive cervical cancer cells and its potential impact on cellular interactions and function.

This project investigated the relationship between centrosomes, KIFC1 expression, and chemotherapy response in triple-negative breast cancer (TNBC) using an ethical chorioallantoic membrane (CAM) xenograft model. MDA-MB-231 TNBC cells were implanted on CAMs, and systemic chemotherapy agents (etoposide and cisplatin) were administered via yolk sac injection. Tumours were harvested, sectioned, and analysed using immunohistochemistry for DNA damage (γH2AX) and KIFC1 expression. Image analysis was performed using QuPath, with H-scores calculated to quantify staining intensity. Acute etoposide treatment increased DNA damage and elevated KIFC1 expression in G2/M cells, while sustained cisplatin treatment reduced tumour size without upregulating KIFC1, indicating that KIFC1 elevation is linked to acute DNA damage rather than prolonged chemotherapy. These results demonstrate the utility of CAM xenografts for studying systemic drug effects and centrosome-associated pathways in TNBC.

This project investigated cohesin dynamics in Saccharomyces cerevisiae, focusing on an intermediary population of cohesin. Yeast strains were engineered with tagged cohesin-associated proteins: CDC45-Halo and Smc4-AID. DNA was extracted, amplified by PCR, and transformed into host strains using lithium acetate/PEG. Colony growth and PCR optimisation revealed strain-specific growth characteristics and highlighted challenges in transformation efficiency. Although the final strains were not fully generated during the studentship, a functional workflow for DNA extraction, PCR, and transformation was established. The student also contributed to complementary live-cell imaging, including DAPI and EdU staining, gaining experience in microscopy and fluorescent quantification. The work laid a foundation for tracking cohesin diffusion and benchmarking intermediate cohesin populations.

This project investigated whether sex steroid hormones alter lymphocyte metabolic pathway usage using the SCENITH assay. Peripheral blood mononuclear cells (PBMCs) from ten healthy donors (5 female, 5 male) were cultured for five days under stimulatory or non-stimulatory conditions, with dihydrotestosterone (DHT), 17β-oestradiol (E2), or ethanol added during the final 24 hours. Metabolic dependencies were profiled by measuring protein synthesis after treatment with pathway inhibitors (2-deoxy-D-glucose, oligomycin, etomoxir). Overall, OPP incorporation, reflecting metabolic output, was not significantly altered by hormone treatment in CD4+, CD8+, or CD19+ cells. Glycolysis inhibition consistently reduced protein synthesis, whereas mitochondrial pathway inhibition had limited effects. No significant sex-specific differences were observed, likely due to the small cohort and assay sensitivity. The study highlights the need for larger donor numbers and further optimisation of SCENITH to detect subtle hormone-mediated metabolic changes.

The project established methods to detect and analyse interactions between peroxisomes and mitochondria, with a focus on neuronal cells. SHSY5Y cells were differentiated into mature neuron-like cells, and neuronal morphology and marker expression were confirmed using immunofluorescence. Peroxisomes and mitochondria were visualised in fixed and live cells using fluorescent dyes, transfected marker proteins, and widefield, confocal, and spinning disc confocal microscopy. Image analysis using ImageJ/FIJI, including the Colocalisation Finder and TrackMate plugins, quantified organelle overlap, movement dynamics, and contact durations. Live imaging revealed distinct behaviours of peroxisomes and mitochondria, including independent and codirectional movement, with colocalisation not affecting movement patterns. Contact duration analysis was fitted to a double exponential model, allowing quantification of long-lasting and transient interactions. SplitFAST reporter constructs were also tested to visualise membrane contact sites, with successful application for mitochondria-ER contacts. The project developed robust methodologies for quantitative assessment of peroxisome–mitochondrion interactions and provided extensive experience in cell culture, imaging, image analysis, and molecular techniques.

This project investigated epithelial-to-mesenchymal transition (EMT) in choroid plexus (ChP) organoids following mechanical injury. Mature day-53 organoids were subjected to scalpel-induced injury, then processed for immunohistochemistry (IHC) and RNA analysis. IHC targeted epithelial markers (TTR, ARL13B) and the stromal marker TAGLN, while qPCR quantified EMT-associated transcription factors (PRRX1, TWIST1, SNAI2) and TAGLN expression. Injured organoids showed a 4–11-fold upregulation of EMT transcription factors and a 4-fold increase in TAGLN, supporting the hypothesis that ChP epithelium undergoes EMT after injury. Treatment with an EMT inhibitor (SB431542 and RI) prevented these changes, confirming the TGF-β/SMAD2/3 pathway’s role. TTR levels did not significantly change, potentially reflecting delayed EMT responses. This work provides preliminary evidence for injury-induced EMT in human ChP organoids, advancing understanding of ChP plasticity and offering a platform for future studies.

This project developed an in vitro neuronal model to study TSC1-associated dysregulation of mTORC1 in Tuberous Sclerosis (TS). Primary cortical neurons were harvested from rat embryos and transduced with lentiviral constructs to knock down endogenous TSC1 and re-express either wild-type or mutant forms, including patient-derived variants. Validation showed that shRNA-mediated TSC1 knockdown (particularly shRNA4) effectively reduced TSC1 protein levels and induced hyperactivation of mTORC1, as indicated by elevated phospho-S6 levels. Re-expression of wild-type Halo-TSC1 partially rescued TSC1 levels and reduced phospho-S6, confirming functional restoration. Mutant constructs I954E and F958R displayed poor expression, while the patient-derived R786X mutant expressed robustly. This system establishes a platform to investigate how specific TSC1 mutations disrupt neuronal signalling and function. The project provides a foundation for future research exploring neuronal phenotypes, autophagy, and potential pharmacological interventions in TS.

This project investigated how the receptor LOX-1 recognises oxidised low-density lipoprotein (ox-LDL), a key interaction involved in cardiovascular disease and cancer development. LOX-1 activation by ox-LDL triggers inflammatory and adhesion-related signalling, but the molecular details of this interaction remain unclear. To address this, the study used a cell-free protein expression system to produce soluble LOX-1 (SLOX-1), allowing rapid protein production without the need for living cells. The protein was successfully expressed and purified as a stable dimer, which is the biologically relevant form of LOX-1. Optimised purification methods selectively enriched the dimeric complex. Binding assays were then used to test interactions between SLOX-1 and ox-LDL, although clear binding was not detected under the conditions tested. Despite this limitation, the project established a reliable and fast workflow for producing functional LOX-1 protein. This provides a strong foundation for future studies aimed at improving binding assays and determining the structure of LOX-1–ox-LDL complexes using advanced imaging techniques.

This project studied how bacteria accurately separate their chromosomes during cell division, a process essential for cell survival. In many bacteria, chromosome segregation is controlled by the ParABS system, involving the proteins ParA and ParB interacting with DNA. Using Vibrio cholerae proteins, ParA and ParB were successfully produced and purified in the laboratory. Experiments showed that ParA can form filament-like structures on DNA in the presence of ATP, supporting existing models of how chromosomes are moved within the cell. When ParB was added, less ParA remained bound to DNA, suggesting that ParB helps trigger ParA release, which may drive chromosome movement. Electron microscopy was used to visualise ParA–DNA filaments, although time constraints prevented clear imaging of complexes containing ParB. Overall, this work developed reliable experimental methods and provided evidence supporting current models of bacterial chromosome segregation, helping to advance understanding of a fundamental cellular process with potential relevance to antimicrobial research.

This project investigated the effects of ascorbic acid (AA) on medulloblastoma (MB) SHH-subgroup spheroids, focusing on DAOY and ONS-76 cell lines. 3D spheroids were generated and treated with low (100 µM) or high (2.5 mM) AA, etoposide (0.4 µM), or combinations. Spheroid growth was monitored over 72 hours using ZOE imaging and quantified with ImageJ, with viability assessed via Live/Dead staining. DAOY spheroids were consistent, whereas ONS-76 spheroids showed high variability and were excluded. In DAOY spheroids, AA alone had no effect, while etoposide reduced growth as expected. Co-treatment with AA (both concentrations) enhanced etoposide’s cytotoxic effect rather than providing protection, indicating that AA’s previously reported “twin effect” with cisplatin does not generalize to etoposide. These findings highlight chemotherapy-specific interactions with AA and demonstrate that AA can modulate drug efficacy.

This project investigated whether the molecular glue VH298 induces interactions between CDO1 and the VCB E3 ligase complex (VHL-Elongin B/C). CDO1 and VCB were expressed and purified from Escherichia coli, including ¹⁵N-labelled CDO1 for NMR studies. Initial ¹H/¹⁵N HSQC NMR spectra showed no intrinsic interaction between CDO1 and VCB. However, addition of VH298 led to signal loss in the spectra, indicating formation of a ternary complex dependent on the ligand. Higher protein-to-ligand ratios enhanced this effect. Comparison with VH032, a previously characterised molecular glue, revealed that VH298 engages CDO1 differently, affecting some residues uniquely. These results suggest VH298 can “glue” CDO1 to VCB but induces a distinct interaction pattern compared to other glues. Future work will involve backbone resonance assignment, control NMR experiments without VCB, 3D structural characterisation, and binding affinity measurements to fully elucidate the molecular mechanism. 

This project aimed to identify perivascular astrocytes in the glia limitans of mice using the nuclear protein ID3 as a candidate marker. Astrocytes regulate brain homeostasis and the blood-brain barrier (BBB), but specialised perivascular astrocytes remain poorly characterised. Using single-cell RNA sequencing, ID3 was highlighted as a potential marker expressed in astrocytes and endothelial cells. Samuel optimised immunohistochemistry protocols on fixed-frozen cryosections and floating slices to visualise ID3 expression alongside GFAP and CD31, revealing ID3-positive nuclei adjacent to blood vessels with astrocytic processes. FFPE tissue was less reliable for staining. Confocal microscopy and slide scanning confirmed ID3 expression in cells with circular nuclei morphology distinct from endothelial cells, suggesting successful identification of perivascular astrocytes. Future work includes using ID3 to isolate these astrocytes for single-nucleus RNA sequencing to define their transcriptional profiles.

This project characterised the biophysical properties of two truncated hemoglobins, Bjgb from Bradyrhizobium diazoefficiens and DsGB1 from Dinoroseobacter shibae, implicated in nitrate assimilation. Bioinformatic analyses using Clustal Omega, AlphaFold3, and PyMOL predicted heme-ligating residues and structural similarities with other bis-ligated low-spin truncated hemoglobins. Both proteins were heterologously expressed in Escherichia coli, purified using Ni²⁺ affinity chromatography, and verified by SDS-PAGE. UV-visible spectroscopy revealed typical low-spin heme features, with DsGB1 displaying an additional 650 nm peak corresponding to a modified heme, confirmed by reconstitution and mass spectrometry. Redox potentiometry determined reduction potentials of +27 ± 5 mV for Bjgb and -59 ± 2 mV for DsGB1, consistent with predicted coordination. Due to time constraints, nitrogen oxyanion binding was not assessed. This work provided high-quality purified proteins and foundational biophysical characterisation, offering tools for future studies on nitrogen oxyanion interactions, heme coordination, and contributions to environmentally relevant nitrogen cycling.