http://theses.ncl.ac.uk/jspui/handle/10443/97 2026-02-04T14:20:06Z http://theses.ncl.ac.uk/jspui/handle/10443/6668 Title: Running the Gauntlet of the Bacterial Cell Wall Authors: Alofi, Amirah Abstract: In nature, bacteria have a variety of shapes, from simple cocci and rods to more complicated spiral or appendage structures. In their life, they need a strong exoskeleton that can maintain their shape during growth and protect them from changes in environmental conditions. This rigid structure is generally provided by the cell wall and is considered an essential component in the bacterial cell. However, the cell wall represents a complex logistical problem in terms of the controlled synthesis and degradation that is required to maintain shape, permit enlargement, and prevent lysis. Significant advances have been made in understanding wall synthesis, and in Gram-negative bacteria synthesis can be integrated with degradation. But, for Gram-positive bacteria the regulation of cell wall degradation is poorly characterised. Genetically the genes encoding the key enzymes are known and the expression of these enzymes are known to be highly regulated. However, understanding how the biochemical activity of these enzymes is restricted and regulated, presumably within the cell wall or on the outside surface of the cell is not clear. The results obtained in this laboratory have indicated that altered cell wall composition or media components modulate the level of the hydrolysis enzyme activity in some way during vegetive growth. This study aimed to extend our understanding of the autolytic systems and how the enzymatic activity may be regulated by components of the cell envelope or minor modifications of the cell wall material. Description: Ph. D. Thesis. 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6460 Title: Manipulation of copper bioavailability in pathogenic bacteria can disrupt metabolism Authors: Stewart, Louisa Jane Abstract: Resistance to traditional antibiotics continues to rise in pathogenic bacteria and the growing worldwide alarm has intensified research within the scientific community seeking alternative antimicrobial therapies. Fundamental to this research is the unravelling of bacterial metabolic pathways to identify proteins susceptible to disruption through either the addition of toxins or the limitation of nutrients and thus reveal novel antimicrobial targets in key pathogens. Copper is an essential micronutrient, required by bacteria as a cofactor in many enzymes, but it can also be toxic when present in excess. Manipulation of the bioavailability and localisation of bacterial copper to create a toxic weapon or to cause nutritional deficiency was explored in pathogenic bacteria through the study of the interactions of copper with key metabolic proteins and with a homologue of a copper storage protein (Csp) that was originally characterised in a strain of Methylosinus trichosporium. Csp1 has been shown to supply copper to the particulate form of methane monooxygenase in a methanotroph, but this copper-enzyme is not found in Neisseria gonorrhoeae, a human pathogen in which a Csp1 homologue has been identified. This organism has undergone extensive genomic reduction, therefore the retention of csp1 suggests a requirement for this protein in this pathogen. In Neisseria gonorrhoeae, potential ‘clients’ for receiving copper from a Csp include metabolic proteins from two alternative respiratory pathways: a nitrite reductase (AniA) and a cbb3 type cytochrome c oxidase (Cco). Using mutant strains of Neisseria gonorrhoeae and manipulation of the copper bioavailability, a growth phenotype was observed in a csp1 knockout, suggesting a role associated with a shift in copper concentration. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the most abundant bacterial metabolic enzymes with a major function in the glycolytic pathway, has been shown to bind copper. Phenotypic investigations of the effect of increasing copper bioavailability in GAPDH-challenged mutants of Staphylococcus aureus showed a weak growth defect, and there was evidence of a change in metabolism, suggesting an increase in copper bioavailability could force the cells to use an alternative energy production pathway; a vulnerability worthy of further exploration. Description: PhD Thesis 2024-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6373 Title: Gene-editing of human pluripotent stem cells for cystic fibrosis modelling Authors: Cuevas Ocaña, Sara Abstract: Cystic fibrosis (CF) is a genetic disorder caused by mutations in the CFTR gene. A defective CFTR protein produces an impaired ion and fluid secretion in the epithelial cells that affects many organs and leads to severe lung disease. Despite recent treatments have been approved for specific genotypes, ~ 44% of people with CF still lack an effective treatment. Therefore, there is a need for more CF research and drug testing in order to find treatments for all CF patients and all relevant affected tissues. Human pluripotent stem cells (hPSCs) hold the potential to provide unlimited production of tissue-specific in vitro live cultures. Geneediting technologies, including TALENs and CRISPR/Cas9 systems, enable introducing or correcting specific mutations for disease modelling, although this is still challenging and time-consuming in hPSCs. This project demonstrates the generation of a robust hPSC geneediting strategy that can be performed in a relatively short time in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) without specialised equipment. This strategy consists of (i) the efficient transfection of engineered nucleases, ssODN and a plasmid containing puromycin resistance into S phase pre-synchronised hPSCs. (ii) Rapid assessment of nuclease activity and integration or correction of the mutation of interest in the initial mixed population, (iii) followed by a rapid isolation and identification of correctly modified clones. This strategy was used to generate a homozygous ΔF508-CF hESC line, and to correct W1282X-CF in three different iPSC lines, with 1-10% efficiency, within 3 weeks. These gene-edited cells hold great potential for the CF research, CF drug testing and CF personalise medicine fields Description: PhD Thesis 2019-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6366 Title: Test anti-senescence interventions to postpone or reverse skin ageing Authors: Low., Evon Abstract: While the role of cell senescence is well-established as a main driver of ageing in various tissues, the impact in human skin ageing remains unclear. Many studies predominantly focused on specific skin compartments or cell types foregoing the importance of cellular crosstalk. This makes cross-model comparisons challenging due to variation in the causes and effects of senescence. Consequently, the existing data do not conclusively establish whether senescence of skin cells is the primary contributor to skin ageing. This study aims to evaluate the role of senescence in skin ageing by assessing changes in morphology during skin ageing in various mammalian models, including human skin biopsies from donors of different ages, mice, therapy-induced premature-ageing mice, and senolytic/senostatic treated mice. We also tested a range of natural compounds and compared their senolytic efficacy against skin fibroblasts with published data. As there was no consensus on which senescent marker best distinguishes senescent cells in the skin, this study was designed to validate as many senescence markers as possible in both in vitro dermal fibroblasts and keratinocytes and in vivo ageing human skin. We also created 3D bioengineered skin equivalents to study the crosstalk between skin layers by incorporating different frequencies of senescent fibroblasts in the dermis. This study showed that senolytic or senostatic did not improve extrinsic skin ageing characteristic in therapy-induced premature -ageing mice. Natural compounds with reported ‘anti-ageing’ activity showed no senolytic efficacy against skin fibroblasts. Senescence markers were well detected in vitro, but they were not as sensitive in in vivo skin samples. Some senescence markers were found to be cell-type specific and could vary over the course of senescence development. However, the enlargement of cell size has shown reliability as a senescence marker in both in vitro and 3D model, potentially in vivo if we have better quality samples. In comparison to published results, 3D skin models revealed effects of senescent dermal fibroblasts onto the epidermis are largely matrix dependent. Description: PhD Thesis 2024-01-01T00:00:00Z