DSpace Collection:
http://theses.ncl.ac.uk/jspui/handle/10443/97
2024-03-29T14:18:13ZExtracellular DNA as a component of dental plaque
http://theses.ncl.ac.uk/jspui/handle/10443/5739
Title: Extracellular DNA as a component of dental plaque
Authors: Abduljalil, Nasrin
Abstract: Therapeutics that can control microbial biofilms while preserving the natural
microbiota represent a promising strategy in managing biofilm-related diseases
such as periodontitis. Extracellular DNA (eDNA) is an important structural
component of many biofilms, including dental plaque. Bacteria within dental
plaque produce deoxyribonuclease (DNase) enzymes that could digest eDNA. This
project aimed to investigate whether a DNase produced by an oral bacterium can
degrade eDNA in oral biofilms, thereby inhibiting the biofilm growth or disrupting
mature biofilms, and compare its activity to a DNase from a non-oral bacterium.
Preliminary investigations were also made into the microbial composition and
immunostimulatory properties of eDNA. NucB, a DNase from a marine isolate of
Bacillus licheniformis, inhibited biofilm growth and dispersed preformed biofilms
of Fusobacterium nucleatum. It also inhibited the growth of model plaque
biofilms, but did not affect preformed biofilms, implying that eDNA is more
important or more accessible during the initial phases of the plaque biofilm
formation. Furthermore, NucB inhibited the growth of anaerobic model plaque
biofilms, but not those cultured in aerobic sucrose-rich conditions indicating that
it may be particularly useful for reducing subgingival plaque, which is essentially
anaerobic. Conversely, SsnA, a DNase from the oral bacterium Streptococcus
gordonii, inhibited the growth of Fusobacterium nucleatum biofilms but did not
disrupt preformed biofilms. SsnA Also lacked any antibiofilm effect with model
plaque biofilms. NucB efficiently degraded various DNA substrates, whereas SsnA
was only effective against single stranded and low molecular weight DNA,
suggesting that eDNA in the plaque biofilm is predominantly double stranded DNA
of high molecular weight. Overall, this work provided key insights into the
activity of a DNase from an oral bacterium and showed that there is a scope to
enhance subgingival biofilm control using exogenous DNases from non-oral
bacteria.
Description: PhD Thesis2022-01-01T00:00:00ZInvestigating the in vivo dynamics of FtsZ and FtsZ-interacting proteins using vertical cell imaging by nanostructured immobilisation (VerCINI)
http://theses.ncl.ac.uk/jspui/handle/10443/5243
Title: Investigating the in vivo dynamics of FtsZ and FtsZ-interacting proteins using vertical cell imaging by nanostructured immobilisation (VerCINI)
Authors: Jukes, Calum Steven
Abstract: Division is arguably one of the most difficult mechanistic tasks that cells face, requiring precise temporal and spatial regulation. Gram-positive bacteria also face an additional mechanistic problem - high turgor pressure. In order to successfully divide, Gram-positive organisms synthesise a peptidoglycan septum which cleaves the cell in two, yielding two daughter cells.
Prior to cell division, the almost universally essential cytoskeletal tubulin homologue FtsZ polymerizes into a highly dynamic, ring-like band of short filaments at mid-cell- the Z-ring. The Z-ring is tethered to the plasma membrane via anchor proteins, following which the Z-ring recruits septal PG synthases forming the mature divisome. These synthases build the septum which partitions the cell in two.
This work investigates the organisation and dynamics of the Z-ring in the Gram-positive model organism B. subtilis. In this thesis, I established a high-throughput approach of Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI) in order to visualise the dynamics of the division machinery around the entirety of the division plane. Using this technique in combination with advanced fluorescence microscopy, I discovered that FtsZ filaments treadmill around the division plane in live bacteria, a phenomenon previously only described in vitro. Treadmilling is a type of motion whereby an asymmetric filament undergoes plus-end polymerization and minus-end depolymerisation. I investigated how the organisation and dynamics of filaments within the Z-ring develops over the cell cycle. My results indicate that FtsZ treadmilling is unstable in nascent Z-rings, but stabilizes during the transition to mature rings, before constriction has been initiated. This shows that both FtsZ filament assembly to midcell and FtsZ filament treadmilling dynamics are actively cell cycle regulated. I examined the dynamics of a number of key FtsZ interacting proteins (EzrA, SepF and DivIVA), observing a range of static and dynamic protein motions in ZIPs imaged, arguing against a single, monolithic divisome complex.
Description: PhD Thesis2021-01-01T00:00:00ZDevelopment of proteomics strategies for the characterisation of endosomal TLR2 signalling
http://theses.ncl.ac.uk/jspui/handle/10443/5236
Title: Development of proteomics strategies for the characterisation of endosomal TLR2 signalling
Authors: Gant, Megan Sara
Abstract: Liquid chromatography- mass spectrometry (LC-MS) is a highly sensitive analytical tool which can be instrumental for a wide range of applications, including use in the clinic to identify disease biomarkers in patient samples and in the laboratory to study the changing proteome from cells. Buffers and HPLC methods for protein separation were developed for the analysis of therapeutic monoclonal antibodies (MAbs) and human serum.
For serum analysis, protein separation methods were created using the MAbPac column with 0.02 % SDS added to the mobile phase. Coupled with basic reverse phase (BRp) chromatography, it is possible to identify >1000 proteins not present when BRp is used alone. This method was also applied to the analysis of serum, as an alternative to protein depletion and as a method of increasing the number of identifications of low-abundance disease biomarkers.
Toll-like receptors (TLRs) are one of the most widely studied groups of pathogen recognition receptors. plasma membrane (PM) TLRs initiate the pro-inflammatory cytokine pathway, and endosomal TLRs initiate the transcription of type I interferons (IFNs). An exception is TLR4, which is a PM TLR but is known to translocate to the endosome after activation, initiating the type I IFN transcription pathway. However, recent research suggests that TLR2 could also signal from the endosome.
The proteome of TLR2-activated TLR2 -/- (KO) and Wild-type (WT) cells were analysed by LC-MS/MS. Basal changes in endosomal proteins were found between KO and WT cells and key signalling differences detected upon TLR2 activation, indicating a role for TLR2 in endocytosis, endosomal signalling, and innate immune response.
The proteomes of phagosomes isolated from TLR2 and TLR4 stimulated WT cells were also analysed by LC-MS/MS. TLR2 was observed on the phagosome even in unstimulated cells, indicating that TLR2 is already present on the phagosome under basal conditions. Changes in phagosomal signalling between TLR2 and TLR4 activated cells shows that TLR2 and TLR4 have different roles in endocytosis and phagosomal signalling.
Biochemistry techniques were employed to investigate downstream endosomal TLR2 signalling, revealing that TLR2 initiation of type I IFN occurs via Interferon regulatory factors (IRF) 1 and 7.
Description: Ph. D. Thesis.2021-01-01T00:00:00ZThe Role of Phosphate Acquisition in Promoting Stress Resistance and Virulence in a Major Human Fungal Pathogen
http://theses.ncl.ac.uk/jspui/handle/10443/5234
Title: The Role of Phosphate Acquisition in Promoting Stress Resistance and Virulence in a Major Human Fungal Pathogen
Authors: Ahmed, Yasmin
Abstract: The ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate (Pi) is regulated by the Pho4 transcription factor, which is important for both virulence and resistance to diverse and physiologically important stresses. A key aim of this work was to investigate the regulation of Pho4, and the roles of Pho80-Pho85 cyclin dependent kinase (CDK) signalling and inositol polyphosphates were explored. As reported in the model yeast Saccharomyces cerevisiae, the Pho80 cyclin functions as a negative regulator of Pho4 in C. albicans. However, in contrast to S. cerevisiae, the CDK inhibitor Pho81 also negatively regulates C. albicans Pho4; Pho4 accumulates in the nucleus in pho81Δ cells and Pi acquisition strategies are activated under Pi replete conditions. With regard to inositol polyphosphates, in contrast to that reported in S. cerevisiae, IP7 synthesis by the Kcs1 inositol pyrophosphate synthase was found to be largely dispensable for Pi homeostasis with Vip1-derived IP7 synthesis playing a more prominent role in C. albicans.
The synthesis of the Pi storage molecule polyphosphate (polyP) is also regulated by Pho4. Previous work found that mobilization of Pi from polyP is one of the first responses evoked in response to Pi starvation and precedes activation of the Pho4 transcription factor. A further aim of this thesis was to investigate the importance of polyP mobilisation in the pathobiology of C. albicans. It was found that two polyphosphatases, Ppn1 and Ppx1, function redundantly in C. albicans to release Pi from polyP. Strikingly, it was shown that polyP mobilisation plays a role in Pho4 activation and stress resistance in C. albicans. Blocking polyP mobilisation also resulted in significant morphological defects. Consistent with these findings, data is also presented illustrating that polyP mobilisation is important for the virulence of C. albicans.
Given the links between Pi acquisition and virulence, a further aim was to explore whether Pi acquisition could be exploited as a novel antifungal strategy. High-throughput screening of compound libraries revealed potential candidates directly targeting the PHO pathway, which present an exciting avenue for future work.
Taken together, the findings presented in this thesis reveal novel insight into Pi homeostasis mechanisms in C. albicans and the potential of targeting this important virulence trait in the development of future therapeutic strategies.
Description: Ph. D. Thesis.2021-01-01T00:00:00Z