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Development and application of a novel high-throughput technique for screening neutrophil extracellular trapsChicca, Ilaria Jessica January 2017 (has links)
Neutrophil extracellular traps (NETs) are antimicrobial web-like structures whose release is mediated by reactive oxygen species (ROS) and purpose is to combat infections. Unbalanced NET production and clearance is, however, associated with auto-antibody production and disease. This thesis aimed to develop a High-Content-Analysis (HCA) approach to study human NETosis and its modulation. Initially, NET-related disease-relevant conditions were studied. Individual periodontal-bacteria generated substantial NET production compared with bacterial biofilms. Calcium or magnesium ions and increases in cell density enhanced NET responses. As part of this study the use of fixation and cell adherence procedures were explored and data indicated that addition of paraformaldehyde prior to centrifugation and the absence of poly-L-lysine provided appropriate conditions for downstream cytological analysis. ‘Compartmental Analysis’ and ‘Tube Formation’ algorithms were initially assessed for HCA, however, it was determined that bespoke ‘NET Detection’ and ‘Nuclear Decondensation’ algorithms provided more accurate analysis of NETosis and peptidyl-arginine-deiminase-4 translocation. The optimised protocol employed for the high-content-screening of a 56-compound library identified 8 NETosis modulators. Further characterisation of compounds’ abilities to modulate ROS and NET production, identified roles for glutathione reductase, Src, molecular-target-of-Rapamycin and mitogen-activated-protein-kinase signalling. These pathways may provide new therapeutic targets for treatment of NET-related inflammatory disorders including periodontitis.
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Exploring the variability in mechanical property testing of dental resin compositesKumar, Naresh January 2011 (has links)
The invention of dental resin based-composites (RBCs) has provided a broad range of materials for the restoration of load-bearing posterior teeth with excellent clinical results and adequate longevity. Currently, a lack of consensus exists among researchers regarding classification of RBCs as a result of slight variations in filler size and associated interchangeable mechanical properties of “microhybrid”, “nanohybrid” and “nanofilled” RBCs. Also, the inconsistency in mechanical property testing of RBCs is evident amongst researchers. This research explored the variability in experimental and statistical testing methodologies of RBCs. The current study identified a wide variation in the bi-axial flexure strength (BFS) of commercial and experimental RBCs with respect to deformation rate with a complex relationship between resin constituents and filler morphology. Experimental unfilled resins revealed deformation rate dependence in BFS following 1 week dry, 1 and 13 weeks wet storage regimes, whereas the addition of fillers modified the deformation rate dependence following 13 weeks wet storage and resulted in the BFS of filled resin composites being independent of testing speed. These findings suggested the need for the development of RBCs with appropriate formulations for clinical situations where variable strain rates may occur, for example, patients with parafunctional habits. To date, the alignment of specimens during storage regimes prior to mechanical property testing has rarely been reported. The effect of specimen alignment on the BFS and surface hardness of RBCs was evaluated and a greater decrease in the both properties were found following wet upright compared with stacked and upper surface exposed alignments. These observations were attributed to a variation in diffusion of water as the result of difference in exposed surface areas of specimens, which may lead to different findings and associated interpretation between investigators. Weibull statistics are used for the analysis of strength data of RBCs, however their applicability to RBCs might be questioned due to some viscous deformation prior to brittle failure. The findings of current study supported the applicability of Weibull statistics for the microhybrid and nanofilled RBCs but not a flowable RBC, which suggested that Weibull statistics may not necessarily be applicable for all RBC types. It was demonstrated that variability and irrelevance in testing methods may cause incorrect interpretation of data among researchers and consequently affect the future research and development of RBCs. Therefore, further standardisation of testing methods is required.
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Biological effects of low-intensity ultrasound on dental stem cellsGao, Qianhua January 2016 (has links)
The value of low-intensity pulsed ultrasound (LIPUS) in regeneration of the dental tissue has not been fully investigated. This investigation aim to research the effects of LIPUS on dental stem cells, and specify the role of signalling pathways in regulating stem cell responses. Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and bone marrow stem cells (BMSCs) were isolated from Wistar rats. LIPUS significantly increased cell growth and proliferation of DPSCs, PDLSCs and BMSCs. In DPSC, ERK1/2 was activated immediately, whereas ERK1/2 inhibition modulated the proliferation effects. In BMSC, JNK MAPK was activated, and the post-LIPUS proliferation was decreased by JNK inhibition. In PDLSC, JNK MAPK was activated immediately post-treatment, and p38 MAPK was activated until 1 hour. Subsequently, specific JNK and p38 inhibition decreased proliferation effects after LIPUS. Piezo1/2 were up-regulated post-treatment in DPSC, but not statistically significant in PDLSC. Ruthenium Red, an inhibitor of Piezo1/2, only inhibited ERK1/2 activation, but not JNK or p38 MAPK. Furthermore, when Piezo1/2 were inhibited before LIPUS exposure, it modulated the increase of proliferation in DPSC, not in PDLSC. In conclusion, LIPUS does promote proliferation of dental stem cells; The proliferation of dental stem cells is dependent on MAPK signalling, and there was variation in different MSC lineages; LIPUS may also have an effect on ion channels such as Piezo1/2. Such mechanically activated mechanisms have implications for future stem cell-based dental tissue regeneration.
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The assessment of ultrasonic instruments in dentistryLea, Simon Christian January 2004 (has links)
Introduction: Ultrasonic scalers are an accepted alternative to hand instruments for the removal of plaque, calculus and stains from teeth. Although the use of these instruments is widespread, the vibrational motion of scalers is not fully understood. Their high frequency, low displacement amplitude oscillations have previously been difficult to analyse. Laser vibrometry is a non-invasive technique for measuring the vibration characteristics of oscillating objects. Aim: The aim of this research thesis was to utilise scanning laser vibrometry to assess a range of dental ultrasonic systems under various operating conditions similar to those employed clinically. … Conclusions: The use of scanning laser vibrometry revealed differences in the operation characteristics of different ultrasonic scalers. Clinicians and researchers should be aware of the variability in the performance of ultrasonic scalers since this may affect both clinical procedures and future research. Further research into the design and operation of ultrasonic scaler tips is needed.
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Nuclear Factor κB transcriptional regulator function in haemopoietic stem cellsSheriff, Lozan January 2012 (has links)
The NF-κB family of transcription factors are essential for different stages of murine haemopoiesis as supported by studies on single or double knockout mice. The function of NF-κB1 and NF-κB2 in haemopoietic stem cells (HSCs) has never been described before. In mice, the bone marrow c-Kit+Sca1+Lin- (KSL) population represent HSCs that have the ability to repopulate the bone marrow of lethally irradiated animals. Using knockout mice technology we were able to study the function of NF-κB1 and NF-κB2 in HSCs by performing amongst others transplantation assays, which is the most precise way to test HSC potential. Nfκb1-/- KSL cells are fully able to reconstitute lethally irradiated recipients indicating that the in vivo self-renewal capacity is unaffected. In contrast, colony assays showed NF-κB1 dependency. Upon serial replating, knockout cells were not able to form colonies and lost their in vitro self-renewal capacity. Transplantation of the nfκb2-/- KSL cells engraft with a proliferative phenotype and a competitive advantage, with changes in the B-cell and myeloid cell lineages. Similar to nfκb1-/-, nfκb2-/- KSL cells lose their function as stem cells upon serial replating. These findings highlight the importance of NF-κB family proteins in HSCs, especially NF-κB2 and should be considered in the development of future cancer treatment that target NF-B proteins.
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Systematic analysis of mineral trioxide aggregate using a model cement systemCoomaraswamy, Kristian Sham January 2017 (has links)
Mineral trioxide aggregate (MTA) is a Portland cement-based material in use in dentistry for over 20 years. Although originally developed as a root canal reparative material, multiple applications have since been found for the cement. Commercial MTA is expensive, and for research, its composition cannot be changed/modified. This investigation endeavored to create and validate an affordable MTA model cement system for future research into the material, and use the model created to analyse the cement with view to further understanding it and improving its properties. An MTA-like model system was established and setting times, compressive strength, relative porosity, apparent and specific densities and radiopacity were measures used to assess cement samples made. Sample storage time, composition, alternative radiopacifiers, material consistency and handling, and the effect of powder-to-liquid ratio (PLR) were all investigated, utilising the model system. 5 wt% Plaster of Paris content was found to provide optimal material characteristics, with Bi2O3 the most effective radiopacifier, requiring a minimum proportion of 20 wt% for adequate radiopacity to meet the required standard. A higher PLR (4.0 – 4.5 g/ml) than recommended for commercial MTA was found to be best for cement workability and material properties, including radiopacity.
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Interactions of 45s5 bioactive glass with the dentine-pulp complexComar, Eisha January 2014 (has links)
The bioactive glass, 45S5, has been proposed for the treatment of dentine hypersensitivity due to its ability to physiochemically occlude dentinal tubules. However, its biological effects on the dentine-pulp complex require further elucidation. The aims of this study were to characterise the ionic dissolution products of bioactive glass and observe their effects on both pulpal cells and the dentine extracellular matrix. The ability of bioactive glass to release dentine matrix components (DMCs) in the presence of a physiological fluid, saline, was investigated. The bioactive glass-released DMCs were characterised biochemically and compared with those released by EDTA, a well-established extractant, using high-throughput proteomic techniques. Results revealed the differential solubilisation of a broad range of proteins, including growth factors and other bioactive molecules, many of which had not previously been identified in dentine. In vitro analysis of dental pulp cells exposed to DMCs released by bioactive glass in saline solution demonstrated a dose-dependent increase in cell proliferation, whilst the direct effects of the ionic dissolution products of bioactive glass were also stimulatory to OD-21 cells. The positive responses of cells to bioactive glass-released DMCs and its ionic dissolution products indicates exciting potential to harness bioactive approaches therapeutically for dentine tissue repair and regeneration.
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Polymerisation kinetics and optical phenomena of photoactive dental resinsHadis, Mohammed Abdul January 2011 (has links)
Globally, several shortcomings of dental resin based composites (RBCs) remain. This may be related to problems associated with incomplete conversion (40-70%), polymerisation shrinkage (1-4% by volume) and the stress generated at the tooth/restoration interface. Additionally, the increased number of technique sensitive incremental steps required to fill relatively large cavities is due to inefficient light transmission at depths greater than 2mm. The current investigation demonstrates the applicability of the exposure reciprocity law in photoactive dental materials in order to try and improve these shortcomings. The development techniques that will allow dynamic monitoring of optical and physical change will aid material development with the goal of improving cure depths. The current investigation has demonstrated the use of several analytical techniques (FT-IR spectroscopy, UV-Vis Spectroscopy and low coherence interferometry) and shown the complexity of optical phenomena within RBCs, which are affected by material composition as well as cavity dimensions. Whilst research continues to develop a novel RBC with reduced shrinkage and improved depths of cure, there is currently no commercially available solution to such problems. Consequently a better understanding of the setting reaction, optical properties and physical properties will aid material development.
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Effect of cation substitutions in an ionomer glass composition on the setting reaction and properties of the resulting glass ionomer cementsKashani, Mitra A. M. P. January 2013 (has links)
This study investigated the effect of Ba\(^2\)\(^+\) and Sr\(^2\)\(^+\) substitutions for Ca\(^2\)\(^+\) in an ionomer glass composition 4.5SiO\(_2\)-3Al\(_2\)O\(_3\)-1.5P\(_2\)O\(_5\)-3CaO-2CaF\(_2\) on the setting reaction and properties of the resulting glass ionomer cements (GICs). Experimental GICs (Ca-GIC, Ca-Sr-GIC and Ca-Ba-GIC) were characterized via various techniques: Diametral tensile strength, compressive strength, flexural strength, Vickers hardness and nano-indentation measurements were conducted at different time points during setting. Real time Fourier transform infrared (FTIR) spectroscopy was used to study the effect of the glass composition on cement setting reactions. A resistance to penetration method evaluated the cement setting time. Additionally, the wear resistance of the experimental GIC was measured by a ball-on-flat wear test. Furthermore, fluoride (F\(^-\)) release and the antimicrobial behaviour of cements were investigated. The compressive, diametral and flexural strength of the cements in which Ca\(^2\)\(^+\) was substituted by Sr\(^2\)\(^+\) and Ba\(^2\)\(^+\) were both statistically significantly higher than the unsubstituted control at 1 hour after setting (P < 0.001). FTIR results confirmed that enhanced metal salt crosslinking occurred in the ion substituted materials, especially from 1 to 60 minutes. Therefore, it can be concluded that replacing Ca\(^2\)\(^+\) with larger cations (Sr\(^2\)\(^+\) and Ba\(^2\)\(^+\)) affects the setting reaction and resulting mechanical properties in the short term. All three experimental GICs inhibited growth of Streptococcus mutans over a period of 48 hours. The F\(^-\)release analysis showed that there was less F\(^-\) release in artificial saliva (AS) than in deionized water over 40 days.
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Potential involvement of epithelial-mesenchymal transition in the pathogenesis of periodontitisAbdulkareem, Ali Abbas January 2017 (has links)
Epithelial-mesenchymal transition is reportedly important in loss of epithelial integrity and cell migration in inflammatory/infectious diseases and cancer. Since Gram negative anaerobic periodontal pathogens are well-recognized to induce intense inflammatory responses; the present study investigated their ability to induce EMT in vitro. A 2D chronic inflammatory model was developed using either the H400 oral keratinocyte cell-line or primary rat oral keratinocytes which were exposed to heat-killed Fusobacterium nucleatum, Porphyromonas gingivalis and Escherichia coli LPS for up to 8-days. EMT-associated changes were determined using semi-quantitative-RT-PCR, PCR-arrays, ELISA, scratch/transwell migration assays, immunocytochemistry/immunofluorescence, and transepithelial electrical resistance. Chronically stimulated cultures increased extracellular levels of the EMT regulatory cytokines, TGF-β1, TNF-α and EGF, whilst subsequent EMT-induction was indicated by up-regulation of mesenchymal markers, including vimentin and N-cadherin, and concomitant down-regulation of epithelial markers including E-cadherin and β-catenin. In addition, intracellular signaling activity of key EMT regulatory transcription factors, Snail-1 and NF-ĸB, increased following chronic bacterial exposure and was associated with enhanced cellular migratory activity and reduced epithelial barrier integrity. These results indicated for the first time that EMT may be involved in the compromised epithelial barrier function observed during periodontitis pathogenesis which may occur in response to prolonged local bacterial exposure.
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