Characterisation of the role of Fas in intestinal inflammation and cancer

Background: The role of Fas (CD95) and its ligand, Fas ligand (FasL/CD95L), is poorly understood in the intestine. Whilst Fas is best studies in terms of its function in apoptosis, recent studies suggest that Fas ligation may mediate additional, non-apoptotic functions such as inflammation. Toll like Receptors (TLRs) play an important role in mediating inflammation and homeostasis in the intestine. Recent studies have shown that a level of crosstalk exists between the Fas and TLR signalling pathways but this has not yet been investigated in the intestine. Aim: The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal cancer cells. Results: Treatment with TLR4 and TLR5 ligands, but not ligands for TLR2 and TLR9 increased the expression of Fas and FasL in intestinal cancer cells in vitro. Consistent with this, expression of Fas and FasL was reduced in the distal colon tissue from germ-free (GF), TLR4 and TLR5 knock-out (KO) mice but was unchanged in TLR2KO tissue, suggesting that intestinal cancer cells display a degree of specificity in their ability to upregulate Fas and FasL expression in response to TLR ligation. Expression of both Fas and FasL was significantly reduced in TRIF KO tissue, indicating that signalling via TRIF by TLR4 and TLR5 agonists may be responsible for the induction of Fas and FasL expression in intestinal cancer cells. In addition, modulating Fas signalling using agonistic anti-Fas augmented TLR4 and TLR5-mediated tumour necrosis factor alpha (TNFα) and interleukin 8 (IL)-8 production by intestinal cancer cells, suggesting crosstalk occurs between these receptors in these cells. Furthermore, suppression of Fas in intestinal cancer cells reduced the ability of the intestinal pathogens, Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8, suggesting that Fas signalling may play a role in intestinal host defence against pathogens. Inflammation is known to be important in colon tumourigenesis and Fas signalling on intestinal cancer cells has been shown to result in the production of inflammatory mediators. Fas-mediated signalling may therefore play a role in colon cancer development. Suppression of tumour-derived Fas by 85% led to a reduction in the tumour volume and changes in tumour infiltrating macrophages and neutrophils. TLR4 signalling has been shown to play a role in colon cancer via the recruitment and activation of alternatively activated immune cells. Given the crosstalk seen between Fas and TLR4 signalling in intestinal cancer cells in vitro, suppressing Fas signalling may enhance the efficacy of TLR4 antagonism in vivo. TLR4 antagonism resulted in smaller tumours with fewer infiltrating neutrophils. Whilst Fas downregulation did not significantly augment the ability of TLR4 antagonism to reduce the final tumour volume, Fas suppression may augment the anti-tumour effects of TLR4 antagonism as neutrophil infiltration was further reduced upon combinatorial treatment. Conclusion: Together, this study demonstrates evidence of a new role for Fas in the intestinal immune response and that manipulating Fas signalling has potential anti-tumour benefit.

Synthesis, functionalisation and characterisation of germanium nanocrystals & their applications

In the last two decades, semiconductor nanocrystals have been the focus of intense research due to their size dependant optical and electrical properties. Much is now known about how to control their size, shape, composition and surface chemistry, allowing fine control of their photophysical and electronic properties. However, genuine concerns have been raised regarding the heavy metal content of these materials, which is toxic even at relatively low concentrations and may limit their wide scale use. These concerns have driven the development of heavy metal free alternatives. In recent years, germanium nanocrystals (Ge NCs) have emerged as environmentally friendlier alternatives to II-VI and IV-VI semiconductor materials as they are nontoxic, biocompatible and electrochemically stable. This thesis reports the synthesis and characterisation of Ge NCs and their application as fluorescence probes for the detection of metal ions. A room-temperature method for the synthesis of size monodisperse Ge NCs within inverse micelles is reported, with well-defined core diameters that may be tuned from 3.5 to 4.5 nm. The Ge NCs are chemically passivated with amine ligands, minimising surface oxidation while rendering the NCs dispersible in a range of polar solvents. Regulation of the Ge NCs size is achieved by variation of the ammonium salts used to form the micelles. A maximum quantum yield of 20% is shown for the nanocrystals, and a transition from primarily blue to green emission is observed as the NC diameter increases from 3.5 to 4.5 nm. A polydisperse sample with a mixed emission profile is prepared and separated by centrifugation into individual sized NCs which each showed blue and green emission only, with total suppression of other emission colours. A new, efficient one step synthesis of Ge NCs with in situ passivation and straightforward purification steps is also reported. Ge NCs are formed by co-reduction of a mixture of GeCl4 and n-butyltrichlorogermane; the latter is used both as a capping ligand and a germanium source. The surface-bound layer of butyl chains both chemically passivates and stabilises the Ge NCs. Optical spectroscopy confirmed that these NCs are in the strong quantum confinement regime, with significant involvement of surface species in exciton recombination processes. The PL QY is determined to be 37 %, one of the highest values reported for organically terminated Ge NCs. A synthetic method is developed to produce size monodisperse Ge NCs with modified surface chemistries bearing carboxylic acid, acetate, amine and epoxy functional groups. The effect of these different surface terminations on the optical properties of the NCs is also studied. Comparison of the emission properties of these Ge NCs showed that the wavelength position of the PL maxima could be moved from the UV to the blue/green by choice of the appropriate surface group. We also report the application of water-soluble Ge NCs as a fluorescent sensing platform for the fast, highly selective and sensitive detection of Fe3+ ions. The luminescence quenching mechanism is confirmed by lifetime and absorbance spectroscopies, while the applicability of this assay for detection of Fe3+ in real water samples is investigated and found to satisfy the US Environmental Protection Agency requirements for Fe3+ levels in drinkable water supplies.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.