Mesenchymal stromal cells (MSCs) and colorectal cancer - a troublesome twosome for the anti-tumour immune response?

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.

Effects of synbiotics on cancer risk biomarkers

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world and second most common (behind lung cancer) in developed countries. In recent years there has been much interest in the potential use of prebiotics, probiotics and synbiotics in the prevention and treatment of CRC. We have previously shown that synbiotic consumption in Azoxymethane treated rats modulates the immune system, influences the genotoxic potential of caecal contents and reduces the number of colonic tumours compared to control rats who did not receive the synbiotic. The aim of the current study was to identify biomarkers suitable for use as cancer risk markers and as intervention markers. A second aim was to determine the influence of synbiotic consumption on cancer risk biomarkers such as in vivo colonic mucosal proliferation and genotoxic damage along with examining the genotoxic, cytotoxic and tumour promoting potential of faecal water (FW). Synbiotic consumption altered the composition of the gastrointestinal flora and reduced in vivo genotoxic damage and the genotoxic potential of FW in cancer and polyp subjects. Synbiotic consumption also reduced the proliferative activity in the colonic mucosa in polyp subjects. In both cancer and polyp subjects gene expression in the colonic mucosa was modulated in synbiotic consuming subjects. In this and other studies the activity of natural killer cells, the level of PGE2 in FW, IL-12 production by PBMCs, genotoxic damage in the colonic mucosa and the tumour promoting activities of FW have been identified as possible biomarkers of cancer risk. Future large scale studies investigating these parameters in healthy and diseased individuals are needed to confirm the suitability of these markers in assessing cancer risk and the role of synbiotics in modulating them.

Plasmid biology of natural Lactococcus lactis strains and molecular mechanisms of bacteriophage-host interaction

Lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P are bacteriocins offering promising perspectives in terms of preservation and shelf-life extension of food products and should find commercial application in the near future. The studies detailing their characterization and bio-preservative applications are reviewed. Transcriptomic analyses showed a cell wall-targeted response of Lactococcus lactis IL1403 during the early stages of infection with the lytic bacteriophage c2, which is probably orchestrated by a number of membrane stress proteins and involves D-alanylation of membrane lipoteichoic acids, restoration of the physiological proton motive force disrupted following bacteriophage infection, and energy conservation. Sequencing of the eight plasmids of L. lactis subsp. cremoris DPC3758 from raw milk cheese revealed three anti-phage restriction/modification (R/M) systems, immunity/resistance to nisin, lacticin 481, cadmium and copper, and six conjugative/mobilization regions. A food-grade derivative strain with enhanced bacteriophage resistance was generated via stacking of R/M plasmids. Sequencing and functional analysis of the four plasmids of L. lactis subsp. lactis biovar. diacetylactis DPC3901 from raw milk cheese revealed genes novel to Lactococcus and typical of bacteria associated with plants, in addition to genes associated with plant-derived lactococcal strains. The functionality of a novel high-affinity regulated system for cobalt uptake was demonstrated. The bacteriophage resistant and bacteriocin-producing plasmid pMRC01 places a metabolic burden on lactococcal hosts resulting in lowered growth rates and increased cell permeability and autolysis. The magnitude of these effects is strain dependent but not related to bacteriocin production. Starters’ acidification capacity is not significantly affected. Transcriptomic analyses showed that pMRC01 abortive infection (Abi) system is probably subjected to a complex regulatory control by Rgg-like ORF51 and CopG-like ORF58 proteins. These regulators are suggested to modulate the activity of the putative Abi effectors ORF50 and ORF49 exhibiting topology and functional similarities to the Rex system aborting bacteriophage λ lytic growth.

The synthesis and biological evaluation of lanostane and cholestane-type natural products

The main objective of this thesis is to outline the synthetic chemistry involved in the preparation of a range of novel lanostane and cholestane derivatives, and subsequent investigation into their biological activity in cancer cells. The biological results obtained throughout the project have driven the strategic synthesis of new compounds, in an effort to optimise the anti cancer potential of lanostane and cholestane derivatives. The first chapter begins with an overview of steroidal compounds and details a literature review of the natural sources of these moieties, as well as their biosynthesis and reported synthetic derivatives. The biological activity of interesting natural and synthetic analogues is also discussed. In addition, an insight into some currently prescribed pharmaceutical compounds, with functional groups relevant to this project, is presented. The second chapter discusses the methods employed for the synthesis of these novel lanostane and cholestane derivatives, and comprises three main sections. Firstly, various oxidation products of lanosterol are synthesised, mainly via epoxidations of the C-8,9 and C- 24,25 alkenes, and also allylic oxidations at these positions. Secondly, amine derivatives of lanosterol are formed by cleaving the lanostane side chain, thereby yielding a new cholestane nucleus, and performing several reductive aminations on the resulting key aldehyde intermediates. Various amines such as piperidine, morpholine, diethylamine and aniline are employed in the reductive amination reactions to yield novel cholestane steroids with amine side chains. Finally, starting from stigmasterol and proceeding with the same methodology of cleaving the steroidal side chain and subsequently performing reductive aminations, novel cholestane derivatives of the biologically active amines are synthesised. The cytotoxicity of these compounds against CaCo-2 and U937 cell lines is presented in terms of percentage viability of cells, IC50 value and apoptosis. The MTT assay is used to determine the percentage viability of cells, and the IC50 data is generated from the MTT results. Apoptosis is measured in terms of fold increase relative to a carrier control. In summary, the compounds formed are discussed in terms of chemical synthesis, spectroscopic interpretation and biological activity. The main reaction pathways involved in the chemistry within this project are various oxidations and reductive amination. The final chapter is a detailed account of the full experimental procedures for the compounds synthesised during this work, including characterisation using spectroscopic and analytical data.

The actinin family of actin crosslinking proteins: natural functions and potential applications in synthetic biology

Actinin and spectrin proteins are members of the Spectrin Family of Actin Crosslinking Proteins. The importance of these proteins in the cytoskeleton is demonstrated by the fact that they are common targets for disease causing mutations. In their most prominent roles, actinin and spectrin are responsible for stabilising and maintaining the muscle architecture during contraction, and providing shape and elasticity to the red blood cell in circulation, respectively. To carry out such roles, actinin and spectrin must possess important mechanical and physical properties. These attributes are desirable when choosing a building block for protein-based nanoconstruction. In this study, I assess the contribution of several disease-associated mutations in the actinin-1 actin binding domain that have recently been linked to a rare platelet disorder, congenital macrothrombocytopenia. I investigate the suitability of both actinin and spectrin proteins as potential building blocks for nanoscale structures, and I evaluate a fusion-based assembly strategy to bring about self-assembly of protein nanostructures. I report that the actinin-1 mutant proteins display increased actin binding compared to WT actinin-1 proteins. I find that both actinin and spectrin proteins exhibit enormous potential as nano-building blocks in terms of their stability and ability to self-assemble, and I successfully design and create homodimeric and heterodimeric bivalent building blocks using the fusion-based assembly strategy. Overall, this study has gathered helpful information that will contribute to furthering the advancement of actinin and spectrin knowledge in terms of their natural functions, and potential unnatural functions in protein nanotechnology.