The immune system comprises highly sophisticated networks of cells and signalling molecules which function in concert to protect the body against pathogens. Within this system a role for the extra-cellular microenvironment as a crucial mediator of immune responses is becoming increasingly apparent. Conventional in vitro cultures lack physiologically relevant extra-cellular cues, such as extracellular matrix (ECM) and shear flow. Tissue engineering can be used to simulate features of the natural microenvironment for the development of biologically relevant platforms. It is anticipated that this will enable the study of the influence of the extra-cellular environment on immune responses. This thesis describes the development and characterisation of tissue-engineered platforms for immune cell culture which incorporate the ECM and shear flow. This work goes on to apply these platforms for the study of the effect of the extra-cellular environment on dendritic cells and their interactions with T cells in the context of immunological stimulation. The ECM defines the three-dimensional architecture of the natural microenvironment. It provides structural support and also promotes cell motility in tissues. This is important for the function of the immune system as it directs the organisation and interactions of immune cells which ultimately contributes to the modulation of immune responses. Candidate synthetic and natural biomaterials were assessed for their suitability to provide an in vitro extracellular matrix (ECM) platform for human immune cell culture. The suitability of these materials to provide an artificial ECM platform was based on the viability, resting immune state and immune competence of the cells. The synthetic biomaterials tested were a thermo-responsive colloidal gel and electrospun PET and PLGA scaffolds coated with a thermo-responsive polymer. An important finding from the work done with the colloidal gel was that the human dendritic cells, which were incorporated into the gel at the beginning of the experiment, could not be separated from the material for flow cytometric analysis. Therefore, characterisation of the colloidal gel for immune cell culture could not be completed. Regarding the characterisation of the electrospun PET and PLGA scaffolds, although they did not significantly impair cell viability of dendritic cells they were found to induce cell maturation. As a result, none of the synthetic biomaterials were found to be a suitable ECM surrogate. A semi-natural biomaterial, gelatin methacryloyl (GelMA) hydrogel, was included in the investigation. The results from the characterisation of GelMA for human immune cell culture indicated that the hydrogel induced a pro-inflammatory immune response due to the profile of secreted cytokines. Based on this, GelMA was also discounted as an appropriate material for the development of the ECM platform. The final ECM candidate was a collagen hydrogel, which is a naturally-derived biomaterial. The collagen hydrogel was shown to support immune cell survival and human dendritic cells maintained an immature phenotype in culture. In addition, typical responses to immunological stimuli by human dendritic cells and T cells were observed in collagen hydrogel cultures. This work demonstrated that out of the biomaterials which were characterised, the collagen hydrogel was the most suitable biomaterial for the development of the ECM platform. The influence of the collagen hydrogel ECM platform on antigen-specific immune responses was investigated in the context of autologous human dendritic cell and T cell co-cultures stimulated with the model antigen Mycobacterium tuberculosis purified protein derivative, also referred to as PPD. The results from these experiments indicated that the presence of the collagen hydrogel increased the sensitivity and specificity of the immune response, compared to conventional tissue culture conditions. An attempt was made at utilising the ECM platform to investigate immune responses to chemical sensitisers to address the requirement for in vitro alternatives to replace current animal testing methods. In this work, innate and adaptive immune responses to sensitisers were detected using the ECM platform. However, the reproducibility of these experiments was low due to large donor variation. Therefore the effect of the ECM platform on immune responses to sensitisers could not be evaluated. This difficulty likely reflects the complexity of the molecular and cellular mechanisms which lead to the acquisition of chemical sensitisation. Shear flow is a type of physiological stress to which immune cells are exposed in vivo due to the movement of blood and lymph fluid. Recent studies have implicated flow as an immunologically relevant stimulus, capable of inducing changes in the expression of receptors and chemokines involved in regulating immune cell migration, and activating immune receptor signalling. A fluidic cell culture platform was developed to recapitulate the effect of shear flow. Two different prototypes were constructed, one of which was taken forward and characterised for immune cell culture applications. The fluidic platform taken forward had a paper-based cell culture scaffold which was coated with collagen hydrogel. The scaffold was found to induce maturation of human dendritic cells which was attributed to the possibility of incomplete coverage of the scaffold by the collagen hydrogel. The viability of dendritic cells was slightly impaired by flow, however not significantly. Interestingly, when exposed to shear flow, dendritic cells maintained a less mature phenotype compared to their static counterparts. Antigen-specific immune responses were studied on the fluidic platform by setting up co-cultures comprising PPD-stimulated autologous human dendritic cells and T cells. Typical T cell activation was observed on the platform and the sensitivity and specificity of immune responses was found to be greater under flow conditions, compared with static cultures. In conclusion, this thesis demonstrates the value of developing biomimetic platforms for studying the influence of the extra-cellular environment on immune responses. Finally, the ability to mimic extra-cellular cues to which cells are exposed in vivo has the potential to generate more realistic immune responses in the lab. This presents huge opportunities for advancing understanding in immunology. It also has implications for methods used in research, drug discovery and safety testing, where currently only animals provide a representative system for the study of immune reactions. It is anticipated that enhancing the physiological relevance of in vitro cell culture will ultimately contribute to the reduction of animals used in research and testing.

In vertebrates, haematopoietic stem cells (HSCs) maintain the blood system throughout life. HSCs are believed to arise during embryogenesis from haemogenic endothelial cells (HECs) that undergo an endothelial-to-haematopoietic transition (EHT). Here, in order to trace the progeny of the embryonic ECs in zebrafish, an inducible CreERT2-LoxP system was used. Following short-term induction of the Cre recombinase during early embryonic stages, Cre reporter gene expression was observed in early larval haematopoietic cells (HCs). At adult stages, PCR revealed the presence of the recombined Cre reporter gene in HCs, demonstrating that adult HCs had originated from embryonic ECs. In zebrafish, HECs of the ventral wall of the dorsal aorta (vDA) are thought to form HCs by basal epithelial-to-mesenchymal transition (bEMT), a process that depends on the transcription factor Runx1. Here, making use of the recently identified gfi1aaqmc551Gt line, confocal microscopy showed that qmc551: GFP+ cells were HECs. While most GFP+ cells underwent bEMT, some displayed a novel type of apical departure. In runx1morphants, bEMT was abrogated and most GFP+ HECs remained in the vDA. Apical departure, however, was still observed in the absence of Runx1, suggesting a fundamental difference in the underlying mechanism. While gfi1aa expression was lost in vDA HECs of qmc551 homozygous embryos, EHT of GFP+ HECs was completely unaffected. Up regulation of its paralogue Gfi1ab suggested functional redundancy. To study this redundancy, the CRISPR/Cas9 system was used to mutate the gfi1ab gene. Here, the mutant gfi1ab alleles qmc552 and qmc553 were identified. Both alleles, as well as a third allele sh320 that was generated by a collaborator, encode truncated, most likely non-functional proteins. Initial data on gfi1aaand gfi1ab double mutant embryos showed a defect in definitive haematopoiesis. Whether HECs were affected and blocked in their ability to undergo EHT remains to be determined.

Human schistosomiasis is one of the helminthic neglected tropical diseases. It leads to serious health problems and imposes a huge burden on communities. All interactions between the parasite and the human body occur at a molecular and cellular level. Therefore, the study of the molecular aspects of infection and the immune response is a very active area of research. It has been known for decades that there is a direct relationship between protection against infection, or reinfection after treatment, and parasite-specific Immunoglobulin E (IgE) antibody in infected individuals. This study aims to investigate the allergenicity of S. mansoni antigens using RS-ATL8 reporter cell line assay. Towards this goal, I first identified the best reporter cell line for allergenicity assessment. This was done by the characterisation of the transgenic human FcԑRIα chain’s gene copy number and by comparing the levels of human FcԑRI receptor surface expression. The second goal was the optimisation of RS-ATL-8 reporter cell line. This was achieved by the optimisation of fundamental conditions such as the cell density, sensitising agent’s optimum dilution and the stimulant optimum concentration. Once a robust standard operating procedure (SOP) had been established, I investigated the allergenicity of the four expressed S. mansoni antigens using the optimised reporter cell line RS-ATL8.

Basic Helix-Loop-Helix proteins are transcriptional regulators crucial for many development processes. Using gain- and loss-of-function analysis in zebrafish, the functional role of two members of this protein family, lyl1 (Lymphoblastic leukaemia 1) and Id4 (Inhibitor of differentiation 4) in stem cell fate was determined. Ectopic overexpression of lyl1 resulted in the expansion of haematopoietic stem cell pool and its progeny promoting erythrocyte differentiation and suppressing myeloid differentiation. TALEN-mediated lyl1-/¬- embryos developed normally but displayed distinct marker gene expression during primitive and definitive haematopoiesis establishing a role for lyl1 in both waves of haematopoiesis. During primitive haematopoiesis expression of scl/tal1 and gata1 was unaltered but expression of pu.1 was increased suggesting that lyl1 antagonises myeloid differentiation. Lyl1-deficiency resulted in reduction of Gfi1aa expression during primitive and definitive haematopoiesis. In addition, a reduction in the expression of c-myb in the caudal hematopoietic tissue and rag1 in the thymus was observed indicating that lyl1 is required to maintain the definitive haematopoietic stem cell pool and to drive T lymphopoiesis. In adult zebrafish lyl1 regulates lineage choice driving lymphopoiesis and suppressing myelopoiesis. Morpholino-mediated knockdown of Id4 alone or in combination with p53 resulted in reduced cell proliferation, increased cell death and premature neuronal differentiation. Phenotypic analysis of TALEN-mediated Id4 mutants confirmed that Id4 plays a crucial role in the expansion of neural stem cells and timing of neuronal differentiation. Inhibition of p38MAPK in Id4 morphants as well as Id4-/- mutants resulted in a phenotypic rescue establishing that Id4 negatively regulates p38MAPK activity to ensure normal neurogenesis.

Stable telomeres play a key role to the survival of cancer cells; therefore, different cancer chemotherapeutic approaches have been developed in order to disrupt or destabilise telomeres or telomerase. One of the newest methods is the disruption of vital protein–protein interactions in the telomere, such as that between shelterin components TRF1 and TIN2. The principal aim of this project was to obtain a novel peptide-like molecule, an analogue of a key interacting region of TIN2 that could compete effectively for the binding sites on TRF1 and so lead to the destabilisation of telomere structure. Molecular modelling and simulations were undertaken as the starting point of the project. Structure-based drug design was applied, starting from the available crystal structure data. A library of peptide analogues of the TRF1-binding motif in TIN2 was designed using the MM-GBSA simulation method to predict binding affinities. Then, a number of the peptide analogues were selected from the library for further investigations. The secondary goal was to investigate the accuracy of the predicted ΔGbinding values and try to optimise them; the latter aim was set out after finding a significant difference in the predicted binding free energy values after repeating the identical protocol for the same complex system. Therefore, different approaches were applied to optimise the predicted ΔGbinding values. Subsequently, selected TIN2 peptide analogues were synthesised in the laboratory using Fmoc solid-phase peptide synthesis. Then, the hTRF1 protein was expressed and purified in preparation for the development of the in vitro assay. Finally, biophysical evaluations and screening of the peptide analogues were performed using fluorescence polarisation assay. One of the peptide analogues developed in this study was identified as an early lead compound. In addition, the findings of this research showed that the ΔGbinding values of the peptide analogues have significantly improved accuracy after optimisation. As a result of these investigations, suggestions were identified for future research.

Forming tissues in the laboratory to replace diseased or dysfunctional tissue or act as models for drug treatment is the goal of tissue engineering. The large intestine epithelium (colon surface) is a tissue which could benefit from both diseased and non-diseased models for the purpose of tackling colon cancer causes and treatments. Scaffolds (cell supports) are a pivotal part in many tissue engineering strategies. This thesis describes the design and production of two separate scaffolds based on the degradable polymer poly(lactic-co-glycolic acid) (PLGA). The first was a two dimensional scaffold to mimic the intestinal basement membrane which was modified with an oxygen plasma. The changes to the surface due to plasma and the degradation properties of the scaffold were extensively studied with SEM, XPS, AFM and GPC. The data showed that the oxygen plasma induced surface porosity and associated changes to surface roughness. The surface chemistry as detected by XPS was unchanged by both plasma treatment and degradation in buffered solution. The plasma treatment did lead to a dramatic loss in molecular weight but the degradation profile of both the untreated and etched films was similar. Extensive cell studies with SEM, live/dead, alamarBlue and Hoechst DNA assays showed that intestinal cells on the plasma treated scaffold was enhanced in terms of morphology, metabolic activity and proliferation. Finally, a two dimensional co-culture model using epithelial and myofibroblasts cell lines on the modified PLGA scaffold was achieved. The second scaffold was a three dimensional scaffold bearing the crypt like architecture of the colon. An accurate mould produced through electron beam lithography using dimensions measured from mouse histological sections. PLGA particles were used to fill the mould and sintered to produce the scaffold. A unique cell seeding approach using cell sheets was used. The cell sheets were produced on plasma polymers of acrylic acid and the discharge power was shown to affect surface wettability, chemistry and cell viability. The cell sheet approach proved to enhance cell attachment to the scaffold compared to individual cell seeding. Finally, a bilayer scaffold with model protein to mimic Wnt protein presence in the lower half of the crypt was studied with ToF-SIMS.

Humans are ubiquitously exposed to peroxisome proliferators including hypolipidemic agents, industrial solvents and atural products. Because of this and the fact that peroxisome proliferators cause non-genotoxic hepatocarcinogenesis in rodents, it is of importance to elucidate the mechanism of action of the peroxisome proliferators in order to provide an assessment of the hazard, if any, of these compounds to humans. It is also known that the peroxisome proliferators begin their actions by inducing hepatic DNA synthesis. Thus, the aim of this thesis was to find genes that could be responsible for triggering the induction of hepatic DNA synthesis caused by peroxisome proliferators, specifically ciprofibrate. First, it was important to indicate when the induction of hepatic DNA synthesis actually happens. This was done with BrdU immunohistochemical procedures. The induction of hepatic DNA synthesis with ciprofibrate in mice was observable only after 4 days making it difficult to specify when the induction actually happened. In rats the induction of hepatic DNA synthesis was found to peak at 24 hours and this system gave the better opportunity to find the genes responsible. The difference in the timing of induced hepatic DNA synthesis betweenmice and rats implied that there could be a species difference in the mechanism of each species’ response to PPAR. With immunohistochemistry it was noticed that there was a difference in the lobular localization of hepatic DNA synthesis in the liver tissues of rats and mice dosed with different inducers, with the rat livers exhibiting periportal distribution while hepatic DNA synthesis in the mice seemed to be distributed throughout the liver tissue. The effects of ciprofibrate or cyproterone acetate on liver gene expression in rats were studied, using cDNA microarrays, transcriptome sequencing and quantitative real- time PCR. A 1- 5 hour treatment period was chosen to detect the immediate early gene response, while a 24 hour time point was chosen to elucidate the confounding effects from the hepatic DNA synthesis seen during the 24 hour stimulation. The results showed that ciprofibrate altered the expression of numerous genes including previously known PPARa agonist-responsive genes involved in processes such as PPAR signalling pathways, fatty acid metabolic pathway, cell cycle, palmitoyl-CoA hydrolase activity, lipid metabolism, inflammatory responses, and stress responses, in addition to a large number of novel candidate genes. Three novel induced genes G0s2, Ccnd1 and Scd1, (and two marker genes CYP4A1 and CYP3A1) were confirmed with quantitative real- time PCR. The G0s2, Ccnd1 and Scd1 were found to be up-regulated at the hours 1 and 3 after dosing and not 24 hours, and the G0s2 and Scd1 were specific for the ciprofibrate suggesting they were involved in a distinct PPARa pathway responsible for the hepatic DNA synthesis. The complete database of the transcriptional response provided here opens doors of opportunity for further research to identify genes responsible for the liver growth induced by peroxisome proliferators.

The induced pluripotent stem cells (iPSCs) technology has revolutionized disease modelling by enabling the generation of patient-specific pluripotent stem cells for the study of complex disorders such as cancer. Somatic cell reprogramming through iPSCs induces global epigenetic reconfiguration of the chromatin which converts cancer cells to an embryonic stem cell-like state with potential reversion of tumorigenicity. Therefore, reprogramming can be used to answer the question as to whether epigenetic alterations alone can be sufficient to induce carcinogenesis, independent of genetic defects. In addition, it can used to dissect the relative contribution of genetics and epigenetics and epigenetics to tumorigenicity. In this study, the triple negative breast cancer (TNBC) cell line BT-549 and oestrogen receptor positive (ER+) cell line MCF7 were successfully reprogrammed by using the non-integrative episomal vectors expressing OCT4, SOX2, L-MYC, KLF4, LIN28, EBNA1, shRNA against TP53, and microRNA-302/367 cluster together with treatment of sodium butyrate. Pluripotency of cancer-derived iPSCs was confirmed by RT-PCR, RT-qPCR and immunofluorescence staining for expression of pluripotency markers. Differentiation potential of iPSCs was also assessed by using in vitro differentiation either spontaneous or directed to the mammary lineage. Functional assays indicated potential loss of tumorigenicity in re-differentiated cells derived from cancer iPSCs. The same approach was applied to study an immortalised, non-malignant mammary epithelial cell line MCF10A and two of its derived isogenic lines harbouring the two most frequent mutations in breast cancer, PIK3CAH1047R (+/-) and TP53(-/-), created by using CRISPR-Cas9 gene editing. Reprogramming induced a tumorigenic phenotype in iPSCs (PIK3CAH1047R (+/-) isogenic line only) and re-differentiated progenies (in both wild type MCF10A and PIK3CAH1047R (+/-) cell lines), suggesting the contribution of PIK3CA mutation in enhancing malignant transformation. Results in this study suggested that epigenetics alone and/or its interaction with genetic defects (e.g. PIK3CA mutation) has significant impact on breast cancer carcinogenesis. The dissection of the molecular mechanisms underlying the loss and gain of tumorigenicity using the iPSC models generated in this study could provide general understandings on breast carcinogenesis, which in turn could have important clinical implications.

Cell instructive biointerfaces are versatile tools to mimic a natural cellular environment and control cell fate in vitro. The particular interest lies in combining information gained from surface and interface analysis tools with biological analysis to explore and understand fundamental processes such as neuronal stem cell differentiation at the biointerface. A major challenge in biointerface design is to mimic and study the complex interactions of the natural processes in the extracellular matrix (ECM) with artificially designed surfaces and interfaces. In the past, peptide surfaces have been used as ECM mimics, however, more research is required in this field to tune the properties of peptide surface to modulate the outcome of stem cell fate. The present work aims to address this challenge by designing new synthetic peptide surfaces with well controlled composition and functionality able to impart have control over the differentiation of neuronal stem cells with the ultimate goal to relate surface properties and stem cell response to understand and control how neuronal networks function. Compositionally well-defined surfaces of two short laminin peptide sequences, Arg-Gly-Asp (RGD) and Ile-Lys-Val-Ala-Val (IKVAV) were prepared of various ratios via the “grafting from” stepwise approach. The surface modification was confirmed with surface analysis techniques to indicate successful peptide functionalization. The neural progenitor stem cells (NPSCs) were set up from embryonic rat hippocampi (E18). Immunocytochemistry (ICC) observed cell viability and differentiation to specific NPSCs lineages for βIII-Tubulin and GFAP. The surface characterizing techniques of WCA, AFM, ToF-SIMS, and XPS validated the successful orthogonal functionalization of controllable peptides composition surfaces with the increase of RGD composition a relative decrease in the IKVAV composition was observed. The increase in the normalized total ion fragments of RGD in the ToF-SIMS measurements can be related linearly to the % area coverage of neurons versus astrocytes observed on the controllable peptide composition surfaces. Well-defined peptide surfaces were designed and successfully demonstrated that the amount of RGD peptide composition present on the surface influences cell adhesion, proliferation and differentiation to a desirable cell fate or controllable cell population (i.e. neurons and astrocytes). Recent technological developments demonstrate a shift from two-dimensional (2D) surfaces to three-dimensional (3D) surfaces, so we used the designed versatile 2D surfaces as the template to design comparable 3D surfaces to examine the biological response of NPSCs to both microenvironments. This study proposes the design of novel 3D nanoparticles (NPs) made of gold and surface-conjugated with differentiation-inducing peptides. The NPs-peptides will guide stem cell differentiation to neurons that self-aggregate around the NPs by cell-cell contacts to form neurospheres. The neural stem cells will establish 3D structures biomimicking the cytoarchitecture of the brain. Successively, they could be used as an alternative 3D in vitro model for neurotoxicity testing of drugs and chemicals. Size-controllable NPs will be surface-conjugated with RGDC and IKVAVC(19) peptides via the SPPS “grafting to” approach. Two different sizes of NPs were characterized as AuNPs and SiO2@AuNPs characterization and validated by TEM, DLS, -potential, EDX; and the surface functionalization on NPs was successfully confirmed by UV/vis spectroscopy and -potential. The NPSCs were set up from E18 rat hippocampi and cell viability and differentiation to specific NSPCs lineages was stained for βIII-Tubulin and GFAP. AuNPs and SiO2@AuNPs peptide immobilized surfaces supported cell adhesion, proliferation, and survival. The confocal light scanning microscope (CLSM) images indicate that the RGDC functionalized AuNPs and SiO2@AuNPs surfaces induced a preferential differentiation towards a neurons cell fate and the IKVAVC(19) functionalized surfaces of AuNPs and SiO2@AuNPs favored an astrocytes cell. The cellular uptake of functionalized AuNPs by the cells in the neurospheres was observed via TEM micrographs, whilst the micrographs of functionalized SiO2@AuNPs surfaces suggest that they were not taken-up into the cells. Hence, indicating that there is a difference in the cellular uptake mechanism for the functionalized AuNPs and SiO2@AuNPs, which might be due to the agglomeration of the nanoparticles than the individual size of the nanoparticles. In conclusion, the relation between the 2D and 3D surfaces may provide new insight in understanding how surface properties affect the regulation of physiological relevance in directing neural cell differentiation, which will be essential to understand how neural networks function.

Cellular microenvironments are an important area of study, and their implications with regard to development, tissue function, and disease, mean that they have particular relevance in tissue engineering. The development of tissue engineered therapeutics is underpinned by the understanding of how the cells exist in their natural environment. A fundamental lack of insight into the signalling mechanisms within microenvironments, due to in part a lack of appropriate technologies, has meant that the therapeutic potential of tissue engineering is limited. To this end, the development of a micropatterning technology that enables control over solute signalling dynamics on the micron scale has been investigated. A bespoke holographic optical tweezers (HOTs) system was used to precisely position cells and controlled release vehicles into three-dimensional arrangements that resemble basic cellular micro-architectures. Via optical manipulation, release vehicles could be patterned to create solute release patterns to mimic signalling events in vitro. A proof of concept was established to demonstrate fluorophore release from microparticles positioned with high precision, into previously unobtainable micron-scale patterns. Such developments required optimisation of the system and protocols, for use with cell and microparticle manipulation and, creating a tool-set suitable for address unsolved biological questions. Biological investigations were completed to demonstrate how the HOTs can be used to control zonal cell differentiation and migration. These processes are paramount to cell microenvironment function, and this study has shown that the HOTs patterning setup is capable of achieving such signalling models in vitro. Herein is presented compelling evidence that optically manipulated release sources can achieve new levels of precision over signalling dynamics, over the length scales suitable for even the smallest cell microenvironments. It is hoped that through the better in vitro modelling of such cellular microenvironments and other signalling events, investigators will be able to elucidate new mechanisms through which cells proliferate and function.