Desmosomes are intercellular junctions found in epithelia and some other tissues. Their primary function is strong cell-cell adhesion. They also link the keratin intermediate filament (IF) cytoskeleton between cells and have roles in cell signalling, tissue morphogenesis and wound repair. Desmosomes are disc-like plaques arranged symmetrically on either side of the plasma membranes of adjacent cells. Each plaque consists of an outer dense plaque (ODP), an electron lucent zone and an inner dense plaque (IDP), the site of intermediate filament attachment. The plaques are joined to each other by the transmembrane desmosomal cadherins desmoglein and desmocollin which are adhesion molecules. These molecules have unique features in their cytoplasmic domains that may be important for desmosomal structure and function. Previous studies have predicted that Dsg extends from the extracellular core domain through the ODP and IDP and possibly bind to the IFs. However, immunogold localization ofthe C-terminus of the Dsg isoform with the shortest cytoplasmic domain, Dsg3, shows that this is not the case and that Dsg3 is entirely folded within the ODP. Dsg 2 has the largest cytoplasmic domain and is the most widespread isoform ofDsg. Its cytoplasmic domain (Dsg2Cyt) consists of483 amino acids divided into 5 sub-domains: the intracellular anchor, intracellular catenin site, internal proline-rich linker, repeat domain (RD) and terminal domain (TD). The aim ofthis project was to localize the RD and the TD within the desmosomal plaque by immunogold-electron microscopy. Recombinant N-terminally His-tagged Dsg2Cyt and GST-tagged Dsg2TD were expressed in E.coli, purified and used to raise polyclonal antibodies in rabbits. Characterization of these antibodies and further characterization of a mouse monoclonal antibody, 33-3D by immunoblotting showed that anti-Dsg2TD reacts exclusively with the TD and 33-3D reacts exclusively with the RD of Dsg2. Quantitative analysis of immunogold labelling of ultrathin cryosections of epidermis indicates that the repeat domain of Dsg2 is localized at the inner face of the outer dense plaque and the terminal domain is localized at the outer face of the inner dense plaque. This work contributes further to the elucidation of the structure of the desmosomal plaque by showing that the largest isoform ofDsg does extend beyond the ODP but it does not extend to the site ofIF attachment at the inner face ofthe IDP as suggested by other investigators.

It is widely accepted that pore interconnectivity and permeability are important characteristics effecting cell migration and cell response as well as the transport of nutrients, oxygen and cellular waste products throughout porous tissue engineering scaffolds. Furthermore, it was hypothesized that limited mass transport throughout three-dimensional structures resulted in diminished cell survival and cell distribution being restricted to the scaffold periphery. Several approaches were described for the quantification of scaffold permeability for liquid systems. Up to date, there are only a limited number of quantitative approaches to determine three-dimensional scaffold interconnectivity. This study aims to investigate interconnectivity and permeability in correlation with pore size and porosity. Therefore, tissue engineering scaffolds were fabricated by solvent casting/particulate leaching, supercritical fluid technology and particle sintering. In order to obtain different scaffold architectures, processing conditions were modified. Pore size, pore size distribution and porosity were quantified by MicroCT, and pore windows were analyzed using SEM. A novel interconnectivity algorithm was developed, which allowed the quantification of interconnectivity in 3D throughout the entire scaffold. Permeability of pre-wet scaffolds was determined. Results suggested that scaffolds with larger pore sizes and porosities also exhibited highest interconnectivities and permeabilities. However, these scaffolds showed a heterogeneous pore structure and pore distribution. The distribution of 3T3 fibroblasts through scC02-foamed scaffolds and particulate scaffolds was investigated by MicroCT and MTT staining. Homogenous cell distributions and largest cell volumes were observed on scaffolds with homogenous pore structure and hence smallest pore sizes, porosities, interconnectivities and permeabilities. This study might enable the tailoring of scaffold interconnectivity and permeability by altering scaffold processing conditions. Further, this study might allow the investigation of a minimum interconnectivity that is required for cell migration and proliferation in to order to generate tissues such as bone and cartilage; as well as to promote vascularization.

Embryonic stem cells (ES cells) are derived by explantation of the embryonic portion of the pre-implantation embryo into culture. These cells have unique properties which have made them invaluable in study of the function of genes <italic> in vivo</italic> and of cell differentiation <italic>in vitro.</italic> They can be grown in culture for extended periods of time in an undifferentiated state and induced to differentiate in vitro. While undifferentiated they can be genetically manipulated. Subsequent reintroduction of these cells into the blastocyst results in the cells being integrated and contributing to all the cells of the animal including the germ line thus leading to designed genetic change. The homology of these cells, however, to their tissue of origin is not unambiguous. The primary aim of this thesis was to apply global transcriptome analysis to investigate the homology of ES cells to the pluripotent compartment of the embryo. Although ES cells can be grown in bulk, the tissue of origin, the embryonic portion of the peri-implantation embryo are small and inaccessible. It was therefore necessary to develop methods which would allow the transcriptome to be amplified without distorting the transcript profile. A linear amplification method proved to give the best result. The best method for fluorescently labelling the cDNA was shown to be enzymatic incorporation of aminoallyl dUTP followed by coupling to monoreactive Cy dyes. With these tools it was then possible to amplify the transcriptome of both colonies of ES cells and the embryonic portion of various peri-implantation embryos and apply the labelled cDNA to microarray slides. Statistical analysis of the results proved that the transcriptome of ES cells most resembles that of the embryonic ectoderm on day 5.5 of development.

Huntington's disease (HD) is a neurodegenerative condition in which the predominant loss of neurons occurs in the striatum. At present there is no treatment for this condition, although neural transplantation may prove to be a viable therapeutic strategy if an appropriate source of donor cells can be identified. A major requirement of these donor cells is that they are able to differentiate into the cells lost to the disease process that is, largely medium spiny projection neurons (MSNs). Currently, suitable donor cells (i.e. those already committed to developing into MSNs) can be extracted from foetal brain and early clinical trials have provided some evidence of efficacy when human foetal-derived striatum is transplanted into the brain of patients with HD. However, there is a major problem of supply and demand with respect to human foetal tissue and so alternative source of donor cells must be identified. However, only a small proportion of animal studies in the literature report differentiation of MSNs from either animal or human stem cell sources and the percentage of mature MSNs is generally low, most cells becoming glia or taking on a 'default' GABA-ergic neuronal phenotype. Thus it is likely that stem cells will need to be 'directed' towards a MSN phenotype. Knowledge of the molecular signals that cause striatal progenitors to differentiate into a MSN phenotype in vivo would help us to understand how to direct the fate of stem cell populations towards this phenotype in vitro. In this thesis I have studied the genetic changes that occur during normal striatal development in the mouse with the aims being (i) to identify genetic markers of stages of differentiation for these cells and (ii) to identify genes important for striatal development with the ultimate aim of using this information to design protocols to direct the differentiation of stem cells towards a MSN phenotype. I have studied the gene expression of the population of cells that make up the whole ganglionic eminence during its period of peak neurogenesis using Affymetrix micro array. I then validated the results of a subset of genes that were found to be significantly up-regulated using in situ hybridisation and then used these genes to characterise either primary cells that were differentiated in vitro, or cells that have been proliferated and then differentiated in vitro. This study has not only provided a gene expression signature of a developing population of striatal precursors, enabling future experiments to compare and contrast expression patterns seen in different in vitro studies, but it has also highlighted Foxpl and Foxp2 that have been shown to have a high degree of association with this period of development. This has encouraged future work in this laboratory in which the developmental functions of these genes in relation to MSN differentiation and development will be studied.

Introduction: Disc degeneration is the primary cause of low hack pain. Due to the main function of the spine, mechanical stimuli play a pivotal role in the development of disc degeneration. Previous studies have shown that cytoskeletal elements are involved in the mechanotransduction pathways between the extracellular matrix (ECM) and cell nucleus. However, the precise mechanism of this mechano-induced signalling is not clear in the intervertebral disc (IVD). Therefore, the effects of tensile strain and compression on the organisation and expression of cytoskeletal elements in bovine disc cells and on ECM metabolism molecules were investigated using confocal microscopy, real-time PCR, Western blotting, gelatin zymography and reverse gelatin zymography. Results: (i) In situ: F-actin filaments were punctate and distributed beneath the cell membrane in both nucleuspulposus (NP) and outer annulus fibrosus (OAF). There was higher p-actin expression in the OAF than NP. p-tubulin filaments formed a meshwork distributed throughout the cytoplasm with more p-tubulin gene and protein expression in the NP. Vimentin filaments formed a meshwork distributed throughout the cytoplasm with lower vimentin protein content in the OAF. There was less vimentin protein, but an increase in partially degraded vimentin in OAF with maturity, (ii) Cyclic tensile strain (CTS): CTS promoted the reorganisation of F-actin and P-tubulin networks and increased both their mRNA and protein expression, whilst reducing vimentin levels. CTS differentially-regidated mRNA expression levels of MMPs and TIMPs suggesting accelerated ECM remodelling processes in IVD cells. OAF cells are more responsive to tensile strain than NP cells. Stretch-induced mechano-respouses in IVD cells are age and strain-dependent. Age delayed the cell's response to tensile force. Low (5%) and medium (10%) strains induced anabolic effects but high strain (15%) induced more catabolic responses. (Hi) Compressive loading: Compressive loading (10%, 1Hz) altered the architecture of F-actin, P-tubulin and vimentin filaments, along with an increase in P-tubulin mRNA and a decrease in vimentin and vinculin gene expression. Compressive loading increased transcription of matrix molecules and decreased mRNA levels of catabolic enzymes in I D cells, suggesting an overall anabolic effect of physiological compression on I T> cell metabolism. Ageing delayed or reversed these mechano-responses in mature IVD cells. Conclusion: Cytoskeletal elements including P-actin, P-tubulin and vimentin are involved in strain-induced and compression-induced mechanotransduction in IVD cells. NP and OAF cells respond differently to the type of mechanical stimulus applied. However physiological loads induce matrix synthesis in NP and OAF cells, whilst non-physiological strains promote a catabolic phenotype. Ageing can delay the cell's response to mechanical load which may induce "abnormal" ECM remodelling events, and increase the potential risk of a loss of tissue homeostasis and the likelihood of disc degeneration.

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell growth and proliferation in tissues - while abnormal control and modulation of the cell cycle are characteristic of cancer cells, particularly in response to therapy. A central theme in cancer biology is to resolve and understand the origin and nature of innate and induced heterogeneity at the cell population level. Cellular heterogeneity - comprising structural, temporal and functional dimensions - is a confounding factor in the analysis of cell population dynamics and has implications at physiological, pathological and therapeutic levels. There is an exceptional advancement in the applications of imaging and cell tracking technologies dedicated to the area of cytometric research, that demand an integrated bioinformatics environment for high-content data extraction and interrogation. Image-derived cell-based analyses, where time is the quality parameter also demand unique solutions with the aim of enabling image encoding of spatiotemporal cellular events within complex cell populations. The perspective for this thesis is the complex yet poorly understood nature of cancer and the opportunities offered by rapidly evolving cytometric technologies. The research addresses the intellectual aspects of a bioinformatics framework for cellular informatics that encompass integrated data encoding, archiving, mining and analysis tools and methods capable of producing in silico cellular fingerprints for the responses of cell populations to perturbing influences. The overall goal is to understand the effects of anti-cancer drugs in complex and potentially heterogeneous neoplastic cellular systems by providing hypothesis testing opportunities. Cell lineage maps encoded from timelapse microscopy image sequences sit at the core of the proposed bioinformatics infrastructure developed in the current work. Through a number of data mining, analysis and visualisation tools the interactions and relationships within and between lineages have provided dynamic patterns for the modulation of the cell cycle in disease and under stress. The lineage data, accessible through databases implemented during the current study, has provided a rich repository for pharmacodynamic (PD) modelling and validation and has thus laid the foundation for fabricating a comprehensive knowledge base for linking both cellular and molecular behaviour patterns. These provide the foundation for meeting the aspirations of systems biology and drug discovery.

Intense excitement and optimism surrounds the rapidly-expanding field of stem cell research, owing to their high capacity for self-renewal and intrinsic ability to differentiate into mature cell lineages. Although it may be envisioned that embryonic stem cells will be of significantly greater therapeutic value than their adult stem cell counterparts, the use of embryonic stem cells is fraught with both technical and ethical challenges and, as such, significant impetus has been placed on adult stem cell-based research. In particular, mesenchymal stem cells (MSCs) present as exciting candidates for potential use in cellular therapies and tissue engineering strategies. MSCs are defined at the functional level in terms of their ability to differentiate into mesodermal derivatives such as bone and fat. However, this functional definition is evolving, and there is considerable evidence to suggest that MSCs have a key role within their niche involving the release and/or uptake of soluble factors and cytokines, significantly influencing the behaviour of other cell types within the niche. Both facets of MSC behaviour are valuable from a clinical perspective, and have been examined in the present thesis. The most obvious and realistically-achievable clinical application of MSCs at present is in the treatment of osseous and adipose tissue defects. However, before the use of MSCs in the clinic becomes more commonplace, it is crucial to gain a more comprehensive understanding of the complex molecular and cellular mechanism(s) by which MSCs commit to a given fate and undergo differentiation to produce mature, fully-functional derivatives. Much of our present knowledge is derived from studies performed on the highly unnatural, 2D environment of tissue culture plastic. The present study assessed the behaviour of MSCs cultured on AlvetexTM, a novel, 3D scaffold manufactured by ReInnervate, with particular emphasis on the ability of MSCs to undergo osteogenic and adipogenic differentiation. Results obtained suggest that AlvetexTM may provide a more realistic and physiologically-relevant system in which to study osteogenesis and adipogenesis, in a manner more pertinent to that which occurs in vivo. Furthermore, the ability of MSCs to influence the behaviour of other cell types via the release of trophic factors and cytokines was examined, with particular emphasis on the nervous system. An in vitro conditioned media model was developed in order to investigate the influence(s) of MSC-derived soluble factors/cytokines on neural development and plasticity, using the adult rat hippocampal progenitor cell (AHPC) line as a model system. Results obtained suggest that, under defined conditions, MSCs secreted a complement of soluble factors/cytokines that induce AHPCs to commit to and undergo astrogenesis. This effect was characterised at both the cellular and molecular level. The specific complement of bioactive factors secreted by MSCs has been investigated using a combination of targeted transcriptional profiling and shotgun proteomics, and several putative candidate factors have been identified for further investigation.

Lamins are type V intermediate filament proteins found at the nuclear envelope. Expression of lamin A in colorectal cancer (CRC) tumours is correlated with poor prognosis and expression of lamin A in CRC cell lines promotes greatly increased cell motility. The aim of this study was to identify proteins that promote cell motility in response to lamin A expression and to investigate lamin A regulated changes in gene/protein expression and cytoskeletal organisation that might underpin the increased cell motility. The effects of lamin A expression were studied using quantitative proteomic and genomic methods using cells from the colorectal cancer cell line SW480 which had been transfected with GFP-lamin A (SW480/lamA) or GFP as a control (SW480/cntl). A biochemical fractionation technique was optimised for the preparation of cytoskeletal fractions which were analysed by 2D DIGE (2D difference in-gel electrophoresis) to reveal accurate and reproducible changes in the representation of proteins within the cytoskeleton in SW480/lamA cells compared to controls. The majority of proteins identified were either components of the actin/intermediate filament cytoskeleton, protein chaperones or translation initiation/elongation factors. Interestingly, tissue transglutaminase 2, a protein which modifies elements of the cytoskeleton and is associated with cancer progression, was highly over-represented in the cytoskeleton fraction of SW480/lamA cells. Ingenuity Pathway Analysis was used to analyse genome-wide Affymetrix microarray analysis of SW480/cntl and SW480/lamA cell lines. A highly significant interaction network was identified which clustered together genes linked to cancer, cellular movement and cellular growth and proliferation. Epithelial markers such as CDH1 were down-regulated and mesenchymal markers such as FN1 were up-regulated in cells expressing GFP-lamin A, which suggested that lamin A over-expression may lead to an epithelial-mesenchymal transition (EMT). As A-type lamins are known to modulate downstream effects of TGFβ signalling, and TGFβ is an inducer of EMT, changes in genes involved in TGFβ signalling were investigated. Knockdown of lamin A using siRNA led to decreased expression of TGFBI and SNAI2 followed by reduced cell motility. The data suggest that expression of lamin A in CRC cells causes changes in the organisation of the actin cytoskeleton and in TGFβ signalling, potentially involving an epithelial to mesenchymal transition, leading to increased cell motility and an increased risk of death from cancer.

Stem cell homeostasis and differentiation are controlled by the complex interplay of a wide range of signalling pathways and small molecules, including all-trans-retinoic acid (ATRA). The endogenous effects elicited by ATRA, have led to its use in numerous in vitro protocols as a tool for cell differentiation. However, ATRA isomerises and degrades under standard laboratory conditions and furthermore, is rapidly metabolised in vivo, which leads to pleotropic effects and a high efficacious dose response. Consequently, synthetic analogues that are structurally and/or functionally equivalent to ATRA have been developed, as alternative pharmacological tools to further the understanding of this molecular pathway and control cell differentiation.In this study a small library of synthetic retinoids were prepared, which were designed to probe structural size, conformation and biological function, while being more resistant to cellular metabolism and isomerisation. Their stability towards fluorescent light was examined along with their activity in four different stem cell models. Two compounds, AH60 and AH61 were found to inhibit cellular proliferation and induce neural differentiation, through acting on the retinoic acid receptor pathway. Compared to ATRA, AH60 was approximately 10-fold more active, while AH61 was 100-fold more active in two of the cell models tested. These compounds are described comprehensively herein, and should be suitable and convenient alternatives to ATRA and 13cRA for use in in vitro studies carried out by cell and molecular biologists. In addition, an unrelated small molecule, neuropathiazol, has been synthesised to further characterise both the chemistry involved in its production and its biological activity in controlling cell differentiation. This compound was highlighted in the literature as an alternative to ATRA, for inducing neural differentiation in neural progenitor cells. We have further investigated its potential to differentiate other neural stem cell types and pluripotent stem cells. In addition potential analogues of neuropathiazol are discussed, as compounds of this nature are potentially highly useful for selectively controlling neural differentiation.

A-type lamins are a group of type V intermediate filaments whose main members are lamin A and C. Lamins A/C are components of the nuclear lamina and are encoded by the LMNA gene. Lamins A/C have a variety of cellular functions, including maintaining the structural integrity of the nucleus and the regulation of signal transduction pathways, transcription factors and DNA replication. Mutations in LMNA give rise to a diverse spectrum of diseases, termed laminopathies, which include premature aging syndromes. In Chapter 3, I sought to understand the role of wild type lamin A in normal cellular aging. Lamin A C-terminal cysteine residues were irreversibly oxidized during the in vitro aging of human dermal fibroblasts (HDFs), which impaired the ability of lamin A to form disulfide bonds, causing loss of function. Furthermore, loss of these cysteine residues induced premature senescence, suggesting that these cysteine residues are important for lamin A function during cellular aging. In Chapter 4, I extended previous findings implicating A-type lamins in the control of cell cycle progression. Loss of A-type lamins or its nucleoplasmic binding partner, LAP2α, caused delayed G1/S-phase progression, reduced cellular proliferation and cell cycle exit. Proliferative defects could not be rescued via treatment with anti-oxidants. In Chapters 5 and 6, I addressed the role of wild type mature lamin A/C in the DNA damage response (DDR). A-type lamins interact with the DDR mediator protein 53BP1 via its Tudor domain. Loss of LMNA caused endogenous DNA damage and loss of 53BP1 protein levels. Furthermore, loss of LMNA resulted in defective DNA repair that ultimately led to increased sensitivity to DNA damage. Together, the data presented here extends previous findings implicating A-type lamins in cell cycle progression and provides novel insights into the cellular roles of A-type lamins in cellular aging and the DNA damage response.