The work discussed in this thesis adds further knowledge regarding the survival of embryonic dopaminergic grafts, derived from the rat ventral mesencephalon, in the rat model of Parkinson’s disease, in terms of the populations of cells involved, their distribution within the grafts, and how these are affected by the donor age and the host environment in to which they are implanted. The current data further reinforce the notion that harvesting ventral mesencephalic tissue at embryonic day 12 (E12) before the peak of dopamine neurogenesis yields more dopamine cells in the grafts and, more importantly, also yields more nigral A9 type dopamine neurons, which are an important determinant for functional recovery. Following on from this, commitment of dopamine neural precursor cells to the two dopamine neuron phenotypes, and how this is affected by the host environment was investigated, by grafting rat E12 and E14 ventral mesencephalon tissue into different cerebral targets. Brain regions were chosen that receive either the nigral A9 type dopamine, ventral tegmental A10 type dopamine or noradrenaline innervation. The yield of A9 type dopamine neurons was found to be influenced both by the environment within the graft and by the host environment in the transplantation site to a higher extent than the yield of A10 type dopamine neurons. Dopaminergic progenitors procured from rat embryos at E12 were shown to have a greater potential to proliferate post-grafting and differentiate into mature dopamine neurons as compared to embryos at E14. In vivo proliferation of younger precursor cells significantly contributed to the higher yields of the A9 type dopamine neurons in the grafts. If this improved yield of the A9 type dopamine neurons could be reproduced in human trials, fewer human donors might suffice to produce functional grafts in Parkinson’s disease patients.

Transglutaminase 2 (TG2) is an enzyme with a predominant role in cell stress response and tissue repair. Dramatically increased production of this enzyme is associated with early changes in arthritis, and the activity of the protein has been shown to directly contribute to both inflammatory and degenerative arthritis, although through distinct molecular mechanisms. Aberrant TG2 activity during joint disease might lead to protein modifications that are not normally present in extracellular matrix components. Those novel epitopes can possibly serve as a qualitative biomarker besides their potential role in disease pathogenesis. TG2 is released from cells via a non-conventional route, and this mechanism controls its extracellular activity. This pathway is likely to be shared with other proteins undergoing alternative secretion, many of which are potent biological signaling molecules. The aim of this project is to investigate whether non-classical secretion of TG2 is mediated by activation of the ligand-gated ion channel 7 (P2X7R) in analogy to interleukin-1β processing and release. Specifically, we are exploring whether ATP, a P2X7R agonist, which might be released from damaged cells at the sites of injury, triggers active release of TG2 from cells. To test this hypothesis we first employed macrophage and breast cancer cell models, where P2X7R is endogenously expressed, to look for involvement of ATP signaling in TG2 externalization through microvesicle shedding. By establishing HEK293 cells stably expressing P2X7R we show for the first time that introduction of functional P2X7R alone is sufficient to reconstitute rapid non-conventional TG2 export in a cell model. P2X7R activation induced time-dependent release of TG2 but not other cytoplasmic proteins, and this response was blocked by a selective P2X7R inhibitor. TG2 release was dependent on Ca2+ influx triggered by P2X7R activation and might be related to P2X7R-dependent membrane pore formation. These results provide a mechanistic explanation for a link between active TG2 release and inflammatory responses.

Peptidases of Leishmania are acknowledged virulence factors. It is hypothesised that peptidases are crucial for the survival of Leishmania in its hosts and that many could be potential targets for new antileishmanial drugs. As such, the investigation of peptidase activity in live Leishmania promastigotes was proposed as a valuable approach by which to increase knowledge on particular peptidases. In order to complete this investigation, it was decided to use short peptidyl fluorogenic substrates, which only fluoresce once the bond linking the peptide to the fluorescent moiety is cleaved. These allow detection of peptidase activity by quantifying the release of the fluorescent moiety. Detection of peptidase activity in live Leishmania using the fluorogenic substrate Bz-R-AMC proved fruitful, enabling study of the activity of the serine peptidase oligopeptidase B (OPB) in live L. major promastigotes. OPB is a member of the Family S9 peptidases, the prolyl-oligopeptidases, which are taxonomically restricted to plants, bacteria and trypanosomatid flagellates. In African and American trypanosomes, OPB has been shown to have important roles: OPB is a virulence factor in Trypanosoma cruzi, mediating entry into host cells, and OPB is released into the serum by African trypanosomes, where it cleaves host blood factors. In this study, the inhibition profile of L. major OPB has been determined and OPB has been localised to the cytosol, the site of hydrolysis of Bz-R-AMC. Immunoprecipitation of OPB confirmed that OPB was the sole peptidase responsible for the hydrolysis of Bz-R-AMC and anti-OPB antibodies were found to inhibit the hydrolysis of Bz-R-AMC. Inhibitors of OPB could also kill Leishmania promastigotes, suggesting OPB could be a valuable drug target. However, genetic manipulation of OPB was successful, with mutants over-expressing OPB and opb null mutants produced. OPB is thus not essential for the growth of promastigote L. major, though the opb null mutants did have a defect in metacyclogenesis, in survival in macrophages and a reduced ability to induce lesions on the footpads of mice. A role in amastigote differentiation or survival in macrophages was also suggested. OPB is thus likely to be a virulence factor, though not essential, and thus not suitable as a primary drug target. A number of avenues require further investigation, including the need for re-expression of OPB in the opb null mutants to confirm that lack of OPB is indeed responsible for the phenotypic deficiencies of the null mutants. Other important areas requiring attention are investigation of the role of OPB in amastigote differentiation or survival, investigation of the reported release of OPB by promastigotes, and identification of the physiological substrate of OPB.

In this thesis, a novel case-based reasoning (CBR) approach to radiotherapy treatment planning for brain cancer patients is presented. In radiotherapy, tumour cells are destroyed using ionizing radiation. For each patient, a treatment plan is generated that describes how the radiation should be applied in order to deliver a tumouricidal radiation dose while avoiding irradiation of healthy tissue and organs at risk in the vicinity of the tumour. The traditional, manual trial and error approach is a time-consuming process that depends on the experience and intuitive knowledge of medical physicists. CBR is an artificial intelligence methodology, which attempts to solve new problems based on the solutions of previously solved similar problems. In this research work, CBR is used to generate the parameters of a treatment plan by capturing the subjective and intuitive knowledge of expert medical physicists stored intrinsically in the treatment plans of similar patients treated in the past. This work focusses on the retrieval stage of the CBR system, in which given a new patient case, the most similar case in the archived case base is retrieved along with its treatment plan. A number of research issues that arise from using CBR for radiotherapy treatment planning for brain cancer are addressed. Different approaches to similarity calculation between cases are investigated and compared, in particular, the weighted nearest neighbour similarity measure and a novel non-linear, fuzzy similarity measure designed for our CBR system. A local case attribute weighting scheme has been developed that uses rules to assign attribute weights based on the values of the attributes in the new case and is compared to global attribute weighting, where the attribute weights remain constant for all target cases. A multi-phase case retrieval approach is introduced in which each phase considers one part of the solution. In addition, a framework developed for the imputation of missing values in the case base is described. The research was carried out in collaboration with medical physicists at the Nottingham University Hospitals NHS Trust, City Hospital Campus, UK. The performance of the developed methodologies was tested using brain cancer patient cases obtained from the City Hospital. The results obtained show that the success rate of the retrieval mechanism provides a good starting point for adaptation, the next phase in development for the CBR system. The developed automated CBR system will assist medical physicists in quickly generating treatment plans and can also serve as a teaching and training aid for junior, inexperienced medical physicists. In addition, the developed methods are generic in nature and can be adapted to be used in other CBR or intelligent decision support systems for other complex, real world, problem domains that highly depend on subjective and intuitive knowledge.

Automatic detection of protein-protein interactions (PPIs) in biomedical publications is vital for efficient biological research. It also presents a host of new challenges for pattern recognition methodologies, some of which will be addressed by the research in this thesis. Proteins are the principal method of communication within a cell; hence, this area of research is strongly motivated by the needs of biologists investigating sub-cellular functions of organisms, diseases, and treatments. These researchers rely on the collaborative efforts of the entire field and communicate through experimental results published in reviewed biomedical journals. The substantial number of interactions detected by automated large-scale PPI experiments, combined with the ease of access to the digitised publications, has increased the number of results made available each day. The ultimate aim of this research is to provide tools and mechanisms to aid biologists and database curators in locating relevant information. As part of this objective this thesis proposes, studies, and develops new methodologies that go some way to meeting this grand challenge. Pattern recognition methodologies are one approach that can be used to locate PPI sentences; however, most accurate pattern recognition methods require a set of labelled examples to train on. For this particular task, the collection and labelling of training data is highly expensive. On the other hand, the digital publications provide a plentiful source of unlabelled data. The unlabelled data is used, along with word cooccurrence models, to improve classification using Gaussian processes, a probabilistic alternative to the state-of-the-art support vector machines. This thesis presents and systematically assesses the novel methods of using the knowledge implicitly encoded in biomedical texts and shows an improvement on the current approaches to PPI sentence detection.

The discovery of stem cells has led to rapid advances in the field of regenerative medicine. Their unique properties, including the ability to self-renew and differentiate make them ideal for the repair/replacement of tissues that have been damaged as a result of disease or injury. Mesenchymal stem cells in particular represent a highly valuable pool of adult stem cells for such regenerative applications due to their accessibility, and potential as an autologous patient derived autologous nature However current methods for the in vitro expansion of high quality autologous mesenchymal stem cells results in spontaneous differentiation of the stem cell population and a loss of differentiation capacity over time. In vivo, it is the stem cell niche that provides stem cells with the appropriate cues required to maintain stem cell self-renewal. It is proposed that by mimicking these cues using biomaterials, that the self-renewal of mesenchymal stem cells can be controlled in vitro. In this study, a novel nanopit topography was investigated for its effects on the maintenance and growth of mesenchymal stem cells in vitro. To investigate this, three main aspects of mesenchymal stem cell state were examined in response to this novel nanotopography: maintenance of the stem cell phenotype over time including expression of stem cell markers and differentiation potential over time, changes in signalling pathways associated with differentiation and lastly, the metabolic profile of stem cells. As a result of this study we have identified a novel nanopit topography, which in the absence of chemical supplements, provides a substrate that is conducive to the maintenance of mesenchymal stem cells. Small RNAs have also been implicated in the regulation of signalling pathways and the metabolic state of stem cells. Furthermore, the ability to produce nanotopographically-patterned substrates using current standard techniques provides an inexpensive, high throughput method for the production of novel tissue culture plastics suitable for the maintenance of mesenchymal stem cells.

In order for cells to proliferate, a certain size has to be reached, which depends primarily on the rate of translation. RNA polymerase (pol) III plays a key role in protein synthesis by catalysing the production of small, untranslated RNA molecules such as transfer (tRNA) and 5S ribosomal RNA (5S rRNA). Indeed, recent evidence suggests that tRNAiMet production is limiting for translation and proliferation in some cell types. Therefore, the rate of pol III transcription plays a fundamental role in cellular growth and proliferation. Regulation of pol III output is mediated via a number of different mechanisms that can alter the activities of the transcription factors which are responsible for directing pol III transcription. Work presented in this thesis aimed at investigating the mechanisms behind the regulation of pol III transcription by the protein kinase CK2.

Nociception involves detection of tissue damage by specialized receptors; nociceptors. These convey information to the first synaptic relays in the dorsal horn of the spinal cord. Within the dorsal horn itself are the dorsal horn neurons which can be divided into two broad classes, based on their axonal projections: projection neurons and interneurons. The neurokinin 1 receptor (NK1r), the main target for substance P, is expressed by most projection neurons and many interneurons in the dorsal horn. These NK1r-expressing neurons show a bimodal size distribution in lamina I. The 1st part of the project tested the hypothesis that large NK1r-immunoreactive cells in this lamina are projection neurons, while the small cells are interneurons. Rats were anaesthetised and received injections of tracers into two supraspinal areas that are likely to label all contralateral lamina I projection neurons. The rats were re-anaesthetized and perfused 3 days later and 1341 NK1r-positive cells were analysed, of which 441 were retrogradely labelled. Cross-sectional soma areas of projection neurons were larger than those of cells that were not retrogradely labelled. This difference was highly significant. Nearly all (99.4%) of the NK1r-expressing cells that were not retrogradely labelled had soma areas <200 microm2, while only 9.8% of the retrogradely labelled NK1r-expressing cells had somata <200 microm2. These results provide a means of distinguishing lamina I NK1r-expressing projection neurons from interneurons based on their soma sizes without the need of retrograde tracing surgeries. Lamina I contains another population of projection neurons that lack or weakly express the NK1 receptor and consists of very large cells: giant cells, which are coated with the glycine and gamma-aminobutyric acid (GABA) receptor associated protein, gephyrin. There is also a group of large NK1r-expressing projection neurons with cell bodies in laminae III-IV and dendrites that pass dorsally to enter lamina I. Extracellular signal-regulated kinase (ERK) is expressed in dorsal horn neurons and is activated (phosphorylated) by noxious stimuli. In the 2nd part of the project, ERK phosphorylation in NK1r-expressing neurons as well as in lamina I giant cells was investigated following different type of noxious stimuli. Anaesthetised rats received noxious cutaneous, deep or visceral stimuli. They remained anaesthetized for 5 min after the end of the stimulus, and were then fixed by perfusion. Spinal cord sections were immunoreacted to reveal NK1r, gephyrin and phosphorylated ERK (pERK). Among the NK1r-expressing lamina I neurons, pERK was detected in both projection (somata >200 microm2) neurons and interneurons, with a significantly higher proportion in the larger cells, after all types of noxious stimulation. There was no significant difference in the frequency of pERK expression between the three morphological classes (fusiform, pyramidal and multipolar) of lamina I NK1r-expressing projection neurons after these stimuli. Most of the giant cells contained pERK after noxious cutaneous stimuli, but few did so following noxious deep stimulation. Only a few of laminae III-IV NK1r-expressing projection cells contained pERK after noxious deep or visceral stimulation, and the labelling in these was very weak. Results from the present study indicate that different types of neurons have different roles in conveying nociceptive information. The superficial dorsal horn (SDH) is also a vital area for modulating nociception and contains large number of excitatory and inhibitory interneurons. Glutamate, released by primary afferents and local excitatory neurons, acts on G-protein-coupled metabotropic glutamate receptors (mGlus). Group I mGlus (mGlu1 and mGlu5) are strongly expressed in the SDH. It has been reported that intrathecal administration of the mGlu1/5 agonist 3,5-dihydroxyphenylglycine (DHPG) induces spontaneous nociceptive behaviours, which are ERK-dependent. In the 3rd part of the project, ERK phosphorylation in mGlu5-expressing neurons following the administration of DHPG was investigated. Anaesthetized rats underwent a laminectomy procedure. DHPG or saline was applied to their exposed lumbar cord for 8 minutes after which they were perfused. Sections from the lumbar spinal cord were immunoreacted to reveal mGlu5, pERK and one of various markers for excitatory or inhibitory interneurons. Following DHPG (but not saline), numerous pERK-positive cells were seen in the SDH, particularly lamina II, and the great majority of these were mGlu5-positive. ERK phosphorylation was detected in both inhibitory and excitatory mGlu5-expressing cells, suggesting that type I mGlus have a complex role in nociceptive processing.

Modern day medicine is on the brink of a new age of therapy, which aims to harness the natural power of molecular biology for disease treatment. This therapy could include replacement of dysfunctional genes that cause disorders such as cystic fibrosis (Lommatzsch and Aris, 2009), or silencing the overexpression of genes that cause disorders such as cancer (Pelengaris and Khan, 2003). In both examples, the treatment of these genetic diseases lies in the delivery of synthetic nucleic acids into diseased cells, the former being called gene replacement therapy (Dobson, 2006a), and the latter being called RNA interference (RNAi) therapy (Whitehead et al., 2009). While these techniques have long been in use as genetic research tools for gene transfection or silencing in vitro, their translation for use in clinical disease treatment has yet to be achieved. The main problem facing the development of these novel therapies is the specific delivery of nucleic acids into diseased cells within the body. It is hoped that nanoparticles (NPs) can be used to overcome this problem, by acting as vehicles to transport nucleic acids through the body for specific delivery into diseased cells. This feat can be aided by the attachment of additional functional molecules such as cell penetrating peptides (CPPs), targeting peptides, additional drug types and molecules for imaging during treatment. Many different NP design strategies are currently under development. It is essential for new designs to be extensively tested for toxicity and efficiency in human cells before they can be successfully released into the clinic. As part of this effort, this PhD project has investigated two different NP design strategies for drug delivery: 1) the use of a magnetic field (MF) and a CPP to increase the delivery of iron oxide magnetic NPs (mNPs) to cells grown in tissueequivalent 3D collagen gels, and 2) gold NPs (AuNPs) for the delivery of siRNA to silence the c-myc oncogene for cancer treatment. In the first investigation, a MF and the CPP penetratin were found to increase mNP delivery to cells grown in 3D. In the second investigation, AuNPs were assessed in a range of different cell types (grown in 2D) for their performance in 4 main areas; cellular toxicity, cellular uptake, c-myc knockdown and effect on the cell cycle.

cAMP is a secondary messenger that is involved in a variety of signalling pathways through its effectors including EPAC, PKA and ion channels. cAMP signalling regulates processes such as memory, muscle contraction and inflammatory responses. PDE enzymes offer a mechanism to negatively regulate elevated cAMP levels elicited by activators of adenylyl cyclase. Studies have shown that cAMP signalling is compartmentalised through binding of PDEs to A-kinase anchoring proteins (AKAPs) that scaffold PKA regulatory subunits. In this study post-translational-modification of PDE4 isoforms is investigated. SUMOylation is a relatively newly identified post-translational modification that is known to regulate the structure and function of its substrates. PDE4 isoforms of the PDE4A and 4D subfamilies are SUMOylated by an E3 ligase, PIASy. SUMOylation alters the rolipram sensitivity and potentiates the PKA mediated activation of the isoforms whilst it confers protection from ERK-mediated inhibition of PDE4 activity. SUMOylation alters the association of PDE4 isoforms with binding partners like β-Arrestin, AKAP18 δ and UBC9. Rolipram is an archetypal PDE4 specific inhibitor. In this study it is shown that in cells expressing a GFP tagged form of PDE4A4 undergoes redistribution into accretion foci upon chronic treatment with rolipram. Data suggests that foci formation requires protein turnover and is regulated by signalling pathways such as PI3 kinase pathway, p38 MAP kinase pathway and PKC pathways. Further, the Immunomodulatory drug Thalidomide® also inhibits foci formation. PDE4 isoforms have isoforms specific N-terminal regions, which play a crucial role in sub-cellular localisation and protein-protein interactions. It is shown here that PDE4D5 interacts with a novel RhoGAP called ARHGAP21 which has been previously reported to bind β-arrestins. This interaction is independent of GAP activity of ARHGAP as well as PDE4 activity. Previous reports have indicated a role of β-Arrestin, PDE4 and ARHGAP21 in regulation of actin cytoskeleton dynamics. Hence complex β-Arrestin-PDE4-ARHGAP21 may play a crucial role in regulating actin dynamics.