Articular chondrocytes reside in a unique and challenging avascular environment in cartilage. During joint disease, O2 and pH levels are further reduced, and may be involved in chondrocyte dysfunction and cartilage matrix breakdown. How fluctuations in these environmental parameters affect factors such as redox balance and mitochondrial function in articular chondrocytes is largely unknown but may provide a link between the extracellular environment to cell signalling pathways that may alter cellular and hence cartilage integrity. Additionally, ROS-regulatory compounds may offer scope for therapeutic intervention in joint disease. In this project, the effects of different O2 levels (<1%, 2%, 5% or 21% O2), pH (pH 7.2 or pH 6.2) and exposure to the pro-inflammatory cytokine, interleukin-1β (10ng/ml) on cell viability and glycosaminoglycan (GAG) release, redox balance (ROS production, GSH:GSSG ratio, NO release, SOD expression) and mitochondrial function (mitochondrial membrane potential (ΔΨm)) in articular chondrocytes cultured in 3-D alginate beads were analysed. The ability of the ROS regulatory compounds N-acetylcysteine (NAC) (2mM) and resveratrol (10μM) to alter these variables was also assessed. This study demonstrates the distinct and interactive effect of O2 tension and pH to influence articular chondrocyte redox balance and mitochondrial function. Cellular ROS levels, ΔΨm and SOD expression all displayed O2 sensitivity, being reduced by hypoxia/anoxia. Articular chondrocytes also displayed pH sensitivity with acidosis modulating ROS levels, ΔΨm and GSH content. Combined conditions (hypoxia/anoxia and acidosis) accentuated responses and further compromised chondrocyte integrity (survival, GAG release, phenotype expression). Treatment with NAC and resveratrol altered these variables through distinct mechanisms and conferred protection against O2 and acid-mediated oxidative stress. Both NAC and resveratrol abolished anoxic-induced inhibition of SOD levels. NAC significantly abrogated anoxic and acidosis-induced GSH inhibition, whereas resveratrol modulated ROS levels and restored O2 and pH mediated mitochondrial depolarisation. This thesis details the powerful effect of the cellular environment to modulate chondrocyte function. Manipulation of this environment or targeting related molecular pathways may hold promise for alleviation of disease-associated oxidative stress and are discussed.

Loss of the signalling protein XLαs in mice leads to a lean phenotype characterised by increased energy expenditure due to elevated sympathetic nervous system (SNS) stimulation of brown adipose tissue. XLαs is the protein produced from the Gnasxl transcript of the imprinted Gnas locus, and has a restricted expression pattern that includes a number of brain regions essential for SNS control of both energy expenditure and the cardiovascular system. However, it is unknown to what degree XLαs influences overall sympathetic tone, or how XLαs signalling in the brain causes these phenotypic changes. Using arterial cannulation, anaesthetised Gnasxl knockout mice had elevated blood pressure, shown to be caused by increased SNS stimulation by a greater blood pressure response to the sympatholytic reserpine in knockouts. Using electrocardiogram (ECG) telemetry, conscious Gnasxl knockout mice had elevated heart rate at night, as well as a significant heart rate response to both reserpine and the parasympatholytic atropine. This supported the previous results showing elevated SNS stimulation of the cardiovascular system, but paradoxically also suggested elevated parasympathetic stimulation. Therefore, autonomic control of the cardiovascular system was investigated in further detail by analysing heart rate variability (HRV). A number of HRV analyses were experimentally validated in wildtype mice. The most reliable method was the fast Fourier transform (FFT); high frequency (HF) power was used as a measure of parasympathetic activity, and low frequency (LF)/HF ratio was used as a measure of sympathetic activity. Gnasxl knockouts had a greater LF/HF response to reserpine, but an equivalent HF response to atropine, suggesting the mice had elevated SNS activity only. Additionally, knockouts had elevated LF/HF ratio at night, suggesting consistently elevated SNS output. Neuronal signalling pathways that may be deregulated in Gnasxl knockouts were investigated by injection of MTII and Exendin-4, agonists to the melanocortin 3/4 and GLP-1 receptors, respectively. Gnasxl knockouts had a hypersensitive heart rate response both to centrally injected MTII in anaesthetised mice and peripherally injected Exendin-4 in conscious mice. The hypersensitivity to Exendin-4 was investigated further by HRV analysis, which showed that Exendin-4 had no effect on the SNS, but caused a potent reduction in parasympathetic activity in both wildtypes and knockouts. Neuronal signalling changes in response to Exendin-4 were investigated by antibody staining for the early response gene c-fos. No significant differences were seen in overall numbers of activated neurones between wildtypes and knockouts in a number of brain regions including the nucleus of the solitary tract (NTS). Interestingly, neurones expressing XLαs showed no c-fos response to Exendin-4, except in the area postrema. In summary, loss of XLαs in mice resulted in elevated SNS stimulation of the cardiovascular system, as well as hypersensitivity to Exendin-4 that was unlikely to be caused by increased activation of XLαs-deficient neurones.

A PDZ domain is a small, ~ 90 amino acid residue region of a protein that acts as a protein-protein interaction module. There are currently 267 known PDZ domain-containing proteins in the human genome, with the predominant function of a PDZ domain being the recognition and binding of C-terminal motifs in partner proteins. Examples of well-studied multi PDZ domain-containing proteins are the postsynaptic density-95 protein (PSD-95) and the synapse-associated protein 97 (SAP97); different PSD-95 and SAP97 PDZ domain-mediated interactions have been implicated in a variety of pathological conditions. The interaction of the PSD-95 PDZ domains with the 5-hydroxytryptamine receptor 2a (5-HT2a) & 2c (5-HT2c) variants is known to be important in inducing hyperalgesia in neuropathic pain; the PDZ-mediated interaction of SAP97 with the human papillomavirus type 18 (HPV18) E6 protein is an important event in a p53-independent pathway of cervical carcinogenesis. As PDZ domains have been shown to bind small organic molecules and that the majority of free energy contributions of the PDZ domain interaction interface to binding, are due to a select few ‘hotspot’ regions; the development of novel, reversible small molecule inhibitors of the PSD-95 and SAP97 PDZ domains was deemed a viable research target. This was the overriding aim of the research programme detailed in this thesis and encompassed biophysical techniques such as: protein production, NMR spectroscopy, isothermal titration calorimetry (ITC), restraint-driven docking and structure determination methodologies. The PDZ-mediated interactions of 5-HT2a and 5-HT2c with the PSD-95 and SAP97 PDZ1 & PDZ2 were confirmed using NMR spectroscopy and then, quantified by ITC. The structure of the physiologically relevant complex formed between PSD-95 PDZ1 and 5-HT2c was determined using NMR solution state spectroscopic determination methods; this is the first example of a PSD-95 PDZ – 5-HT2x complex structure. A systematic investigation into the interaction between the SAP97 PDZ2 domain and the HPV18 E6 protein was carried out by ITC; this process involved binding experiments utilising commercially acquired peptides of varying lengths and sequences, containing natural and non-natural amino acids. A variety of small organic molecules that bind to the PSD-95 PDZ1 domain have been developed by an NMR screening process. The PSD-95 PDZ1 binding affinity of two of the small molecules was determined by NMR spectroscopy and their inhibitory effect on the native PSD-95 PDZ1 – 5-HT2c interaction was quantified using ITC. The structure of the individual PSD-95 PDZ1 – small molecule complexes formed were determined by NMR restraint-driven docking, using the high-ambiguity driven docking (HADDOCK) programme; this is the first instance of HADDOCK being utilised to determine a PDZ domain – small molecule complex structure. The research detailed in this thesis alludes to the possibility that PSD-95 and SAP97 PDZ domain-mediated interaction inhibition by a small molecule compound may not be a feasible research aim and that future PDZ domain inhibition research may need to be focus on other inhibitor structures i.e. β-strand peptidomimetics.

Initial predictions of drug response and drug-drug interactions (DDIs) are made following high-throughput in vitro screening. Such assays are indispensible in the pharmaceutical industry to determine the metabolism, transport and pharmacokinetics of new chemical entities. However, they often fail when extrapolated to in vivo response due to unsuitable pharmacokinetic or pharmacodynamic prediction. The primary aim of this thesis was to investigate and understand the differences in the expression profiles of drug disposition genes, between transformed hepatic cell lines and primary human hepatocytes. Primary human hepatocytes were also analysed to determine uptake contribution, induction and genotype of key drug disposition-relevant genes. The loss of hepatic phenotype in HepG2 and Huh7 cells is partly due to the altered expression of transcriptional regulators including; chaperones, co-chaperones, co-activators and co-repressors. Indeed, Chapter 2 of this thesis shows lower levels of the Gadd45β and PGC1α gene expression in HepG2 cells corresponds to a deficient expression and activity of cytochrome P450 3A4 (CYP3A4), with the levels reducing further as cell passage increases, in comparison to primary human hepatocytes. HepG2 cells were transfected with a novel complex transfection of Gadd45β and PGC1α with the aim to improve CYP3A4 activity in Chapter 3. CYP3A4 activity was improved by 54% and induction response was enhanced in comparison to control cells with no off-target effects. Over the last decade it has become apparent that transporters can play a significant role in the disposition of many drugs. Organic anion transporting polypeptide (OATP) transporters have received considerable recent attention since they mediate sodium-independent uptake of a broad array of xenobiotics. A method to determine the specific contribution of OATP1B1 in the hepatic uptake was successfully optimised and applied for 5 therapeutic drugs in Chapter 4. Future application of this strategy is likely to have broad importance in determining relative contribution that individual transporters play in drug disposition. To prevent accumulation and toxicity of xenobiotics, biotransformation and transport of foreign compounds occurs. However, these processes can be altered by induction or inhibition mechanisms. Rifampicin is a first line drug in tuberculosis (TB) treatment but it is a potent inducer of CYPs and transporters. DDIs during TB treatment are common but the induction potential of different rifamycins has not been comprehensively ranked. Chapter 5 investigated the induction potential of rifampicin, rifapentine and rifabutin. Rifampicin significantly induced CYP3A4, ABCB1, OATP1B1 and ABCC2 in primary human hepatocytes. Induction by rifabutin was observed for CYP3A4, OATP1B3 whilst rifapentine only significantly induced OATP1B1. This work serves as a basis for further study into the extent to which rifamycins induce key metabolism and transporter genes. Nuclear receptors (NR) regulate the expression of CYPs and drug transporters influencing pharmacokinetics. PXR and VDR have been found to synergistically increase CYP3A4 expression and activity in intestinal cell lines. This effect has been observed in vivo with seasonal variations apparent for CYP3A4 substrates. In Chapter 6, novel associations between vitamin D receptor polymorphisms and expression of it and its target genes involved in drug disposition were shown in D2 intestinal biopsies. This thesis reports generation of model systems and their application to enable many questions to be answered relating to pharmacokinetics and DDIs. The thesis forms a solid platform from which to further investigate these issues in future studies.

Ageing is a phenomenon common to almost all living organisms and is characterised by the accumulation of changes with time that are associated with the ever-increasing susceptibility to disease and inevitably death. The rate of ageing is species-specific, indicating a strong genetic component. It is now widely accepted that genes involved in basic cellular processes such as stress resistance, metabolic regulation and genomic stability determine longevity in divergent organisms from yeast to mammals. This suggests that there may be conserved universal mechanisms involved in ageing. Furthermore, lifespan can be increased in all these model organisms by reducing the nutrients consumed - a phenomenon known as dietary restriction (DR). Saccharomyces cerevisiae is a useful model organism in which to study ageing and has been at the forefront of recent pioneering work on the molecular mechanisms underlying lifespan extension by DR. DR can be studied by reducing the glucose concentration from the standard 2% down to 0.5% or below, or by using genetic mimics. However, the exact mechanisms of lifespan extension by dietary restriction remain unclear and highly controversial. Research in our laboratory has identified two proteins in yeast that are induced in response to DR and thus correlate with longevity. Both proteins, Hsp12 and Hsp26, belong to the small heat shock protein (sHsp) family. Previous work by the Morgan laboratory has found that Hsp12 is essential for the longevity effect of DR and have solved the structure of the protein by NMR. Further studies have revealed a genetic interaction between HSP12 and HSP26, as hsp12/hsp26Δ double knockouts show a strongly reduced mean and maximum replicative lifespan. Despite this, the hsp12/hsp26∆ double knockout is not defective in various processes associated with yeast ageing, including stress resistance, rDNA silencing or protein aggregation. To shed light on the mechanisms by which Hsp12 and Hsp26 affect longevity, we employed unbiased approaches of synthetic genetic array (SGA) and quantitative fitness analysis (QFA) to identify genetic interactions of HSP12 and HSP26 on a genome-wide scale. This involved the generation of thousands of double mutant strains and analysis of their growth under various conditions, including DR. Results from the SGA analysis have revealed genetic interactions between HSP12 and HSP26 with the regulation of transcription from RNA polymerase II and processes associated with the mitochondria and vacuoles. QFA data is still to be analysed but ultimately we hope that QFA will re-confirm the genetic interactions identified by SGA analysis. We hope both SGA analysis and QFA data will provide insight into the cellular functions of Hsp12 and Hsp26 and how these proteins affect ageing, in particular lifespan extension by DR.

Deubiquitylases (DUBs) have been implicated in the regulation of cell signaling processes. However, the role of DUBs in the regulation of the Epidermal growth factor receptor (EGFR) signaling is not completely understood. This study has aimed to identify DUBs involved in the regulation of EGFR signaling and downstream cascades. The first part of this study has characterized the role of the DUB USP15 in the regulation of the mitogen activated protein kinase (MAPK) cascade, a pathway downstream of EGFR. An interaction between USP15 and the MAPK negative regulator BRAP had been previously reported. When we tested the USP15 depletion phenotype on MAPK signaling we observed a paradoxical decrease in MAPK activation. Examination of upstream components of the MAPK cascade revealed a decrease in the levels of the CRAF kinase following USP15 depletion. Concordant depletion of CRAF also caused a reduction in MAPK activation, showing that depletion of CRAF phenocopied that of USP15. This work demonstrated that USP15 has a dual role in the regulation of the MAPK through BRAP and CRAF. The dominant signaling effect in the cell lines studied is through maintenance of CRAF levels. We employed a previously characterized GFP-DUB library to identify DUBs that exhibited EGF dependent distributions. One such DUB, USP46, exhibited MAPK dependent recruitment onto multi-vesicular bodies (MVB). To further characterize USP46 we generated a set of cell lines expressing GFPUSP46 and catalytically inactive GFP-USP46-C44S using the Flp-in system. While the Flp-in cells lines did not exhibit the same EGF dependent recruitment onto the MVB compartment, they did localize to Saponin resistant punctate structures. Furthermore, I observed differential activation of downstream EGFR signaling pathways that USP46 may play an undetermined role in EGF signaling. We combining stable isotope labeling of amino acids in culture (SILAC) with immuno-precipitation (IP) to quantitatively identify interactors of USP46 using mass spectrometry. We identified a 13 number of candidate interactors and confirmed a novel interaction between USP46 and FBXO11 using western blotting. Next we aimed to identify DUBs that regulate the retrograde trafficking pathway from the MVB to the trans Golgi network (TGN). We used the localization of the cation independent mannose 6-phosphate receptor (CIM6PR) as readout of the retrograde trafficking. CI-M6PR constitutively recycles from the TGN to the endo-lysosomal pathway, delivering newly synthesized acid hydrolases, required for degradative action of the lysosome. Depletion of USP8 trapped CI-M6PR in aberrant endosomes and caused a concomitant missorting of the acid hydrolase, Cathepsin D. Cathepsin D is activated through limited proteolysis in the acidic environment of the endolysosomal pathway. Depletion of USP8 caused a decrease in the mature cellular form of Cathepsin D. The mislocalization of CI-M6PR could be rescued by re-expression of GFP-USP8. The activated EGFR is degraded via the lysosome and depletion of USP8 has been demonstrated to cause a delay in the degradation of EGFR. The results presented here suggest that the decrease in active acid hydrolases observed in USP8 depleted cells, may contribute to the delay in EGFR degradation.

Non-union fractures are defined as fractures that do not heal after 6 months of conventional treatment. They usually require multiple surgical treatments, autologous bone grafts or treatments with growth factors or Bone morphogenetic proteins (BMPs). There is a clinical need for a material which can be used to replace autologous bone transplantation in the treatment non-union fractures that negates the problems associated with autologous grafts. This thesis aims to consider and develop a coating that can be used on a readily available polymer biomaterial to induce a response from mesenchymal stem cells, which are found in abundance at fracture sites, and facilitate repair by their differentiation into osteogenic cells. The use of a synthetic chemical coating rather than a growth factor or peptide aims to cause similar effects at a greatly reduced cost Plasma application techniques were used initially to screen potential terminal groups on a 3D system. Amine groups were found to be osteogenic (which was confirmed by positive Von Kossa and Alizarin red staining), and hydroxyl groups were found to be chondrocytic (which was confirmed by positive Van Geison and Alcian blue staining). The osteogenic effect of the amine group was investigated further, but in the form of silane SAMs, which were more easily definable. The presentation of the terminal group was investigated using varying carbon chain length, to see if this had an effect on osteogenicity) This was explored using both MSC and primary osteoblast-like cell models on glass initially, then on PLGA films and finally a 3D PLGA system. The results of this showed positive expression of osteogenic markers for the MSC and osteoblast-like cells when on glass and PLGA films. There was an expression of the osteogenic marker osteocalcin and a positive mineralisation stain (Von Kossa) at 7 days. This effect however was not transferred to a 3D platform as further optimisation will be required to achieve this goal-an essential progression on the way to the development of an injectable 3D system suitable for clinical application.

The endothelial barrier is essential for vascular function, and its disruption may play a major role in the development of cardiovascular diseases. An increase in cAMP levels tightens the endothelial barrier by enhancing junction formation, and this is, in part, mediated by activation of exchange protein directly activated by cAMP (Epac). Vascular endothelial cells express vascular endothelial cadherin (VE-cadherin), and connexins 37, 40 and 43. Re-distribution of VE-cadherin in human coronary artery endothelial cells (HCAECs), induced by EGTA treatment or blocking VE-cadherin-VE-cadherin interactions through pre-treatment with anti-VE-cadherin primary antibody, triggered a subsequent re-distribution of Cx37 that was reversible. Epac activation with the Epac-selective agonist 8-pCPT induced a re-distribution of VE-cadherin and connexin 37 to sites of cell-cell contact in HCAECs, increasing the co-localisation of these two proteins, as detected by immunocytochemistry. This increased co-localisation was completely blocked by Epac1 siRNA. To test whether the re-distribution of Cx37 induced by Epac activation resulted in the formation of new functional gap junction channels, Lucifer yellow dye transfer was examined in HCAECs under varying conditions. Epac activation in HCAECs enhanced gap junction intercellular communication (GJIC), and this increase was completely blocked by the Epac inhibitor HJC0197. 8-pCPT addition was also shown to induce a transient increase in the intracellular calcium concentration in HCAECs, as detected with the calcium indicator Fluo-4. This calcium transient was independent of protein kinase A (PKA), and occurred in the absence of extracellular calcium, but was inhibited by the presence of the endoplasmic reticulum calcium ATPase inhibitor cyclopiazonic acid (CPA), indicating that Epac primarily mediated the increase, and that intracellular stores were the predominant source of calcium. Furthermore, the calcium transient induced by 8-pCPT was considerably reduced by the Epac inhibitor ESI-09 and completely inhibited by the Epac inhibitor HJC0197. Together, these data suggest that Epac strengthens the endothelial barrier through re-distribution of VE-cadherin and Cx37. In addition, GJIC is enhanced by Epac activation and there is a rise in intracellular calcium, a second messenger that can be transferred to adjacent cells through GJIC. It may be possible, therefore, that these effects of Epac promote endothelial cell- smooth muscle cell communication and therefore Epac could play a role in the regulation of vascular tone.

Human embryonic stem cells (hESCs) are undifferentiated cells arising from the inner cell mass of the blastocyst, which are able to self-renew or differentiate in vitro into specialised cell types. These pluripotent cells are a powerful tool to study human embryonic development and have great potential in the field of regenerative medicine. Human ESC pluripotency is governed by an intrinsic transcriptional network composed of the three well-known transcription factors OCT4, SOX2 and NANOG, whereas the role of extrinsic cell/microenvironment interactions in the maintenance of hESC stemness has been neglected to some extent. The aim of this work was to develop a systems biology approach oriented on these extrinsic factors and their links with the transcriptional network, in order to uncover some of the fundamental mechanisms underlying the stemness state. The thesis is divided into two complementary approaches: a top-down in silico study and a bottom-up in vitro study. The top-down in silico approach consists of a meta-analysis of hESC transcriptional data, leading to the construction of a hESC transcriptome. These mRNA data served as proxy for proteins in a protein-protein interaction database to build a hESC interactome. This interactome (or protein-protein interaction network) was structurally defined to identify the likely cell surface and extracellular proteins regulating hESC stemness by revealing the ’module organiser’ or hub proteins and the ’module connector’ or bottleneck proteins, along with the extracellular/transcriptional links. The bottom-up in vitro approach was the study of five of the previously identified cell surface/extracellular proteins in hESC fate decision. These candidates, together with OCT4, were stably knocked down using short hairpin (sh)RNAs and lentiviruses. The optimisation of the shRNA lentivirus production led to the development of a method for the direct quantification of these lentiviral particles. The effects of shRNA-mediated knockdown on hESC phenotype were investigated by assessing cell morphology and by determining the expression levels of the following groups of mRNAs: candidate stemness mRNAs, pluripotency mRNAs, as well as trophectoderm, endoderm, mesoderm and ectoderm mRNAs. We found that the candidates could modulate each other’s expression and appeared to regulate hESC commitment into different lineages. Furthermore, the expression levels of some of the candidates were regulated by OCT4. Taken together, these results suggest that by using the novel in silico approach developed during this project, it is possible to identify new stemness factors that could potentially have a role in either maintaining hESC self-renewal or in regulating lineage specification.

The reversible covalent conjugation of the small highly conserved ubiquitin protein modifier to selective substrates plays central roles in countless proteolytic and non-proteolytic cellular functions. Substrate protein ubiquitination is co-ordinated by the sequential activity of three distinct classes of proteins: (i) E1-activating enzymes, (ii) E2-conjugating enzymes, and (iii) E3-protein ligases. Really Interesting New Gene (RING) proteins represent the largest family of E3-proteins comprising over half of predicted human E3-ligases. As such, E3-RING proteins play pivotal roles in controlling both specificity and functionality within the ubiquitin system. E3-RING proteins function as catalytically inactive molecular scaffolds that position Ub~E2 and substrate proteins in close proximity for ubiquitination to occur. Within the active ligase complex, E3-RING proteins and E2 conjugating enzymes are believed to select protein substrate(s) and the form of conjugated ubiquitin upon them, respectively. Whilst E3-RING/E2 partners have been investigated in recent HTP screen approaches, a key area of data paucity exists for integral membrane E3-RING (TM-E3-RING) proteins. As such, high throughput yeast-two-hybrid assays were performed for the entire complement of TM-E3-RING proteins and E2-conjugating enzymes. A broad subset of TM-E3-RING/E2 positive and negative Y2H interactions was re-tested in secondary luciferase protein complementation assays (PCAs), which increased confidence in Y2H-derived interactions and extended network coverage. Data from these studies was collated with previously published binary TM-E3-RING/E2 interaction data to provide a high-confidence TM-E3-RING/E2 network consisting of 312 unique binary interactions. In vitro auto-ubiquitination assays were employed to assign functional activity to TM-E3-RING/E2 protein pairs, revealing high verification rates for both positive and negative Y2H or PCA binary interaction data. Furthermore, novel trends in the generation of different forms of ubiquitin modifications were identified between selective TM-E3-RING/E2 pairs. Finally, Y2H screens were also performed to identify TM-E3-RING dimerization events, which represent an emerging theme in ubiquitin system regulation. In total 71 TM-E3-RING/TM-E3-RING interactions were reported demonstrating high incidence of these binding events. Novel data was combined with known interactions to generate a TM-E3-RING network containing >500 binary interactions, encompassing both components of the core ubiquitin cascade and non-ubiquitome proteins. This TM-E3-RINGcentric network provides a valuable tool for the investigation of specificity and regulation of TM-E3-RING proteins and specific ubiquitin cascades.