The ability of many filamentous fungi, such as Aspergillus niger, to secret a high level of homologous proteins has led to their consideration as hosts for the production of heterologous proteins. However, the levels of some secreted heterologous proteins have often been low. Although many strategies have been developed to improve the level of secreted heterologous proteins, further studies into the remaining bottlenecks are required. One common strategy used to improve secreted protein production from filamentous fungi is to express the target protein under the control of a highly-induced native promoter. One major bottleneck in the secretion of heterologous proteins is caused by the folding of proteins within the lumen of the endoplasmic reticulum (ER). Recent studies have shown that expressing some heterologous proteins could subject A. niger to ER-stress. In this study, A. niger was subjected to different environmental conditions and ER stress responses were examined under each of these environmental conditions to further investigate the regulation of the gene encoding glucoamylase (glaA). Treating A. niger with dithiothreitol (OTT), a reducing agent that causes the formation of unfolded proteins, caused the down-regulation of transcription of the glaA but not the gene encoding the non-secreted protein y-actin. The OTT-treated fungal cells also showed evidence of induction of the UPR because expression of bipA was up-regulated and splicing of hacA, the gene encoding the transcription factor responsible for induction of the Unfolded Protein Response (UPR), occurs allowing the production of an active HacA protein. This is the first study to show clearly by nuclear run-on studies that the transcriptional down-regulation effect occurs at the level of transcription, rather than mRNA stability, and is found to be mediated through the promoter of the glaA gene (PglaA) in a region more than 1.192 kb upstream of the translational start. As a preliminary attempt to investigate if the transcriptional downregulation effect was mediated through HacA (i.e. part of the UPR), the ER stress was induced through antisense technology to lower the level of POI in the ER of A. niger. Although the transcription of glaA was attenuated in that strain of A. niger, UPR was not evident, suggesting that the transcriptional down-regulation mechanism is controlled differently from the UPR. Furthermore, activation of the ER-Associated Degradation (ERAO) mechanism in OTT-treated A. niger cultures was demonstrated by detecting transcriptional up-regulation of the putative gene encoding the RpnG, a homologue of the yeast Rpn7p subunit of the 26S proteasome.

The Wharton’s Jelly (WJ) of human umbilical cord (HU) contains multipotent stem cells (WJ-MSC) which express mesenchymal markers but not hematopoietic markers. WJ-MSC are increasingly being tested for use in stem cell therapy, with intravenous delivery being the preferred route. Fetal stem cells from embryonic germ layers are present in maternal blood and can home to damaged maternal tissues. This study investigates how WJ-MSC cross the fetal and adult endothelial barriers; including the cellular and molecular mechanisms employed. WJ-MSC were isolated from HU (n=27) which were taken from normal term pregnancies after elective Caesarean section. Flow cytometry and immunofluorescence were used to check presence/absenc of mesenchymal versus haematopoietic markers. Cells were induced to become adipocytes, chondrocytes and osteocytes by using specific induction medium. Isolated WJ-MSC were added after labelling with PKH26 to confluent monolayers of isolated human umbilical vein endothelial cells (HUVEC) or commercially bought human uterine microvascular endothelial cells (HUtMEC) at a 1:5 ratio. Cell-cell interactions were monitored with real time microscopy for 24 to 40h. Fluorescence and confocal scanning microscopy, after vascular endothelial (VE) cadherin immunocytochemistry were used for detailed analysis of VE-cadherin junctional occupancy and spatio-temporal location of stem cells. Tyrosine phosphorylation status of VE-cadherin, whether at Tyr685 or Tyr731, at different time points were investigated by immunoblotting whilst levels of vascular endothelial growth factor (VEGF) in the conditional media (CM) were measured by ELISA. Three different isolates were tested, with 3 experimental repeats for all expermints. Statistical analyses were performed with ANOVA (One or Two way). Cells (>95%) from each passage were positive for the mesenchymal markers CD 29, CD 105, CD 90, CD 73 and CD 44. <2% cells showed positivity to the haematopoietic markers CD 34, HLA-DR, CD 14, CD 19 and CD 45. WJ-MSC differentiated into adipocytes, osteocytes and chondrocytes. WJ-MSC displayed exploratory behaviour for a minimum of 30 min on HUtMEC or 60 min on HUVEC with interrogation of paracellular openings before crossing rather than replacing endothelial cells. By 2h, half were found at sub-endothelial positions, with a majority reaching this within 16-22h. There was accompanying loss of junctional VE-cadherin (64.9 + 3.7 %; p<0.001 in HUVEC; 63 + 4.6%; p< 0.001 in HUtMEC) in the endothelial monolayers followed by a return at 16h and increased continuity by 22h (p<0.01 in HUVEC; p<0.001 in HUtMEC). Junctional disruptions were found close to overlying or migrating WJ-MSC. Confocal microscopy confirmed paracellular extravasation. VE-cadherin protein levels matched controls in the early hours (0-2h) and increased after 22h co-culture in both fetal and uterine endothelium. VE-cadherin showed a 2-fold increase in phosphorylation at Tyr685 from 30 min to 2h. P-Tyr731 remained unchanged, similar to untreated endothelial layers, then decreased at 2h and 22h. VEGF levels in WJ-MSC – HUtMEC co-culture supernatants was highest at 2h (88 + 3 pg/ml) and decreased by 22h, reaching negligible levels by 48h. Anti-VEGF blocked Tyr685 phosphorylation but did not affect the decrease in P-Tyr731; this was accompanied by a 25% decrease in transmigration of cells in the first two hours and a 43% decrease in total by 22h. in WJ-MSC – HUtMEC co-cultures. However, in HUVEC-WJ-MSC co-cultures, no VEGF were detected and anti-VEGF did not block Tyr685 phosphorylation and Tyr731 de-phosphorylation. WJ-MSC from term umbilical cords can be easily isolated and expanded in culture. They retain mesenchymal stem cell properties for the passages tested (up to P5) making them a valuable model for studies into mechanisms underlying extravasation. The data obtained suggest that WJ-MSC can influence expression of VE-cadherin, with perturbation during transmigration followed by upregulation and repair once the adlumenal side is reached. There was a similarity in the cellular and molecular mechanisms employed by WJ-MSC in their paracelluar migration across fetal and uterine endothelium, although VEGF may not be the key player in HUVEC interactions. For both endothelial types, WJ-MSC appear to induce phosphorylation events linked with paracellular permeability and de-phosphorylation events normally associated with leukocyte extravasation. The data from the uterine endothelial investigations suggests that fetal stem cells are able to influence paracellular junctional dynamics and strengthens the growing hypothesis that they may also play a role in re-modelling the uterine circulation for fetal advantage. The extra-embryonic WJ-MSC holds the promise of use in restoring junctional maturity and vascular repair in future therapeutic applications.

Reactive Oxygen Species (ROS) are known as important intracellular signaling molecules. These are also well known for their role in oxidative stress and cellular damage, leading to their involvement in several pathologies. Despite the widespread postulation of ROS mechanisms, little is actually known about the immediate response in living cells to the generation of these highly reactive compounds. The development of nanoplatforms incorporating photosensitizers would permit the generation of ROS at specific sub-cellular locations and determine the in situ cellular response. The work presented in this thesis describes the development of porphyrinic nanoplatforms for the controlled generation of ROS and investigates their impact on the surface marker expression of human Mesenchymal Stem Cells (hMSCs). Surface tailoring of polyacrylamide nanoparticles with alkyne and amine functionalities were exploited to achieve stable reactive chemical groups for further conjugation. Nanoplatforms surface was also modulated with trimethylammonium functionalities for the development of nanosystems for sub-cellular targeting and facilitated uptake. Physicochemical characterization of alkyne and alkyne/trimethylammonium functionalised constructs showed sizes in the range of 40 nm with a positive surface charge. Alkyne/trimethylammonium nanosystemswere found to be stable over long periods of time, whilst amino functionalized nanosystems were found to be prone to aggregation. Mechanisms of conjugation were exploited to create covalent linkage of porphyrinic photosensitizers to mono and dually functionalised constructs. Conjugation through "click chemistry" allowed stable coupling with alkyne and alkyne/trimethylammonium nanosystems. To overcome aggregation associated with amino functionalised nanoplatforms, porphyrin conjugated monomers were synthesised which resulted in stable polyacrylamide nanoparticles. The developed conjugated nanosystems showed final sizes in the range 40-100 nm, while conjugates with surface charges greater than + 20 mV have led to sizes higher than 100 nm. The effect of surface charge on cellular delivery was investigated and nanosystems with a surface charge in the range + 13 mV to + 18 mV proved optimal in terms of cell delivery and viability. It was found that highly charged nanosystems (above + 20 mV) remained attached to the cellular membrane and had a negative effect on cell viability. In addition, intracellular co-localisation studies showed preferential mitochondrial targeting of the delivered nanosystems. Production of ROS in nanoparticle treated hMSCs was achieved by exposure to light at wavelength of 575 nm. For porphyrin conjugated nanosystems a single light dosage resulted in a "blast zone" in the irradiated area where significant production of hydrogen peroxide was also observed. Titration of the amount of porphyrin conjugated at the surface of nanoparticles resulted in systems with different levels of ROS production. Control of ROS generation allowed development of a nanoplatform that was used to expose cells to repeated exposure of ROS over a time period of 100 minutes. The surface marker expression of hMSCs treated with porphyrin conjugated nanosystems was investigated. In the absence of light the surface marker expression of hMSCs was maintained, positive for CD29 and CD105 and negative for CD34 and CD45. Increased generation ROS in hMSCs did not produce alterations in the surface marker expression of cells, and over two generations of treated cells (light and nanoparticles) no changes were detected in surface marker expression. The developed nanoplatforms have the potential to be applied as a tool to investigate the cellular mechanisms and metabolism associated with different levels of oxidative stress. In addition, these nanosystems could also represent an innovative platform for theranostic applications (drug delivery/diagnostic).

The pharmaceutical industry has been criticised for a lack of innovation associated with the drug discovery and development process, for example when compared with the computer or music industries. In fact, bringing a new medicine to the market requires, on average, the screening of up to 10 000 molecules, an expense in the range of $500 million-$2 billion and roughly 10-15 years of research. Such a situation not only has a direct impact on the health and life expectancy of every single human being on the planet, but also indicates that alternative strategies for drug development should be investigated. In this thesis, studies of direct formulation-membrane interactions, both in a high throughput (HT) manner and at a nanometre scale, were initially identified as an important approach that could offer advantages for in vitro-in vivo correlations of in-man drug behaviours. Subsequently, supported lipid bilayers (SLBs) of physiologically-relevant lipid compositions were indicated as experimental models of preference for pre-clinical drug development. For that reason, the characterisation and assessment of physicochemical and behavioural properties of the model SLBs at a nanometre scale, as well as development of an SLB microarray for HT applications were the focus of this research. Here, the optimisation and characterisation of model lipid films was performed using atomic force microscopy (AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Additionally, the AFM-investigated assessment of the interactions between model SLBs and formulation components (e.g. Pluronics®, siRNA, DNA polyplexes) enabled both the correlation of in vitro observations with literature-reported in vivo performances of the components of interest and the development of hypotheses with regards a number of phenomena in biology. Furthermore, the development of a SLB microarray prototype suitable for HT applications is reported. Directly, this research improves: the understanding of SLB behaviours and experimental investigation at a nanometre scale of the mechanisms of interactions between membranes and: Pluronics®, nucleic acids and their complexes, as well as the technology of SLB microarray development. Indirectly, this research contributes towards the progress in a number of research areas within pharmaceutical sciences, potentially resulting in new scientific disciplines, such as immunolipidomics or nanopharmacology.

Telomeres are repetitive elements of DNA which are located at the ends of chromosomes. During cell division, telomeres on daughter chromomeres shorten until the telomere length falls below a critical level. This shortening restricts the number of cell divisions. In this thesis, we use mathematical modelling to study dynamics of telomere length in a cell in order to understand normal ageing (telomere shortening),Werner’s syndrome (a disease of accelerated ageing) and the immortality of cells caused by telomerase (telomere constant length maintenance). In the mathematical models we compared four possible mechanisms for telomere shortening. The simplest model assumes that a fixed amount of telomere is lost on each replication; the second supposes that telomere loss depends on telomere length; for the third case the amount of telomeres loss per division is fixed but the probability of dividing depends on telomere length; the fourth cases has both telomere loss and the probability of division dependent on telomere length. We start by developing Monte Carlo simulations of normal ageing using these four cases. Then we generalize the Monte Carlo simulations to consider Werner’s syndrome, where the extra telomeres are lost during replication accelerate the ageing process. In order to investigate how the distribution of telomere length varies with time, we derive, from the discrete model, continuum models for the four different cases. Results from the Monte Carlo simulations and the deterministic models are shown to be in good agreement. In addition to telomere loss, we also consider increases in telomere length caused by the enzyme telomerase, by appropriately extending the earlier Monte Carlo simulations and continuum models. Results from the Monte Carlo simulations and the deterministic models are shown to be in good agreement. We also show that the concentration of telomerase in cells can control their proliferative potential.

The natural compound cordycepin (3’-deoxyadenosine) causes a reduction in breast cancer cell viability. Microarray analysis showed that growth related genes are down-regulated by cordycepin. Indeed, mTOR, ERK and AMPK signalling was shown to be altered by cordycepin, but the effect was too fast to be mediated by transcriptional changes. It was hypothesised that cordycepin affected signal transduction through translation. However, polysome profiling did not identify clear candidates for the effects of cordycepin on signal transduction but unveiled that cordycepin leads to translation repression on 5’ terminal oligopyrimidine (TOP) mRNAs. As TOP mRNAs are known to be regulated by mTOR signaling, this result consistently suggests mTOR signaling is inhibited by cordycepin treatment. To test if it is possible that cordycepin affects gene expression via signal transduction, we compared its effects to various signal transduction inhibitors and an activator. So far, Pictilisib, a pan-PI3K inhibitor, is the only inhibitor that mimics both the gene expression and signal transduction effects of cordycepin, indicating the PI3K-PDK1-AKT axis is affected by cordycepin. The RNAs upregulated by cordycepin were highly enriched in a group of non-coding RNAs, which are also appeared to induce during serum withdrawal. Knockdown of poly(A) polymerases induced these RNAs, indicating that they probably are degraded by the PABPN1 and poly(A) polymerase dependent nuclear RNA decay pathway. Thus the data suggest that cordycepin affects gene regulation by two distinct pathways, one affecting signal transduction and growth related mRNA expression and another affecting polyadenylation mediated decay of non-coding mRNAs.

Cordycepin (3′-deoxyadenosine) is a medicinal bioactive component of the caterpillar fungi (Cordyceps and Ophicordyceps). It is reported to have nephroprotective, antiapoptotic, anti-metastatic, hepatoprotective (Yue et al. 2013), inflammatory effects, antioxidant, anti-tumor, immunomodulatory and vasorelaxation activities. Cordycepin is well known to terminate and inhibit polyadenylation, both in vitro and in vivo. Other proposed mechanisms of action of cordycepin include activation of adenosine receptors, activation of AMP dependent kinase (AMPK) and inhibition of PARP1. The purpose of this study is to elucidate the biological and pharmacological effects of cordycepin on cancer cell lines such as MCF-7 cells. In this study I found that cordycepin reduces the cell proliferation in all examined cell lines without always exerting an effect on 4EBP phosphorylation and protein synthesis rates. Therefore, the effects on protein synthesis via inhibition of mTOR, which were previously reported, are not only the sole reason for the effect of cordycepin on cell proliferation. Knockdown of poly (A) polymerases reduces cell proliferation and survival, indicating that poly (A) polymerases are potential targets of cordycepin. I studied different adenosine analogues and found that 8 aminoadenosine, the only one that also consistently inhibits polyadenylation, also reduces levels of P-4EBP. It also inhibits the expression of specific genes indicating that the effects on polyadenylation, mTOR signalling and gene expression are linked. Also consistent with polyadenylation inhibition as the major mode of action is the fact that the effects of cordycepin on gene expression are predominantly post-transcriptional. However, knockdown of poly (A) polymerases did not have the same effects on gene expression or on polyadenylation, indicating that cordycepin may act as a dominant negative rather than as a null mutant. This is consistent with the fact that cordycepin is known to arrest a normally transient polyadenylation complex. We performed microarray analysis of cordycepin treated MCF-7 cells and found that the downregulated mRNAs were predominantly involved in transcriptional regulation, cell proliferation, cell cycle and cell migration. These data show that cordycepin is a promising new drug for cancer and indicates that the mode of action it is likely to be through the inhibition of polyadenylation.

The unlimited replicative potential of cells is one of the hallmarks of cancer. Telomeres, DNA structures found at the ends of chromosomes have attracted a great deal of interest in recent years as potential anti-cancer drug targets since they play an important role in cancer cell immortality. The repetitive TTAGGG sequences of telomeres are complexed to a group of six indispensible proteins, one of which is the protection of telomeres 1 (POT1) protein. This specialised protein binds to a ten nucleotide single stranded DNA sequence at the ends of chromosomes and plays an important role in telomere capping and length regulation. It has recently been proposed that the key function of POT1 is to suppress a potent DNA damage response at telomeres thereby protecting chromosome tips from being recognised as sites of DNA damage. Deletion of POT1 from telomeres in a variety of organisms including humans results in cytogenetic aberrations, senescence and cell death. These results indicate that POT1 is an integral telomere end-protection protein which is necessary for continued cellular proliferation and therefore POT1 is becoming a promising new target in cancer. Using a structure-based approach, several small molecule inhibitors of POT1 have been designed to affect telomere integrity by disrupting the binding interaction of human POT1 with its target DNA sequence thereby driving cancer cells into senescence/apoptosis. Using a range of computational tools, a suitable drug binding pocket in POT1 has been identified and the de novo design of a specific class of POT1 inhibitor was completed. Using this novel scaffold, a small focussed library of hit-like compounds were synthesised and screened in a new POT1 fluorescence polarisation displacement assay developed by scientists at the University of Nottingham. In total, over 90 small molecule inhibitors based on two different scaffolds: pyrido[1,2-a]pyrimidines and sulfathiazoles have been synthesized with some inhibitors effectively decreasing POT1-DNA binding between 10-54% at 100μM ligand concentration. The biological results have established that electron-withdrawing substituents on the pendent phenyl ring of the pyrimidine core are essential for strong binding. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of cancer.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has proven its ability to characterise (in)organic surfaces, and is increasingly used for the characterisation of biological samples such as single cells. By combining ion imaging and molecular depth profiling it is possible to render 3D chemical images, which provides a novel, label-free way to investigate biological systems. Major challenges lie, however, in the development of data analysis tools and protocols that preserve the cell morphology. Here, we develop and employ such tools and protocols for the investigation of neuronal networks. One of the reasons 3D ToF-SIMS imaging of cells is underused is the lack of powerful data analysis tools as 3D ToF-SIMS measurements generate very large data sets. To address this issue, we developed a method that allows the application of principal component analysis (PCA) to be expanded to large 3D images making 3D ToF-SIMS image processing of whole, intact cells and cellular networks with multivariate analysis now accessible on a routine basis. Using this method, we are able to separate cellular material from the substrate and can then correct z-offsets due to the cells' topography resulting in a more accurate surface heightmap. The method also facilitates differentiation between cellular components such as lipids and amino acids allowing the cell membrane, the cytoplasm and the extracellular matrix (ECM) to be easily distinguished from one another. These developments permit us to investigate the intracellular localisation of specific native and non-native compounds label-free, not just in single cells but also in larger cellular networks. The visualisation of the cellular uptake of non-native compounds, namely fluorescent dyes, in primary rat cortical neurons and the chemical differentiation between cell types, namely primary rat cortical neurons and retinal pigment epithelium (RPE) cells, are presented as applications. Even though the dyes have distinct fragment ions in the high mass range, it was not possible to detect the fluorophores by 3D ToF-SIMS imaging of freeze-dried cells. However, it was possible to detect distinct differences in the kind of ions detected for freeze-dried primary rat cortical neurons and RPE cells albeit in the low mass range. To obtain meaningful results, however, it is paramount that sample preparation does not induce significant physical or chemical changes. We present the first comprehensive comparison between large 3D ToF-SIMS images of freeze-dried and frozen-hydrated cells using PCA to facilitate the data analysis of these large data sets. A higher degree of colocalisation of the K+ signal with cell regions is observed for frozen-hydrated cells, which indicates a lower degree of membrane damage and migration of diffusible chemical species. Frozen-hydrated cell samples are therefore considered to best reflect the native cell state, but freeze-dried cell samples allow far easier sample handling. The mass spectrum of frozen-hydrated cellular material also has increased ion intensities for higher-mass fragments, which is an additional advantage, because the poor signal-to-noise ratio of molecular species with m/z > 200 is a major bottleneck in the advancement of ToF-SIMS imaging as a diagnostic tool.

Polyadenylation is a universal step in the production of all metazoan mRNAs except histone mRNA. Despite being universal, previous experiments have implicated it in the regulation of inflammation. An inflammatory system using RAW 264.7 murine macrophage cells was established with bacterial lipopolysaccharide (LPS) used as a stimulus. After improving the poly(A) tail test (PAT) method of measuring poly(A) tail lengths, it was applied to inflammatory mRNAs during the inflammatory response. Poly(A) tail length was shown to vary over the course of the inflammatory response, and for Tnf, this was even true of initial poly(A) tail size, which is widely believed to be uniform for the majority of mRNAs. The adenosine analogue cordycepin (3’-deoxyadenosine) was shown to have anti-inflammatory effects on mRNA, in line with existing literature, and is likely to be the anti-inflammatory component of Cordyceps militaris ethanol extract. Inhibition of either import of cordycepin into cells or phosphorylation of cordycepin was sufficient to abolish its anti-inflammatory effects. Adenosine treatment led to repression of Il1b mRNA, but did not repress other mRNAs tested that were cordycepin-sensitive. This suggests that cordycepin does not simply act by mimicking the effect of adenosine, and that the two compounds have distinct modes of action. Inhibiting deamination of cordycepin potentiated its effects. We also observed that pre-mRNA levels of inflammatory genes were decreased by cordycepin treatment, indicative of effects on transcription. Other groups have reported that cordycepin interferes with NF-B signalling. As NF-B is an important transcription factor for the induction of inflammatory genes, this would provide a basis for explaining our observation that cordycepin represses at the transcriptional level. However, we did not observe any changes in NF-B signalling, with degradation of IB completely unimpeded by cordycepin treatment. Notably, cordycepin did shorten the Tnf poly(A) tail, and the observed inhibition of polyadenylation is consistent with observations that cordycepin led to decreased efficiencies of mRNA 3’ cleavage and transcription termination for Tnf. Such effects on polyadenylation and 3’ processing of mRNA were hypothesised to particularly affect unstable mRNAs that depend on longer poly(A) tails for avoiding decay and/or mRNAs with a high rate of transcription. However, comparison of microarray data to data from RNA-seq of RNA from 4-thiouridine labelling experiments showed that cordycepin-sensitivity did not correlate with mRNA stability or transcription rate. Long noncoding RNAs (lncRNAs) were found to be enriched in cordycepin-treated cells. If some of those lncRNAs have regulatory roles in inflammation, cordycepin’s effects may be mediated through them. Lastly, cordycepin significantly altered pain behaviour in a rat model of osteoarthritis (OA), supporting its continued use as a lead compound for exploration of new OA therapeutics.