β-Arrestins are essential adaptors for G protein-coupled receptor (GPCR) trafficking. Evolutionary ancestors of the β-arrestins – dubbed α-arrestins – are present in yeast/fungi and, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking. Mammalian α-arrestins include five largely uncharacterised arrestin domain-containing (ARRDC1-5) proteins that display a predicted arrestin structure; the current study focuses on human ARRDC2. Confocal microscopy of exogenous, fluorescent protein-tagged ARRDC2 in U2OS cells in combination with compartment-specific markers indicated that ARRDC2 is dynamically distributed throughout the plasma membrane and endocytic system, predominantly to late endosomes/lysosomes. Anti-ARRDC2 immunostaining in several primary cell lines broadly supported this conclusion. ARRDC2 contains two proline-rich (PPxY) motifs that in other α-arrestins have been reported to mediate interactions with WW domain-containing NEDD4 family E3 ubiquitin ligases. Coimmunoprecipitation indicated that ARRDC2 is able to interact with several NEDD4 E3s via its PPxY motifs, and confocal microscopy suggested that this interaction may influence the subcellular targeting of the ligases. Ubiquitination of ARRDC2 was detected by coimmunoprecipitation, although this modification was independent of ARRDC2 interaction with NEDD4 E3s. ARRDC2 colocalised with agonist-stimulated, internalised GPCRs (β2-adrenergic receptor (β2AR) and δ-opioid receptor (δOR)) and colocalisation analysis indicated that this involved compartmental redistribution of ARRDC2 to receptor-containing early/recycling endosomes, suggesting a specific effect. Interaction of ARRDC2 with δOR was detected using coimmunoprecipitation, and confocal analysis suggested that ARRDC2 may influence δOR and β2AR intracellular trafficking. ARRDC2 was also found to oligomerise with itself and the β-arrestins. Confocal microscopy showed that ARRDC2 overexpression can induce the redistribution of β-arrestin1 to ARRDC2-positive vesicles, and a punctate bimolecular fluorescence complementation (BiFC) signal was detected between ARRDC2 and β-arrestin2. From this, it is speculated that α-/β-arrestins may function cooperatively or competitively to mediate discrete GPCR sorting events in the endocytic pathway.

In the absence of telomerase, Saccharomyces cerevisiae telomeres erode leading to senescence. Rare cells can survive after this stage as they can elongate their telomeres utilizing homologous recombination. Two different types of survivors can be easily distinguished by Southern blot. Type I survivor cells, elongate the telomere by amplifying Y elements and require RAD51, RAD54, RAD55 and RAD57 for establishment. Type II survivors elongate their telomere by amplifying TG1-3 repeats, however, they require the following genes to be established: RAD50, MRE11 and XRS2, RAD59, SGS1 and KU80 in some cases. Both types require the gene RAD52. In this study several candidate genes were deleted individually in diploid type II survivor strains. The main aim of this work was to see if these genes were required for type II telomere maintenance. Most of these genes are not required for type II telomere maintenance at least until ~150 generations after deleting these genes. The exceptions were KU80 and RPB9. Ku80Δ strains switched to a new survivor type that is similar to type I and continued for the long term. RPB9 was required for two independent type II survivor strains to survive, whereas the third type II strain did not require this gene at ~150 generations after deleting the gene. After many generations (~ 350), this strain switched to type I. At long term propagation (~500 generations) after deletion of the candidate genes, all type II strains displayed telomere shortening until the propagation was stopped. However, Rad50Δ strains switched to type I after long term. Finally, the absence of the candidate genes did not affect the sensitivity of type II survivor strains to temperature. On the other hand, type II survivor strains with some genes deleted displayed sensitivity to UV.

Background: RNA splicing is a crucial process for delivering the appropriate message for protein synthesis. Most genes are affected by alternative splicing, and among these is CHRNA7. This gene encodes for the nicotinic acetylcholine α7 receptor subunit that is involved in the cholinergic anti-inflammatory pathway. This anti-inflammatory pathway is considered an important part of the human body’s defence line against tissue injury or infection and causative mechanism in COPD. Aim: The aim of the present study was to investigate the role of alternative splicing on the nature of the transcripts generated by CHRNA7 gene and its partial duplicate, CHRFAM7A. Methods: Airway epithelial cell lines, A549 and BEAS2B, were mainly used as targets for testing alternative splicing. RT-PCR, TA cloning and gel extraction methods were used for testing CHRNA7 and CHRFAM7A transcripts. Following RT-PCR, the resulting product band intensities were analysed using densitometric analysis tools. This was followed by the use of several bioinformatics analysis tools to predict the protein structure for the resulting transcripts. For one of the detected transcripts, minigene methods were used to test for the source of expression. Results: A novel transcript missing exon 9 is reported for the first time. Both genes showed the expression of full length and the novel transcripts (missing exon 9) at similar ratios (~2:1). These results could be detected in immortalised cell lines from human alveolar and bronchial epithelial cells (A549 and BEAS2B, respectively) and in BE (2)-c cells (neuroblastoma cells with bone marrow metastasis). The same results were shown when primary human peripheral blood monocytes cells (PBMC) were tested. This means that the effect of missing exon 9 is not tissue-specific, and is not only found in cancerous cells, indicating that it could be a common feature of splicing for these two genes. Furthermore, another novel transcript was detected which is inserted exon 9b. The initial RT-PCR experiments seemed to suggest that this was derived from CHRFAM7A only. The use of minigene methods showed that this transcript could be expressed from both genes, CHRNA7 and CHRFAM7A, but a single nucleotide base within the inserted sequence (at position 77 from the 5` end) could play a role in enhancing of exon 9b in the mRNA transcripts. This base is C allele in CHRFAM7A sequence of exon 9b, while its corresponding base in CHRNA7 is G allele that has less prominent effect on exon 9b inclusion. Conclusion: CHRNA7 and CHRFAM7A express novel transcripts in different human cells that are missing exon 9. This could be due to inactive splicing factors that are required for recognition of exon 9 as a constitutive exon. For exon 9b transcripts, these lie within the common sequence of CHRNA7 and CHRFAM7A, and it seems that the presence of C allele at position 77 could enhance the inclusion of exon 9b in CHRFAM7A more than the presence of G allele in CHRNA7 sequences. The results shown in this study implicate a possible regulatory role of the transcripts detected on the control mechanism exerted by CHRFAM7A on CHRNA7. These results help to suggest a possible role of in the development of COPD in the form of inflammatory/anti-inflammatory control imbalance.

The G1uN3 subunits of the NMDA receptor are thought to reduce the Ca 2+ permeability and Mg2+ sensitivity of NMDA receptors. cRNA for rat NMDA receptor subunits were injected into Xenopus oocytes and responses were recorded using two electrode voltage clamp at -100, -75 and -50 mV. G1uN1-1a/2A, GluN1-1a/2A/3A and G1uN1-1a/2A/3B containing receptors were characterised using Mgz+, memantine, philanthotoxin-343, methoctramine and MK-801. IC50 values were calculated and generally showed significant increases between those containing G1uN1-1a/2A/3 subunits and G1uN1-1a/2A, while those with G1uN3B were found to be significantly higher than G1uN3A. Activity was also typically shown to be partially restored with mutations at the N and N+1 site asparagines of G1uN3A. As the ICS0 was only partially restored the changes cannot be attributed to the loss of the N-site alone. Further differences may be due to a constricted threonine ring within the M3 vestibule region, or due to continued reduced flux through the channel. Another possibility is that to restore block it may require both the double N and N+1 mutation at the N-site. Multi-target-directed ligands combine two pharmacophores to produce drugs which retain the properties of the constituents. Memantine has been approved for use in Alzheimer's disease and there is a search for drugs that have similar actions. A range of multi-target compounds were tested to determine if NMDA receptor blockade activity was obtained. The pharmacophores explored were tacrine, donepezil, lipoic acid carvedilol and dimebon. The most promising compounds were carbacrine(3) (tacrine and carvedilol) and lipocrine (lipoic acid and tacrine), and it was found that the former was equipotent and the latter more potent than memantine. Potency was likely due to the tacrine moiety. These compounds should be further categorised to determine if they retain the kinetics that gives memantine its favourable side effect profile.

Despite current control measures, plant parasitic nematodes are estimated to be responsible for > $100 billion of damage to worldwide crop production per annum. Current nematicides are highly toxic, and due to health and environmental safety concerns, many are being withdrawn from the market under directive 914/414/EEC. Alternative control strategies are urgently required. The cysteine proteinases papain, actinidain and recombinant endoproteinase B isoform 2 (R.EP-B2) have been demonstrated to affect the mobility of M. incognita J2s; 50.7 μM R.EP-B2, 101.7 μM papain and 200.3 μM actinidain immobilised 50% of the M. incognita population. Papain has also been demonstrated to affect the infection of plants by M. incognita, 5 μM papain reduced the attraction to and invasion of A. thaliana by 41.2 + 25.6% and 80.4 + 10.5% respectively. M. incognita J2s showed extensive damage to and removal of the cuticle when treated with 100 μM papain. MALDI-TOF analysis identified a number of M. incognita proteins affected by the papain treatment; of particular interest were a cuticle preprocollagen and a rhodopsin-like GPCR chemoreceptor. Proteins of these types are essential for movement and host location, disrupting their function helps to explain the loss of mobility and reduction in A. thaliana infection observed in the bioassays. Finally transgenic A. thaliana was generated with the barley cysteine proteinase endoproteinase B isoform two under the control of the root cap specific MDK4-20. The preliminary testing of these plants showed a reduction in root invasion similar to that obtained with papain.

This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in transcript levels of genes encoding enzymes involved in plant cell wall degradation (CAZy) as well as of genes encoding lipases, expansins, hydrophobins, G-protein coupled receptors and transporters when mycelia were cultivated in the presence of a lignocellulosic substrate (wheat straw). The encoded non-CAZy proteins were proposed to have accessory roles in carbohydrate deconstruction. A model for solid substrate recognition in T. reesei was described, based on the comparison with the one proposed for Aspergillus niger. Post-transcriptional regulation mediated by regulatory RNAs was identified for nearly 2% of all T. reesei genes, including genes encoding cell wall-degrading enzymes. Transcriptional regulation studies confirmed that transcription patterns of genes encoding enzymes involved in polysaccharide degradation differed between different carbon sources and that they are fine-tuned and dependent on factors such as culture conditions, consumption rate, assimilation of glucose and the presence of several transcription factors. The analysis of the structure of chromatin in the promoter and coding regions of one of these genes, cbh1, revealed different nucleosome positioning patterns under repressing (glucose) and inducing (sophorose, cellulose) conditions. CRE1, the carbon catabolite repressor in T. reesei was shown to be involved in the repression of many CAZy and non-CAZy encoding genes. Furthermore, CRE1 was also shown to be important for nucleosome positioning within the cbh1 coding region under repressing conditions and proposed to do so by interaction with (a) yet unidentified protein(s).

One hallmark of embryonic stem cells (ES) is their ability to renew themselves indefinitely and still retain the potential to develop into any specialized cell type once triggered by specific exogenous signals. This very versatile nature has made them an attractive model to study developmental events occurring during embryogenesis and also to employ them for regenerative medicine. The idea to exploit their developmental potential for intended therapeutic applications requires a detailed knowledge of the molecular regulation of differentiation. Thus pluripotent embryonic stem cells can be employed to investigate the molecular framework regulating pluripotency. The major aim of this research endeavour is to explore the role of DNA methylation in regulation of endogenous Sox2 transcription factor in context of mouse ES cells following their transition from the pluripotent to a differentiated state. An insight into molecular regulatory mechanisms will shed light on developmental programming and also aid in refining of methodologies for differentiation and nuclear reprogramming increasing their chances of success and efficiency. Mouse embryonic stem cells were differentiated towards osteogenic and neural cell types through the formation of embryoid bodies (EBs) – cellular aggregates partially recapitulating the early embryonic development. These EBs were then disaggregated and single cells plated in medium containing supplements to promote osteogenic or neural differentiation while control cells were grown in medium lacking those factors. Cells were harvested undifferentiated and at different time points during differentiation. Molecular characterization was carried out by expression profiling of lineage specific genes and proteins using RT-PCR and immunofluorescence respectively. DNA methylation analysis of two regulatory regions of Sox2 i.e. SRR1 and SRR2 was carried out by MS-PCR and bisulphite sequencing. Embryonic stem cells were observed to be differentiating as evidenced by changes in cellular morphologies and lineage-specific markers expression. Two regulatory regions of Sox2, namely SRR1 and SRR2, were found to be methylated by methylation sensitive PCR at all time-points chosen for analysis in differentiating cells. Three individual CpGs in SRR2 region were then analysed further by bisulphite sequencing which appeared unmethylated in both undifferentiated and differentiated embryonic stem cells. This hints towards the possible role of DNA methylation in regulating the expression of Sox2 in differentiating embryonic stem cells and need further investigation.

In recent years, two G protein coupled receptors have been de-orphanised which respond to long chain free fatty acids (FFAs), and so are able to mediate the signalling of these important nutrient molecules. FFA1 (GPR40) is predominantly expressed in pancreatic -cells, while the expression profile of GPR120 includes gut endocrine cells and adipose tissue. These distributions, together with the potential of both receptors to stimulate insulin and incretin hormone secretion, singled them out as potential drug targets for type 2 diabetes and obesity. The aim of this thesis was to evaluate the pharmacology of these receptors and their signalling properties, including the development of fluorescent FFA receptor ligands to evaluate agonist binding using imaging techniques. GPR120 has been identified to exist as two splice isoforms in humans, differing by a short insertion in the third intracellular loop, but no full isoform specific characterisation of receptor signalling and trafficking had been undertaken. This work therefore studied the GPR120S and GPR120L isoforms in terms of both G protein dependent and arrestin dependent signalling, and trafficking. It was found that the long GPR120L isoform exhibited reduced G protein signalling, but similar arrestin recruitment and lysosomal intracellular trafficking profiles as GPR120S. Potentially, expression of the long GPR120 isoform provides a mechanism to direct signalling to the arrestin pathway, for example to produce anti-inflammatory effects in macrophages. As the expression profile of GPR120 overlaps with that of FFA1, for example in colonic endocrine cells, a series of constrained GPR120 homo-dimers and GPR120:FFA1 heterodimers were created using irreversible bimolecular fluorescence complementation, and the potential for novel pharmacology was investigated by monitoring dimer internalisation. However, there was no evidence that such dimerisation altered the pharmacology of the ligand tested. Second, a model of the GPR120S ligand binding site was tested using point mutagenesis of the receptor. This mutagenesis validated key features of the model, including the role of Arg99 in co-ordinating the agonist carboxylate group, and interactions of the agonists with the conserved transmembrane VI Trp “toggle-switch” involved in receptor activation. Another mutation (Asn215) provided evidence for ligand-specific binding modes within the pocket. This study showed the complexity of testing mutants designed to interfere with ligand binding indirectly through signalling assays and highlighted the requirement for a FFA receptor binding assay to measure ligand affinity directly. In the absence of radioligands of suitable selectivity and affinity, a novel fluorescent ligand, based on the FFA1/GPR120 agonist GW9508, was used to successfully develop a whole cell FFA1 competition binding assay for the first time, obtaining FFA1 affinity estimates for a range of synthetic ligands. Fluorescent ligand binding was further investigated using fluorescence correlation spectroscopy and photon counting histogram analysis, defining the diffusion characteristics of FFA1 receptors in the membrane of single living cells, and providing preliminary evidence for their dimerisation.

Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to be effective against GI nematodes of rodents, chickens, pigs and sheep. The current study extended evaluation of different plant extracts and the factors affecting the efficacy of papaya latex supernatant (PLS) as an anthelmintic against GI nematodes in a mouse model system and formulation and delivery for use in ruminants. The study started with purification and concentration of CPs in PLS using different methods to determine which of them would provide high yield of CPs. It was found that concentration by dialysis provided a high yield of active enzyme in PLS. Storage of PLS at -200C and -800C retained more active enzymes for prolonged period of time than at ambient temperature and 4oC. Motility assay conditions showed to have no influence on enzyme activity. While the in vitro experiment results showed significant detrimental effect of pineapple fruit extract, stem bromelain and little effect of kiwi fruit extract against Heligmosomoides bakeri motility. In vivo experiments showed less efficacy of these enzymes than expected when compared with PLS. The first factor to be assessed in this study was the effect of fasting on the anthelmintic efficacy of PLS. The results showed that PLS was equally effective in reducing worm burdens whether mice were fasted before treatment or not, and by avoiding fasting the side effects of treatment were minimized. Comparison of efficacy in a range of mouse strains indicated that efficacy varied between mice of different genotype. At the dose used, the treatment was most effective in C3H mice ranging from 90.5% to 99.3% in reducing worm burdens and less effective in NIHS, CD1 and BALB/c strains (7.9%, 36.0% and 40.5% reduction respectively). However, host sex and body size were shown not to have any influence on the anthelmintic efficacy of PLS. Since CPs are particularly sensitive to pH, variation between mouse strains in gut pH was investigated but no significant differences in pH were found along the GI tract of the poor (BALB/c) and high responder mice (C3H) to PLS treatment and concurrent administration of the antacid cimetidine also did not improve efficacy. The study also explored the potential of formulation and delivery of PLS as an anthelmintic drug for ruminants. In vitro studies involving both immediate and slow release dosage formulations simulating the physiological conditions (pH, temperature and peristaltic movement) in the GI tract of the animal were conducted. In the slow release experiments, two hydrophilic matrices were tested, the xanthan gum and hydroxypropyl methylcellulose (HPMC) (both Methocel-LVCR and Methocel-CR). Methocel-CR provided better slow release results compared to the others. In the immediate release experiments 3 disintegrants (Primojel, L-HPC and Ac-Di-sol) were investigated and Ac-Di-Sol® was found to produce the faster immediate drug release rate. Preliminary in vitro studies also showed that PLS was highly effective against equine GI nematodes. Finally the empirical findings in this study provide useful information for improvement of formulation and delivery of these naturally occurring plant-derived enzymes for treatment of intestinal worm infections in humans and livestock, while achieving maximum efficacy and minimal side-effects.

Significant opioid- and endocannabinoid-dependent changes occur within the periaqueductal grey (PAG), rostroventral medulla (RVM) and spinal cord (DH) during postnatal development of the rat (Sprague Dawley). These changes are involved in the differential descending control of spinal excitability between young and mature rats. Microinjection of the µ-opioid receptor (MOR) agonist DAMGO (30ng) into the PAG of rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120ng, MOR antagonist) which lowered mechanical thresholds and increased spinal reflex excitability. Spinal adminstration of DAMGO inhibited spinal excitability in all ages yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan RT-PCR and immunohistochemistry. Proopiomelanocortin (POMC) peaked at P21 in the ventral-PAG, and MOR increased significantly in the DH as the animals aged. CB1/CB2 receptor activation by WIN55212 (4µg, CB1/CB2 agonist) and HU210 (4µg, CB1/CB2 receptor agonist) in the PAG, RVM and DH was anti-nociceptive in both young (P10, P21) and adult rats, but GPR55 receptor activation by LPI (12µg, endogenous GPR55 agonist) and AM251 (2.77µg, CB1 antagonist, GPR55 agonist) was exclusively inhibitory in young rats. Micro-injection of LPI into the adult RVM facilitated spinal reflex excitability, suggesting that GPR55 receptor activation in mature animals is pro-nociceptive. The expression of cannabinoid receptors and endocannabinoid-synthesising enzymes was investigated with immunohistochemical and TaqMan RT-PCR techniques. Overall the expression of CB1 receptors and the anandamide synthesising enzyme NAPE-phospholipase D (NAPE-PLD) increased within the descending pain pathway with age, whereas the expression of the 2-AG synthesising enzyme Diacylglycerol lipase α (DAGLα) decreased. These results illustrate that profound differences in the endogenous-opioidergic and endocannabinoid signalling systems occur within the descending pain pathway throughout postnatal development.