This thesis described the isolation and structure elucidation of secondary metabolites from three medicinal plants selected on the basis of their traditional use in the treatment of infectious diseases. The work also focused on the evaluation of the plant extracts and some of the isolated compounds for activity in vitro against Mycobacterium tuberculosis. Compounds obtained in suffient yield were further tested for activity in vitro against Methicillin-resistant Staphylococcus aureus. A total of 27 pure compounds and two mixtures were isolated from the three plants investigated: Arctium lappa, Tussilago farfara and Verbascum thapsus. Phytochemical investigation of the aerial parts of A. lappa led to the isolation of four terpenoids (taraxasterol, taraxasterol acetate, isololiolide and melitensin), two steroids (sitosterol/stigmasterol mixture and daucosterol), three flavonoids (quercetin, kaempferol and kaempferol-3-O-glucoside), two phenolic acids or derivatives (caffeic acid and 1, 3-dicaffeoylquinic acid) and one alkane (n-nonacosane). Isololiolide, melitensin, kaempferol-3-O-glucoside and n-nonacosane are reported for the first time from this species, and daucosterol and kaempferol are first reported from the aerial parts of this plant. Phytochemical investigation of T. farfara aerial parts led to the isolation of a monoterpene lactone (loliolide), two steroids (sitosterol/stigmasterol mixture and daucosterol), three flavonoids (quercetin, kaempferol and kaempferol-3-O-glucoside), and six phenolic acids or derivatives (p-coumaric acid, p-coumaric acid/4-hydroxybenzoic acid mixture, caffeic acid, 3, 4-dicaffeoylquinic acid, 4, 5-dicaffeoylquinic acid and methylcaffeate). Among them, loliolide is reported for the first time from this species. The aerial parts of V. thapsus afforded two pheophorbides (pheophorbide A and pheophorbide A ethyl ester), two pheophytins (pheophytin A and pheophytin B), one steroid (a-spinasterol), one known flavonoid (luteolin), one phenylethanoid glycoside (verbascoside), three simple pheonolic acids (trans-cinnamic acid, 4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid) and one fatty acid (1-monoacylglycerol). All compounds, except for a-spinasterol, luteolin and verbascoside, are reported for the first time from this species. a-Spinasterol is first reported from the aerial parts of this plant. When screened for activity against M. tuberculosis in the SPOTi assay, A. lappa n-hexane extract and dichloromethane phase of methanol extact, and T. farfara n-hexane and ethyl actate extracts were active at MICs of 62.5 μg/mL; A. lappa ethyl acetate extract and T. farfara methanol extract were active at MICs of 125 μg/mL; V. thapsus ethyl acetate extract was active at the concentration of 250 μg/mL. Among the tested compounds isolated from active extracts, p-coumaric acid displayed the highest activity (MIC=31.3 (So(Bg/mL, 190.7 μM); p-coumaric acid/4-hydroxybenzoic acid mixture showed good activity (MIC=62.5 μg/mL); sitosterol/stigmasterol mixture exhibited moderate activity (MIC=125 μg/mL); loliolide, caffeic acid and trans-cinnamic acid revealed weak activity (MICs=250 μg/mL, or 1273.9, 1387.6 and 1687.4 μM, respectively). This is the first time that the antitubercular acitivity of A. lappa, T. farfara and V. thapsus has been investigated. The anti-TB activity of all tested compounds is also first reported in the SPOTi assay. When initially screened for activity against M. tuberculosis in the MABA assay at the highest concentrations of 25 or 50 μg/mL, all plant extracts and tested compounds were identified as inactive at such concentrations. This is the first report of the screening of A. lappa, T. farfara and V. thapsus extracts and of all tested compounds in the MABA assay. Among the compounds screened for activity against Methicillin-resistant S. aureus, luteolin exhibited good activity with an MIC valueof 62.5 μg/mL (218.3 μM), and a-spinasterol had an MIC of 500 μg/mL. No other compound was active at the highest concentration (500 μg/mL) used in this assay. This is the first report of the investigation of the anti-MRSA activity of kaempferol, a-spinasterol, 1, 3-dicaffeoylquinic acid, 3, 4-dicaffeoylquinic acid, 4, 5-dicaffeoylquinic acid, 1-monoacylglycerol, pheophorbide A ethyl ester, pheophytin A, pheophytin B and verbascoside.

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP4) and an important immune regulator. It specifically dephosphorylates the MAP kinases ERK and JNK to influence pro- and antiinflammatory cytokine production. MKP-2 has recently been shown to play a significant role in controlling Leishmania mexicana infection (Al-Mutairi et al., 2010) primarily by influencing macrophage activity. However, information on the effect of MKP-2 deletion at the molecular level on macrophage development and function is limited. This project utilised a novel DUSP4 gene knockout mouse and investigated the effects of MKP-2 deletion on M-CSF induced MAPK signalling and macrophage development as well as macrophage gene expression. Experiments in bone marrow derived macrophages demonstrated that in response to M-CSF macrophage, proliferation was reduced following to MKP-2 deletion. This was correlated with ERK phosphorylation, the expression of CD115 and CD34 on macrophage progenitors as well as the induction of genes related to macrophage differentiation and proliferation, colony stimulating factor-2 (Csf2) and monocyte to macrophage differentiation associated (Mmd) genes. In addition a comparative microarray gene expression analysis was conducted on MKP-2-/- and MKP-2+/+ macrophages following (LPS) or (IL-4) activation. As demonstrated previously, and associated with a role for MKP-2 in antimicrobial activity, arginase-1 expression was up-regulated in MKP-2-/- compared with MKP- 2+/+ macrophages. Surprisingly, and in contrast, we found that other alternative activation markers Ym1 (Chi3l3) and Fizz1/Retnla (Relm-a) were significantly reduced in MKP-2-/- macrophages when compared with their wild-type counterparts. As both Ym1 and Fizz1 have been implicated to play a major role in extracellular matrix disposition this suggests a significant role for MKP-2 in wound healing. Collectively, the findings in the current study have established that MKP-2 plays an important role in macrophage development and immune function.

Based on ethnobotanical information collected from Cuban diabetic patients in Cuba, medicinal plants such as Allophylus cominia (L.) Sw. (A. cominia) was identified as a possible source of new drugs that could be used for the treatment of type 2 diabetes mellitus. Type 2 diabetes mellitus or non-insulin dependent diabetes mellitus (T2-DM) is a chronic disease, and when associated with obesity (a condition known as diabesity) leads to an increase in the risk of a number of comorbidities, e.g. cardiovascular, kidney and liver diseases. A. cominia is a Cuban plant used traditionally by diabetic patients for the treatment of their diabetes symptoms. Preliminary studies of its leaves (Veliz et al., 2003; Veliz et al., 2005 Sanchez et al., 2014) have shown potential anti-diabetic activity and it is therefore being further investigated in the search for a novel, nontoxic, and efficacious anti-diabetic agent. The present project investigated the in vitro hypo-glycaemic activity of A. comin ia extracts. Chemical characterisation of the extracts was carried out using different phytochemical methods. Fatty acids, tannins, pheophytins (A and B), and a mixture of flavonoids were detected. The identified flavonoids (42.1 mg) were mearnsitrin, quercitrin, quercetin-3-alloside, and naringenin-7-glucoside. Some of these compounds have been reported in the literature as potent hypo-glycaemic agents. Separation of the mixture of quercitrin and mearnsitrin was carried out by high performance liquid chromatography using an amino column. Extracts from A. cominia were tested for their ability to inhibit the activity of four enzymes. DPPIV plays an essential role in glucose metabolism. PTP1B is important in inhibiting downstream signalling of the insulin and leptin receptors. Alpha-glucosidase is one of the enzymes responsible for the breakdown of carbohydrates into monosaccharides, and alpha-amylase breaks down large, insoluble starch molecules into soluble starches, producing successively smaller starches and ultimately, maltose. The flavonoids produced a concentration-dependent inhibition against DPPIV with a Ki value of 2.6 « 0.2 (So(Bg/ml. The flavonoids fraction from A. cominia revealed a competitive inhibition using DPPIV substrate comparable to the inhibition by the commercial (P32/98) inhibitor. In addition, PTP1B enzyme was 100 « 5% inhibited by the flavonoid mixture and 65 « 2% inhibited by pheophytin A and 61 « 1% inhibition by pheophytin B at 30 (So(Bg/ml respectively. The flavonoid mixture elicited a significant concentration-dependent inhibition against PTP1B with a Ki value of 3.2 « 0.09 (So(Bg/ml, as well as with pheophytin A with a Ki value of 0.64 « 0.05 (So(Bg/ml and pheophytin B with a Ki value of 0.88 « 0.03 (So(Bg/ml; both were lower than that of TFMS inhibitor, with a Ki value of 1.1 « 0.03 (So(Bg/ml. Both flavonoid and pheophytin A extracts from A. cominia revealed a competitive inhibition of PTP1B enzyme using DiFMUP as substrate . Competitive inhibition was also shown with TFMS inhibitor. On (Sa(B-glucosidase enzyme, a 79 « 1% inhibition was produced by the flavonoid mixture at 30 (So(Bg/ml. The flavonoid fraction from A. cominia showed a concentration-dependent pattern against (Sa(B-glucosidase, with a Ki value of 1.7 « 0.5 (So(Bg/ml that was lower than that of acarbose inhibitor (190 « 0.5 (So(Bg/ml). These extracts have shown a competitive inhibition using 4-nitrophenyl-glucopyranoside as substrate. Acarbose also produced a competitive inhibition against (Sa(B-glucosidase. No significant effect was found with any of the extracts from A. cominia at 30 (So(Bg/ml against (Sa(B-amylase enzyme. After separation of the flavonoids, mearnsitrin and quercitrin did not produce any effect (at 30 (So(Bg/ml) on any of the enzyme activities (DPPIV, PTP1B, (Sa(B-glucosidase and (Sa(B-amylase). Quercitrin and mearnsitrin were active only in synergy. On a glucose uptake assay using HepG2 cells, the crude methanolic extract from A. cominia enhanced insulin activity by increasing 2-NBDG uptake by two-fold (2-NBDG is a fluorescently-tagged glucose derivative). The 2-deoxy-D-glucose uptake by differentiated 3T3-L1 cell line showed an increase of the glucose uptake in the presence of 100 (So(Bg/ml of flavonoids by enhancing insulin activity (100 nM), whereas the uptake was increased in the presence of 100 (So(Bg/ml of pheophytin A without enhancing insulin activity. The effect of different compounds from A. cominia on 3T3-L1 cell differentiation was also confirmed by quantifying GLUT4 transporters in the pre-treated cells with flavonoids and pheophytin A. GLUT4 transporters in th e pre-treated cells were similar to those of the differentiated normal 3T3-L1 adipocytes. 2-NBDG glucose uptake assay was also performed using L6 myotubes. The uptake was significantly increased by two-fold in the presence of 100 nM insulin, and by four-fold in the presence of both 100 nM insulin and 100 (So(Bg/ml flavonoids. A significant increase was also shown in the presence of 100 (So(Bg/ml pheophytin A and 100 nM insulin with a 10-fold increase (P<0.05) of glucose uptake by L6 cells. An increase of 2-NBDG uptake by L6 cells was shown in the presence of flavonoids and pheophytin A in addition to 100 nM insulin. Both flavonoid and pheophytin extracts (100 (So(Bg/ml) blocked the differentiation of 3T3-L1 fibroblasts into adipocytes by decreasing the fat accumulation by two-fold (more than the TNF-(Sa (Binhibition at 10 ng/ml). A significant difference was shown (P<0.05) compared to the control. Troglitazone significantly enhanced 3T3-L1 differentiation by two-fold. Exposing 3T3-L1 cells to both extracts from the third day of the differentiation induction did not alter the adipogenesis. Exposing 3T3-L1 adipocytes to the extracts from A. cominia containing flavonoids and pheophytin A showed a significant decrease in the fat accumulation after five days of incubation with the extracts (P<0.05). However, no fat accumulation was observed after withdrawal of the extracts from the cell growth medium. These compounds may be responsible for the pharmacological effects observed in experimental diabetic models in Cuba. Therefore, all these results strongly suggest that this plant could be a new and promising candidate for treating diabesity with natural sources.

In the search for new, safe and efficacious uterine active agents, the plant Ficus exasperata was subjected to phytochemical screening and pharmacological analysis. Ethyl acetate and methanolic leaf extracts of F. exasperata were fractionated and purified by a series of chromatographic techniques. The isolation process was guided by in vitro functional uterine assays involving the use of C57Bl/6 female mice. Identification of the active chemical constituents was performed by several spectroscopic techniques which included 1D and 2D nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). The uterine effects of these compounds were investigated on lidocaine-induced, spontaneous, oxytocin-induced and high KCl-induced contractions using isolated uterine segments of non-pregnant female mice. The activity of different compounds on the amplitude (maximum tension above basal force) and frequency of uterine contractions were simultaneously measured and then statistically analysed. The structure-activity relationships were also examined where possible. These studies led to the identification of some new phytochemical derivatives. Pharmacological assays revealed the presence of both uterine stimulatory and inhibitory constituents. The new pheophytin/pheophorbide derivatives, flavonoids, fatty acids and glycerol derivatives significantly reduced the frequency and amplitude of uterine contraction, while KCl salt, pyrimidine and pheophorbide-b derivatives significantly augmented both spontaneous and agonist-induced contractions. This study has demonstrated that F. exasperata generates secondary metabolites which have proven effective in the significant inhibition of uterine contractions and thus a potential source of new tocolytic agents. Additionally, uterine stimulatory constituents were also generated some of which may be potential drugs for contraception and/or labour facilitation. Lead compounds generated from this study are the pheophytin/pheophorbide derivatives, pyrimidine derivatives and flavonoid derivatives. The rather low yield of compounds frequently experienced in the course of bioassay-guided phytochemical screening has made it almost impossible to investigate the possible mechanism(s) of action of active fractions and/or compounds. This necessitated study into the development of high throughput analytical tools combining metabolomics and pharmacology in the investigation of the function of drugs. This study involved the application of liquid chromatography coupled to high resolution Fourier transform mass spectrometry (LC-HRFTMS) and proton NMR (1H-NMR) as analytical platforms in the determination of myometrial function. An initial study was performed to determine the success of the method and this was achieved by assessment of mouse myometrial metabolites altered in response to oxytocin and ritodrine. The myometrial tissues and bath fluids were extracted at the peak of activity and subjected to LC-HRFTMS analysis. The use of the bath fluids in this study was an innovative approach in sampling. The resulting data were preprocessed and analyzed via a pair-wise chemometric comparison model. Pathway analyses following metabolite identification confirmed previously known mechanism(s) thus validating the method while revealing new insights and creating knowledge-driven hypotheses for future research. This study therefore enabled the development of a technique which combines metabolomics with in vitro pharmacology for the rapid detection of compelling myometrial metabolites in drug function and was successfully applied in the determination of possible mechanism(s) of the active constituents of F. exasperata.

The biosynthesis of anthocyanin compounds involves type III polyketide synthases (PKSs): chalcone synthase (CHS), which is the first enzyme of the flavonoid synthetic pathways leading to synthesis of various anthocyanins. The European red raspberries are a commercially important fruit that needed both for fresh fruit consuming and food manufacturing. The fruits are a valuable source of nutraceuticals, notably anti-oxidants by anthocyanins that are pigments conferring impressive red colour to the fruits. To improve the quality in red raspberry through increasing anthocyanin content, the main objective was focused on investigation of the PKS genes that are an essential for the biosynthesis of anthocyanins. The aim of this thesis was to characterize the PKS/CHS gene regions within the three Bacterial Artificial Chromosome clones (BACs), which originated from the genome of the European red raspberries (Rubus idaeus) cv. Glen Moy (SCRI), using 454 sequencing technology assisted by a systematic construction of fosmid libraries. The two BACs (29M05 and 24B12) always presented similarities in almost analyses resulting from the presence of a 53 kb overlap that was revealed by assemblies of resulting 454 sequencing as it may be possible that there is no PKS/CHS gene in another one, BAC31B12. The assembly of BAC24P12 was completed, with an entire DNA length of 117 kb but BAC29M05 could not be completed along all its length of ~170 kb as the longest contig was 96 Kb provided the most important information of this BAC whilst containing a complete PKS1 gene sequence, currently identified as Naringenin chalcone-PKS. The alignments of assembled sequences also strengthened the similarities between these two BACs both at nucleotide and amino acid sequences. All four PKS/CHS gene regions found in the both BACs presented being multiple PKS/CHS gene family with conservation of an intron and two exons of gene structure as variations of those sequence confirmed being PKS/CHS pseudogenes in plant genome. The two informative sources generated in this thesis, including the fosmid sub-libraries, which are availably manageable sources for validating the BAC sequences, and physical map drafts provided better information of this chromosomal segment in this thesis, as they will be useful available tools for further studying in red raspberry genome.

Blood glucose homeostasis is highly regulated and is essential for survival. A key process is the insulin-stimulated recruitment of the facilitative glucose transporter GLUT4 to the plasma membrane in adipocytes and muscle cells; defects in this pathway can cause insulin resistance and type 2 diabetes. The insulin signalling and GLUT4 trafficking pathways in these cells have been extensively characterised, and a prominent role for protein phosphorylation has been uncovered. In contrast, relatively little is known about the role of other post-translational modifications (PTMs) in these pathways. The aim of this study was to expand existing knowledge of how palmitoylation, a PTM involving reversible attachment of fatty acids onto cysteine residues, affects components of the insulin signalling and GLUT4 trafficking pathways. For this, palmitoylated proteins were isolated from 3T3-L1 adipocytes by resin-assisted capture of S-acylated proteins (acyl-RAC), and screened to identify novel palmitoylated components of the insulin signalling and GLUT4 trafficking pathways. This approach successfully identified the following novel palmitoylated proteins: GLUT4, insulin-responsive aminopeptidase (IRAP) and caveolin-2. Furthermore, click-chemistry confirmed palmitoylation of caveolin-2 and IRAP and also enabled identification of the palmitoylation sites on these proteins. Palmitoylation has been shown to regulate proteins in many different ways, in particular by modulating protein trafficking, protein stability and protein-protein interactions. Mutation of the palmitoylation sites in caveolin-2 and IRAP had no obvious effect on protein localisation. However, palmitoylation-deficient mutants of caveolin-2 exhibited: (i) a deficit in conversion of monomeric caveolin-2 into low molecular weight oligomeric complexes, and (ii) decreased oligomer stability, revealed by a loss of SDS-resistant caveolin-2 complexes. Overall, this work has identified novel palmitoylated proteins in 3T3-L1 adipocytes, mapped the palmitoylation sites of these proteins, and determined the effect of this PTM on the localisation and oligomeric status of these proteins. These findings have thus expanded existing knowledge on the potential regulation of insulin signalling and GLUT4 trafficking pathways by PTMs.

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) effecting millions of people worldwide. The pathology of MS is characterised by the demyelination of neurons. Though the root cause of MS remains elusive a combination of genetic, environmental and infectious factors are thought to contribute. IL-33 has previously been shown to be protective (e.g. atherosclerosis), or detrimental (e.g. asthma), in certain disease states. IL-33 and ST2 (IL-33R) are highly expressed within the CNS and have been shown to exacerbate Experimental Autoimmune Encephalomyelitis (mouse model of Multiple Sclerosis). In-situ staining was used to indicate expression of IL-33 and ST2 in the CNS at different stages of EAE disease. IL-33 remained unchanged throughout the time course of disease. However, ST2 expression was upregulated during cellular infiltration. Within naïve and EAE mice IL-33 and ST2 were both expressed on astrocytes and neurons. In acute and chronic MS tissues IL-33 was highly expressed by neurons and axons. Within the lesions IL-33 was present on damaged axons as well as microglia and ODCs. ST2 displayed a diffuse staining in control and MS tissues, however within the lesion site of acute and chronic MS samples ST2 surrounded damaged axons and was present on several ODCs. The potential for IL-33 to affect myelination was investigated using an in-vitro culture system. IL-33 significantly reduced myelination within rat cultures, however no significant effect was observed within the mouse culture system. The main findings from EAE tissues were corroborated in human MS tissue and the implications this may have on understanding the disease course of MS are discussed.

Mitogen-activated protein kinase phosphatases (MKPs) belong to the dual-specificity phosphatase (DUSP) family and are negative regulators of mitogen-activated protein kinases (MAPKs). Recently, MKP-2 has been shown to play a novel role in development, the immune system and cancer. However, our knowledge in relation to its function in the central nervous system (CNS) is limited. Hence, we have utilised novel MKP-2 knockout mice to investigate the role of this phosphatase in CNS function. The effect of MKP-2 deletion on CNS was first investigated using primary hippocampal culture by examining astrocyte proliferation and neurite length using immunocytochemistry techniques performed on cultures 1-7 days in vitro (DIV). To investigate the functional consequence of MKP-2 deletion on CNS at 3, 7 and 11 DIV, intracellular calcium concentrations were determined by fluorescence imaging using Fluo-4 dye. Na+, K+ current, synaptic transmission was also investigated using patch clamp electrophysiology. To examine the effect MKP-2 deletion in whole tissue, standard western blotting techniques were utilised to examine the consequence of MKP-2 deletion on ERK activity in the hippocampus of 3 week old mice. The functional effect of MKP-2 deletion on basal synaptic transmission and paired pulse facilitation (PPF) on acute hippocampal slices, field excitatory postsynaptic potentials (fEPSPs) were recorded from acute hippocampal slices of 3 week old mice. Astrocytes number and neurite length were reduced in 1-3 DIV in MKP-2-/- compared to MKP-2+/+. However, there is no difference in astrocyte proliferation and neurite length in 4-7 DIV. Further investigation into the reduction of neurite length, it was due to impairment in astrocyte function and not account of reduction in astrocyte proliferation. Investigating further into the functional consequences, astrocytic intracellular calcium was reduced at 7 DIV however neuronal intracellular calcium was increased at 11 DIV in MKP-2-/- primary hippocampal culture. Furthermore, Na+ and K+ current were also reduced at 7 DIV. However, spontaneous excitatory postsynaptic current (sEPSC) and synapse number has been shown to increase at 7 and 11 DIV. The mechanism underlying this seems to be not related to ERK phosphorylation as no difference in ERK activation was evident when compared between MKP-2+/+ and MKP-2-/-. In further experiments in acute hippocampal slices, MKP-2 deletion leads to a reduction in ERK activity within the hippocampus. In contrast, increased ERK activity was observed in the heart and liver. Basal synaptic transmission was enhanced in MKP-2-/- mice at high stimulus intensities compared to MKP-2+/+ but PPF was unaltered at all in inter-stimulus times tested (10-500 ms). In summary, in this thesis I demonstrate that even though MKP-2 deletion reduced astrocyte growth and neurite length at 3 DIV, it doesn't affect their functional properties at early development. However, alterations of functional activity at 7 and 11 DIV and acute hippocampal culture suggest that MKP-2 deletion might play a role in functional activity when the neuron is fully developed. This data suggest a novel physiological role for MKP-2 in the brain and might reveal valuable insight for the drug development.

Recombinant human erythropoietin (r-HuEPO) administration stimulates the increase in red blood cell (RBC) mass and ultimately the oxygen carrying capacity of the blood, which results in its ergogenic benefits. EPO and its related forms are banned by the World Anti-Doping Agency. The main aim of this thesis was to apply an untargeted liquid chromatography-mass spectrometry based approach to investigate a possible phenotypic response to r-HuEPO in the plasma, urine and RBC (from whole blood and centrifuged blood residue) metabolomes of twenty physically active non-smoking males, as a potential method to detecting abuse. Participants underwent a ten week sampling protocol consisting of two weeks of baseline, four weeks of treatment (50IU.kg-1 every second day) followed by four weeks of wash-out. Significant metabolites (p-value < 0.05) were identified from a diverse range of metabolic pathways. Most interesting are as follows: orotate from urine, as well as linoelaidyl and elaidic carnitines, glycine, ergothioneine, thymine, 2-oxoglutarate, L-arginine, homoarginine, deoxycholic acid 3-glucuronide, D-glucuronate, D-glucosamine, 4-aminobutanoate, muramic acid and [PR] tretinoin/all-trans retinoic acid from plasma. The whole blood investigation highlighted significant metabolites which could be responsible for the generation of glutathione (namely 2-oxoglutaramate) which is an essential component to RBCs, because of its antioxidant capacity. Whereas the RBC residue obtained following removal of plasma showed significant changes in L-carnitine and acyl-carnitines (linoelaidyl, elaidic, hydroxybutyryl, heptadecanoyl, stearoyl, tetradecanoyl carnitines). As a result of the absence in a control group as well as training frequency of the participants, in the primary study, a small pilot investigation was carried out with the same untargeted methodology to investigate the phenotypic response of an hour of aerobic exercise in the urine metabolome of three physically active non-smoking males. Main significant systems affected include: purine pathway, tryptophan metabolism, carnitine metabolism, cortisol metabolism, androgen metabolism, amino acid oxidation and the gastrointestinal microbiome. It was concluded that a more robust investigation into r-HuEPO abuse would require a placebo controlled cross-over study, in order to ensure that the findings were truly related to the administration itself and not to other external stimuli, such as training.

Aim: This study aimed to develop a grounded theory to explain the experience of patients with neurodegenerative diseases and their carers, following an episode of residential respite care. Background: Neurodegenerative diseases are incurable, debilitating and result in progressive deterioration of the patient which present as problems with cognitive functioning (dementias) and/or physical functioning (ataxias). To allow quality of life and purpose for the patient and caregiver, as well as providing value for money, respite is an area of care which could offer the patient rehabilitation, maximisation of functionality and quality of life; relief from caregiving duties for the carer and signposting for additional assistance and support. Methods: This was a qualitative, grounded theory study conducted across south east England. An initial audit, attendance at support groups and specialist clinics provided scoping of the scale of the problems encountered by this patient population. Data collection included 17 semi structured interviews conducted with patients and carers who had recently received residential respite, non-participant observation at a hospice offering dedicated respite care and 4 hospice staff interviews. Findings: A successful respite depended on the patient and carer identifying the need for respite and the information required to determine where, what, when and how respite could be accessed. The logistics of the referral process, preparation for respite and the handover of care needs to professionals were key factors for a therapeutic admission. Conclusion: The outcomes from the respite admission should be mutually acceptable to both the patient and carer and be able to demonstrate acceptance and adaptation to a new normalcy, influenced by disease progression and reflecting on time present and time past. The onward journey sees a transitioning which involves restoration and building up a level of resilience for the carer, which all contribute to sustainability and being able to continue in the caring role.