The work described in this thesis had the main aim of understanding protein solution rheology. This was from a biopharmaceutical perspective, with account of the biophysical properties of proteins and in particular their level of aggregation. Molecular interactions influencing the rheology of a range of protein solutions were studied. Proteins were selected to relate directly to the diversity of protein types used in biopharmaceuticals. In addition, the roles of a surfactant formulation additive and synthetic amphiphilic polymers in the flow behaviour of protein solutions were studied. The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of a recombinant albumin (rAlbumin) was studied. The effect of the level of protein aggregation, variation in protein concentration and its impact on solution viscosity was revealed. Theoretical models predicting the increase of viscosity with concentration were applied to these data. A recent model that accounts for multiple protein species in solution, predicted the experimental data best. The rAlbumin study, although a relatively simple system, represented a 'real-life' formulation with results highlighting the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentration of protein solutions. Beta-lactoglobulin (b-LG) excipient-free solutions were characterised by bulk and interfacial shear rheology. Solutions at various concentrations, characterised using conventional rheology instrumentation, evidenced an apparent yield stress behaviour at a low shear rate range (0.01 - 10 1/s), whilst showing constant viscosities throughout higher shear rates. Comparing interfacial shear rheology, air-water interface-free bulk rheology measurements, and tensiometry results, it was demonstrated that the complexity of this protein's solution rheology was due to the formation of a protein viscoelastic film at the air-water interface, as present in conventional rheometry. This is in agreement with literature. Further studies considered the effect of insoluble b-LG aggregates on the solutions' rheology, linking with their characterisation in size and quantication. The presence of insoluble proteinaceous particles was suggested to have an impact on the solution's flow behaviour, particularly at the lower shear rates. Excipient-free monoclonal antibody (mAb) solutions were studied with the aim of generating protein aggregates (soluble and insoluble) to explore their impact on solution rheology. mAb samples were subjected to thermal stress and were characterised for their purity, aggregate content and size. The change in species content did not alter the original protein's yield-stress behaviour at low shear rates. An increase in aggregate content was related to the increase of viscosities observed at high shear rates. Establishing a relationship between species content (in volume fraction) and viscosities, as for the rAlbumin study, was not possible due to this mAbs specific aggregation behaviour. However, from the b-LG and mAb case studies, our results highlight the importance of detailed characterisation of protein solutions with orthogonal biophysical techniques so as to better understand protein solution rheology. An additional study looking at the effect of polysorbate-80 upon protein rheology was made. In agreement with literature, this commonly used excipient in biopharmaceuticals was demonstrated to affect the rheological measurements of globular protein solutions. Amphiphilic brush-like poly(ethylene glycol) methacrylate polymers were also synthesised and tested as novel additives with b-LG and mAb solutions, for their potential effects on protein solution rheology, similar to those observed with polysorbate-80. Preliminary results showed that the effects of these polymers are likely related to competition for the air-water interface, between these and the proteins involved. This competition leads to changes in the yield-like behaviour at low shear rates.

Campylobacter is currently the most frequently isolated food-borne bacterial pathogen worldwide. Infections caused by C. jejuni may be self-limiting enteritis or chronic conditions such as Guillain-Barre' syndrome (GBS). Although the mechanisms by which C. jejuni causes disease are not clearly understood, the activation of the transcription factor NF-KB, which controls pro-inflammatory responses, is thought to be an important contributing mechanism for initiating the host's immune responses to C. jejuni infection. Signaling pathways leading to NF-KB by pathogens and/or their products include transmembrane Toll-like receptors (TLRs) and intracellular receptors nucleotide-binding oligomerization domain proteins (NODs). This study was carried out to: 1) investigate NF-KB activation by C. jejuni, 2) provide structural details regarding the NF-KB activating component(s) in C. jejuni boiled cell extract (BeE) and 3) investigate the role of TLRs (TLR2 and TLR4) and NODs (NODI and NOD2) in mediating NF-KB activation by C. jejuni. By means of measuring reporter gene activity, NF-KB activation and subsequent cytokine production by live or heat-killed C. jejuni, or BeE were observed in a range of tissue cultures cell lines. Structural characterisation of the NF-KB activating component in BeE indicated that the bioactive structure is an alpha-linked linear oligosaccharide composed of glucose where the activation by the oligosaccharide is suppressed upon pre-treatment of BeE with amyloglucosidase. NF- KB activation was observed to be augmented in cell lines transfected with TLR2 but not with TLR4. This activation is reduced upon transfection of cells with the dominantnegative versions (DNV) of TLR-adaptor molecules MYD88 or lRAKl. Additionally, NF-KB activation by C. jejuni was observed to be independent of NOD 1 and NOD2 in cells transfected with DNV of these receptors.

Novel and sensitive CE methods coupled with LIF and LINF detection using a series of separation modes have been developed for profiling organic metabolites containing amines and carbohydrates in mammalian and plant biofluids. In this study, metabolites containing an amine group were derivatized with 4-Fluoro-7-nitro- benzofurazan (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection (LIF). Under the optimized conditions most of the amine-containing metabolites in human biofluids such as plasma, urine and saliva could be identified by reference to standard compounds and the concentrations measured were found to be in agreement with literature values. Furthermore, 17 carbohydrates including mono-, di- and oligosaccharides are also simultaneously derivatized via a two-step reaction involving reductive amination with ammonia followed by condensation with NBD-F. Under the optimized derivatization conditions all carbohydrates were successfully derivatized within 2.5 h and separated within 15 min using borate buffer (90 mmol L−1, pH 9.2). The method was applied to measure sugars in nanoliter volume samples of phloem sap obtained by stylectomy from wheat and to honeydew samples obtained from aphids feeding from wheat and willow. Finally, an on-line sample concentration technique, sweeping-micellar electrokinetic chromatography (sweeping-MEKC), in conjunction with UV laser-induced native fluorescence detection (LINF) was developed and applied to the detection of native fluorescent analytes in biofluid samples such as plant phloem sap, human plasma and urine samples. The concentration limits of detection of analytes were in the range 7–100 nmol L−1, which were 250–3600-fold improvement for dopamine, DOPA and epinephrine compared with conventional capillary zone electrophoresis (CZE). The results indicated that a long-term limitation of relatively low detection sensitivity in CE-UV analysis arisen from the small injection volume and short optical path-length could be much improved, while no apparent loss in separation efficiency occurred.

Mucosal surfaces offer a potential for non-invasive delivery of proteins. The role of these surfaces, however, is to limit the movement of material from the external environment (mucosal lumen) into systemic circulation. Mucosal absorption of protein therapeutics is constrained through several physiological barriers such as mucus and mucociliary clearance, protease enzymes, epithelial tight junctions (TJs) and cellular membranes. This work explores different strategies with the view to improving the transport of macromolecules (proteins and protein drug models) across polarised epithelial cell layers in vitro, which could potentially be a reflection of improved mucosal absorption and bioavailability in vivo. The Calu-3 cell line was used in this work to produce such layers, serving as an in vitro model of the airway epithelium. Following growth of Calu-3 cells on filters under air-interface culture conditions polarised layers of closely packed cells were formed. The cell layers exhibited a TEER ≥500 Ωcm2 and cells showed structural features similar to the native epithelium, including the TJs, the microvilli and the secretory granules. Cell layers presented a barrier to the permeability of FITC-dextrans (FDs, paracellular markers) and nanoparticles (NPs). The first class of tested compounds, namely alkylglycoside (AG) surfactants, exhibited severe toxicity at concentrations considerably lower than those used in the literature. Data indicated that the cellular toxicity of AGs possibly results from a membrane effect. Investigation of calcium depletion as a proposed strategy to improve mucosal absorption of protein therapeutics, revealed that calcium depletion on the apical side produced limited TJ opening, as demonstrated by reversible decrease in transepithelial electrical resistance (TEER) and modest enhancement of permeability of macromolecules. Although combined apical and basolateral calcium exhaustion produced significant effects on TJs, this scenario becomes irrelevant in an in vivo situation. Application of chitosan in the form of solution and NPs to Calu-3 layers demonstrated that chitosan NPs formulated by the ionic gelation method, exhibit a similar TJ-opening effect to solution. This was shown by similarities in measurable indicators of TJ opening such as reduction in TEER and enhancement of dextran permeability across the cell layers. Furthermore, chitosan NP and solution exhibited similar effects on the TJ protein, Zonula Occludens-1. These results therefore indicated that chitosan NPs could potentially be used to carry and protect fragile therapeutic proteins to the mucosal surface(s) of interest and at the same time promote their absorption through TJ opening. TJ opening as a strategy to improve mucosal absorption of macromolecular therapeutics is rather inefficient for larger proteins such as antibodies, or for nano- sized drug carriers following their mucosal administration; this led to investigation of the IgG/neonatal Fc receptor (FcRn) transcytotic pathway. Immunostaining data demonstrated that Calu-3 cells express FcRn. IgG was shown to traverse the Calu-3 layers and studies characterizing this transport indicated FcRn involvement in this process. Confocal microscopy revealed that IgG- or Fc-adsorbed NPs were taken up by Calu-3 cells. Adsorption of Fc on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. Characterisation of cell uptake and transport of Fc-NPs revealed data that strongly suggested FcRn involvement in these processes.

The prevalence of food allergy is increasing in westernized countries, affecting 5-8% of children and 1-3% of adults. Although innumerous proteins are encountered in normal diets, only few proteins are commonly implicated as food allergens. In this vein, the major focus in allergy studies falls into intrinsic features of allergens; however, it is known that extrinsic factors can play a role in allergic processes. The allergenicity of Ber e 1, the major allergen from Brazil nuts, is well established and it has been shown that natural lipids from Brazil nuts are essential for the development of an immune response towards Ber e 1. The present study aimed to characterize the humoral response induced by recombinant (r)Ber e 1 alone or in the presence of lipids, and to investigate the mechanism(s) by which natural lipids influence the development of an immune response. BALB/c mice were sensitised intraperitoneally with rBer e 1 alone or in the presence of different lipid fractions. It was found that rBer e 1 alone did not induce an immune response and only one specific fraction of Brazil nut lipids (SPC fraction C), composed of a mixture of lipid classes, was able to induce a Th2-type humoral response, with the presence of Ber-specific anaphylactic antibodies, high levels of Ber-specific IgG1, and low levels of Ber-specific IgG2a. CD1-restricted natural killer (NK)T cells recognize lipids and therefore to test the hypothesis that NKT cells may be involved in the response, the sensitisation protocol with rBer e 1 and SPC lipid fraction C was tested in mice lacking these cells (J18 KO mice). These animals presented significantly lower titers of Ber-specific anaphylactic antibodies, Ber-specific IgG1, and total IgE than sensitised wild type mice, indicating that one of the pathways by which lipids triggered an immune response involved NKT cells. In conclusion, the present work found that lipids from Brazil nuts were essential for the development of a Th2-type humoral response to rBer e 1 and that the immune response induced by lipids involved NKT cells.

Embryonic stem cells are of great interest to scientists as they can differentiate into any somatic cell lineage making them excellent candidates for tissue regeneration and cell based treatment therapies. Currently, human embryonic stem cells (hESCs) are cultured using feeder fibroblasts or protein substrates such as matrigel, fibronectin or laminin in conditioned media. hESCs are then subcultured using enzymes to detach them from the culture substrate. However, the use of the xenosupport systems makes the hESCs therapeutic applications difficult due to cross-contamination with animal pathogens from the animal derived feeders, matrix or conditioned media to the hESCs. Moreover, the use of enzymes to recover hESCs can damage these cells. For the mentioned reasons, development of completely synthetic surfaces is desirable for hESCs culture. Thermo-responsive surfaces have been extensively studied for cell culture using the well know thermo-responsive polymer poly (N-isopropylacrylamide) (PNIPAAM) which has a switchable properties across its lower critical solution temperature (LCST) at 32°C. However, more biocompatible polymers that have similar thermo-responsive properties to PNIPAAM and biocompatible properties, such as PEG based materials, have been proposed for use as switchable surfaces. Within this thesis, thermo-responsive copolymer brushes composed of 2-(2- methoxyethoxy) ethylmethacrylate (ME02MA) and oligo (ethylene glycol) methacrylate (OEGMA) that have LCST close to body temperature (37°C) were investigated for use as a cell culture surface for temperature sensitive passaging. Poly (ME02MA-co-OEGMA) brushes were grafted from the surface using atom transfer radical polymerisation (ATRP). Hydroxyl plasma-polymer functionalised glass slides were prepared using plasma polymerisation and then an ATRP initiator was reacted to these surfaces. ATRP of the copolymers was then performed from these initiation sites. These surfaces were characterised using X-ray photoelectron spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), atomic force microscopy (AFM) and water contact angle (WCA). Using 3T3 fibroblasts as a model cell type for initial studies, it was demonstrated that these cells adhered and proliferated on the poly (ME02MA-co-OEGMA) thermo-responsive surfaces at 37°C when the polymer brushes are in their hydrophobic state. Subsequent detachment assays were conducted when the temperature was lowered to 20°C i.e. the hydrated conformation of the copolymer brushes. Mouse embryonic stem cells (mESCs) were then cultured on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for 3 days. Passaging experiments were performed for 10 passage cycles and the cells analysed for retention of the undifferentiated stem cell status. These thermoresponsive polymer/fibronectin surfaces are found to be suitable for mESCs culture but evidence of differentiation was observed. Attachment of a novel gelatine based peptide to the poly (ME02MA-co-OEGMA) was also investigated for mESCs culture to avoid the use of fibronectin (which was thought to be a contributing factor to the stem cell differentiation seen). mESCs adhesion was observed both to the peptide adsorbed on TCPS and on the peptide coupled to the thermo-responsive poly (ME02MA-co-OEGMA) surface. This research indicates that these smart stimuli biomaterials have promise as a new generation of culture surfaces for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.

Osteoarthritis (OA) is expected to become the fourth leading cause of disability worldwide by 2020. There is no cure, and joint replacement surgery becomes a final resort. Chronic pain associated with OA is poorly controlled by current treatments, and often involve chronic use of non-steroidal anti-inflammatory drugs (NSAIDs), which is associated with serious side-effects. OA is associated with alterations in endocannabinoid (EC), an attractive target for the control of pain. ECs are rapidly degraded by a number of enzymes, including cyclooxygenase-2 (COX- 2), the major target of NSAIDs. However, the role of COX-2 in EC-mediated effects on nociceptive transmission is not fully understood. The aims of this thesis were to investigate peripheral and spinal pain responses in a model of OA pain, understand the role of COX-2 inhibition on neuronal responses and the potential role of ECs in mediating these effects, and to establish the functional effects of the EC system in a model of OA pain. Effects of spinal and peripheral administration of the COX-2 inhibitor nimesulide (1-1 OOlJgin 501JL)on mechanically evoked responses of dorsal horn neurones in the naive, anaesthetised rat were measured, and the contribution of the CB1 receptor was determined with the antagonist AM251 (11Jgin 50IJL). Effects of nimesulide on spinal levels of ECs and related compounds were quantified using liquid chromatography-tandem mass spectrometry. Spinal, but not peripheral, injection of nimesulide significantly reduced mechanically evoked responses of dorsal horn neurones. Inhibitory effects of spinal nimesulide were blocked by the CB1 receptor antagonist AM251, but spinal EC levels were not elevated. Indeed, both anandamide and N-oleoylethanolamide were significantly decreased by nimesulide, highlighting a putative role for other oxidative enzymes of ECs in the generation of CB1-active metabolites. The monosodium-iodoacetate (MIA) model of OA pain has recently received much interest, but is not yet fully defined. Work in this thesis sought to further characterise this model. Cytokine levels in synovial fluid, spinal cord and hindpaw skin at early time-points post- intra-articular injection (1mg MIA in 50IJL, P.O. 3-24hr) were measured, and the later (P.O. day 28-31) effects on neuronal responses and pain behaviour were determined. Intra-articular injection of 1mg MIA produced stable and robust changes in two measures of pain relevant to clinical OA, and evidence for the presence of central sensitisation was demonstrated. It was also demonstrated that early-stage painful responses in this model are not associated with changes in cytokines in the joint. Effects of spinal and systemic administration of nimesulide (3-100IJg in 501JL)on mechanically evoked and post-stimulus responses of dorsal horn neurones in MIA-treated rats were also measured, as were the effects of spinal cannabinoid receptor antagonism with AM251 (0.1- 10IJg in 501JL)and the CB2 receptor antagonist SR144528 (0.001-0.1IJg in 50IJL). Spinal and systemic COX-2 inhibition in the MIA model attenuated spinal neuronal responses to both noxious and innocuous stimuli, demonstrating the importance of both spinal and peripheral COX-2 products in mediating neuronal responses in this model. Antagonism of the spinal cannabinoid receptors resulted in elevated spinal neuronal responses in MIA-treated rats, demonstrating a functional role for spinal EC-mediated modulation of nociceptive transmission in the MIA model, expanding on work in this lab which showed elevated spinal ECs in the MIA model of OA pain. This work therefore demonstrates that the central EC system may be an important target for the treatment of OA pain.

Eukaryotic Initiation Factor (eIF) 4A is the most abundant initiation factor and the prototypical member of the DEAD-box family of helicases. Once recruited to the cap-binding complex, eIF4F, eIF4A unwinds inhibitory RNA secondary structure in the 5’ untranslated region (UTR) of mRNAs, promoting efficient ribosomal scanning to the start codon. The requirement for eIF4A in translation initiation correlates with increasing 5’ UTR length, suggesting that regulating the activity of eIF4A may affect the translation of particular mRNAs. It is well established that the transcripts of genes involved in cell cycle control and proliferation have long 5’ UTRs; therefore altering the activity of eIF4A may affect these genes specifically. A cross-discipline approach was used to investigate eIF4A helicase activity to obtain information regarding both the mechanics of helicase activity and the biological impacts of its inhibition. Recombinant eIF4A helicase activity, the stimulatory effect of eIF4B and the effect of known eIF4A inhibitors was first analysed using an ensemble helicase assay. Due to the limitations of this method a single molecule technique utilising optical tweezers was developed to investigate helicase activity at a higher force resolution. Optical tweezers were used to ‘trap’ and manipulate a dual-labeled RNA:DNA construct containing a central stem-loop hairpin known to be inhibitory to ribosomal scanning attached to functionalised microspheres. Although instrumental failure prevented the completion of these experiments, initial force extension curves using this molecule were obtained. Once established, this single molecule system may be used to observe eIF4A activity with its accessory protein eIF4B and known eIF4A inhibitors. 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2) is a newly identified natural inhibitor of eIF4A activity which induces apoptosis and is implicated in the resolution of inflammation. The translation of specific mRNAs affected by 15d-PGJ2 treatment of HeLa cells was analysed by translational profiling coupled to microarray analysis. No correlation, however, was seen between those transcripts that were translationally repressed and their 5’ UTR length or composition.

Selenium (Se) is an essential element for humans, which is derived primarily from dietary sources. Habitual suboptimal dietary Se intake is associated with reduced Se status and adverse health outcomes including cardiovascular disorders, impaired immune functions and some cancers. The global extent of suboptimal dietary Se intake is difficult to estimate, but is likely to be widespread where food choices are narrow, for example, in subsistence agricultural contexts. This study aimed to: (1) characterise the likely contribution of maize grain to dietary Se intake in rural Malawi; (2) test the dependency of maize grain Se concentrations on soil factors; and (3) identify agronomic methods to improve Se concentration in maize grain. 88 field sites across Malawi were sampled across Malawi in 2009 and 2010 before determining maize grain, total soil and KH2PO4-extractable soil Se concentrations by inductively coupled plasma-mass spectrometry (ICP-MS). Dietary Se intakes from other food sources were estimated from the literature. The median maize grain Se concentration in Malawi was 0.019 mg Se kg-1 (range 0.005-0.533), representing a median intake of 6.7 µg Se person-1 d-1 from maize. Suboptimal (<30 µg d-1) dietary Se intake is therefore likely to affect most of the rural population in Malawi. Maize grain Se concentration was c. 10-fold higher in crops grown on high pH (>6.5) soils (Vertisols), probably because the dominant species of Se at high soil pH Se(VI) is more available to crops than Se(IV), as evidenced by the KH2PO4-extractable soil concentrations recorded. Total soil Se concentration ranged between 0.0521 and 0.6195 mg kg-1 but provided a poor index of Se availability. The results showed that KH2PO4-extractable Se concentrations >0.01 mg kg-1 and soil pH values >6.5 produced grain Se concentrations exceeding 0.15 mg Se kg-1, a value above which rural populations in Malawi would attain adequate Se intake. Field experiments in which three Se application methods (Na2SeO4 (aq), granular compound (NPK+Se) and granular calcium ammonium nitrate (CAN+Se) were applied were conducted at up to six sites in 2008/09 and 2009/10. Application of Se significantly increased grain and stover Se concentrations and the response was approximately linear for all sites and application methods in both years (R2 >0.90). The results showed that application of Se at 5 g Se ha-1 to maize would deliver adequate intakes for much of the population in Malawi. As total plant recovery of Se ranged from 3-45%, further work is required to identify and address the sources of this variation. In more detailed experiments, the fate of applied Se was investigated at two sites using the stable 74Se isotope. Recovery of applied Se was 0.65 and 1.08 g Se ha-1 at the Chitedze and Mbawa, sites respectively, representing 6.5 and 10.8% of the applied 10 g Se ha-1 by the maize crop; 0.2 g Se ha-1 of native soil Se was also absorbed, leaving 9.35 and 8.92 g Se ha-1 unaccounted. Of the total soil and applied fertiliser Se, fertiliser-derived Se (74Se-labelled) comprised 71 and 82% of plant-Se recovery at Chitedze and Mbawa, respectively. The residual effects of Se application on grain Se in maize crops grown in the subsequent cropping season were 0.3025 and 0.5858 µg kg-1 g-1 applied Se at Chitedze and Mbawa respectively. Residual Se detected as KH2PO4-extractable Se ranged from 0.0029 to 0.106 µg kg-1 g-1 applied Se between sites. Further studies are required to quantify the amount of Se immobilised in the soil pool or lost due to leaching or volatilisation. A further experiment examined how traditional processing procedures for maize grain affected Se concentration in maize flour. At Se fertilisation levels which would increase dietary Se intake to appropriate levels, there was no evidence that traditional milling produced any significant loss of Se from maize flour. Assessment of the contribution of maize to the dietary supply of other nutrients showed that calcium concentration, and hence intake from maize, were very low. Maize grain was low also in K, Cu and Zn but provided a good source of Fe, Mg, Mn and Mo. There is a need to monitor the concentrations of trace metals such as Cd, Co, Ni and Cr as these might exceed the daily allowance and pose a risk to human health.

This project was aimed to elucidate the sub-cellular and molecular regulation of Lipopolysaccharide (LPS) induced muscle protein turnover (protein synthesis (PS) and protein degradation) in two in vitro models, C2C12 murine myotubes and avian primary skeletal muscle cell line. In addition, the effect of natural challenge of chicken with Salmonella serotypes gallinarium or Enteritidis on mRNA expression levels in skeletal muscle was assessed. LPS (1 μgml-1) transiently decreased PS rate by 50% compared with control cells. This effect was mediated via decreased phosphorylation of translation initiation mediators (p70S6K, 4E-BP1 and eIF-4E). This effect was preceded by decreased Akt and mTOR phosphorylation. Although, LPS significantly increased p38, Erk1/2 and their down stream target Mnk1, however, this effect was not sufficient to abolish LPS-induced decreased PS. The role of Akt and MAPKs (p38 or Erk1/2) was verified using specific pathway inhibitors. Inhibition of Akt by LY0294002 (PI3-K/Akt inhibitor) dramatically decreased PS by 80% compared with control cells. Incubation of C2C12 myotubes with SB203580 (p38 inhibitor) or with PD098059 (MEK/Erk inhibitor) alone significantly decreased the PS rate at the 3 h time point by -63 ± 12.48% and -64 ± 5.05% respectively compared with control cells (P < 0.01). In contrast, LPS (1 μgml-1) significantly increased the chymotrypsin-like enzyme at all the time points. This effect was preceded by a significant increase in the IkB-α phosphorylation and nuclear translocation of NF-kB, and significant increase in TNF-α, atrogin-1, MuRF1 and TLR4 mRNA expression. Of note, increased atrogin-1 mRNA is the prominent feature of our septic model. The data presented in chapter 4 and 5 showed that, there is no absolute correlation between the expression levels of atrogens (atrogin-1 and MuRF1) and the overall proteolytic activity in LPS-stimulated C2C12 myotubes. The beneficial roles of the curcumin were evaluated LPS-stimulated C2C12 myotubes for 3 h. Incubation of C2C12 myotubes with LPS (1 μgml-1) and curcumin (25 μM) significantly decreased the LPS-induced chymotrypsin-like enzyme activity. This effect was mediated via decreased p38 and IkB-α phosphorylation. Although, curcumin blocked LPS-induced decreased Akt and p70S6K phosphorylation and significantly increased Erk1/2 phosphorylation, however, curcumin still had no effect on LPS-induced decreased protein synthesis. The effect of the LPS on the muscle protein turnover in the avian primary skeletal muscle was summarised in chapter (7). Incubation of avian primary skeletal cells with LPS (1 μgml-1) for 3 h, significantly decreased the proteasomal activity and increased PS rate. The difference in response to LPS between C2C12 myotubes and avian primary skeletal muscle cells could be attributed to the different incubation parameters mainly the presence of insulin in case of avian primary cells. Finally, the effect of natural challenge of chicken with S. Gallinarum or S. Enteritidis on skeletal muscle mRNA expression was summarised in chapter 9. Natural challenge of chicken with S. Gallinarum or S. Enteritidis had no effect on the expression of many atrophic genes in chicken skeletal muscle (gastrocnemius and pectoral muscle). The data collected from this project showed that, LPS is a strong catabolic stimulus significantly decreased PS along with increased protein breakdown rates in skeletal muscle. This effect was mediated via two main pathways PI3-K/Akt and MAPKs (p38 or Erk1/2) and the cross talk between them is exists. The better understanding of these signalling cascades and their cross talk will be the starting point for developing the appropriate and safe therapeutic intervention in order to decrease the sepsis-induced muscle proteolysis.