An interruption of the blood supply to the brain, as occurs during ischemic stroke, results in a rapid decline of ATP levels and a subsequent loss of neuronal function and viability. Under physiological conditions the brain reuses ATP degradation metabolites, such as hypoxanthine, via the purine salvage pathway, to restore its ATP pool. However, the massive degradation of ATP during ischemia results in the accumulation and loss of diffusible purine metabolites and thereby leads to a reduction in the post-ischemic ATP pool size, leaving the brain more vulnerable to secondary ischemic insults (recurrent strokes) and less able to deploy reparative mechanisms. The aim of this study was to improve the recovery of post-ischemic ATP levels by enhancing the purine salvage pathway, with substances that are already known to be tolerated in humans. Using acute hippocampal rat brain slices, I found that 1 mM Ribose (Rib) and 50 μM Adenine (Ade), two main metabolites of the purine salvage pathway, significantly increased the tissue ATP levels under basal conditions. Rib/Ade pre-treatment results in accelerated decline of synaptic transmission after onset of oxygen/glucose deprivation (OGD), due to increased adenosine release. However, this intervention does not delay the onset of anoxic depolarisation, or improve the recovery of synaptic transmission after prolonged ischemic periods. Pre-treatment of brain slices with 1 mM creatine, which increases phosphocreatine levels and thereby buffers the rapid decline of ATP levels upon energy shortage, significantly delays the onset of AD and helps to improve the recovery of synaptic transmission. By using cultured cerebellar granule cells, for more protracted studies on cell viability after OGD, I show that addition of Rib/Ade after ischemia helps to improves cell viability. Therefore my results suggest that both, delaying the decline of ATP upon onset of OGD (pre-treatment with creatine), or enhancing the post-ischemic recovery of ATP (post-treatment with Rib/Ade) are useful strategies to improve cell survival and function after in vitro ischemia.

The maritime environment is one of the harshest and most dangerous in which to work, particularly for professional users (e.g. military and rescue services). The aims of this research were to: a) investigate the effects of marine transits conducted in high speed craft (HSC) on the subsequent physical performance of military passengers, b) assess mitigation techniques to limit any degradation in human physical performance.

Load carriage (19.3 Jan, 280 min, 31 kg load) in the field elicited a cardiovascular strain of 72 ± 5 %HRmax and caused neuromuscular impairment (7 ± 8% decrease in jump height (P0.05). The differences between conditions appeared to relate to changes in substrate oxidation, neuromuscular impairment and reduced mechanical efficiency. Neuromuscular function was measured in study 5 at 0, 24, 48 and 72 hours after the treadmill walking conditions described in study 4. L W caused no changes in neuromuscular function. Isometric knee extension force decreased immediately after LWLC (15 ± II %, P<0.05) and DWLC (16 ± 17 %, P<0.05), recovering by 72 hours for DWLC only. VA decreased after LWLC only, douhlet half relaxation time and 20:50Hz decreased after LWLC and DWLC (P<0.05). LWLC and DWLC were associated with decreases in isokinetic peak torque of knee, trunk extensors and flexors and shoulder flexors (P<0.05) with complete recovery by 72 hours. Regression models developed in study 6 indicated that participants with the most efficient metaholic and neuromuscular performance during 120 minutes of load carriage (25 kg backpack) on a level gradient had high body mass and high absolute V O, max with strong trunk, shoulder and knee flexors. To examine nutritional interventions to reduce the metabolic cost during, and neuromuscular impairment following, load carriage, participants consumed Placebo [PLA], Carbohydrate [CRO] or Whey Protein [PRO] beverages during 120 minutes ofload carriage and for three days of recovery. During load carriage (study 7), there were no differences in V 0,. RER, or EMG RMS between conditions at minute 5 (P>0.05). The increase in V 0, between 5 and 120 minutes was less during CRO (8 ± 5 %) than PLA (14 ± 6 %, P<0.05) or PRO (17 ± 4 %, P<0.05). RER decreased between minutes 5 and 120 during PLA and PRO only. Peak RMS did not change over time in In. rectus femoris, m. vastus latera/is, m. semitendinosus, and m. biceps femoris. Attenuation in V 02 drift during CRO could not entirely be accounted for by higher carbohydrate oxidation rates. During recovery, neuromuscular function was measured 0, 24, 48 and 72 hours after load carriage (study 8). There was no difference between PLA, CRO or PRO in the decrease in peak torque of knee and trunk extensors and flexors at 0 h. Peak torque of the knee extensors and flexors returned to pre-exercise values at 24 h during PRO followed by CHO at 48 hand PLA at 72 h (P>0.05). Trunk flexors returned to pre exercise value at 24 h for CHO and PRO but 48 h for PLA (P>0.05). Faster recovery of neuromuscular function was probably due to CRO and PRO improving protein balance, thus enhancing repair of muscle tissue damaged during exercise. In conclusion, load carriage increases V 0, and V O, drift whilst walking and causes neuromuscular impairment, which can last for up to 72 hours following exercise. Nutritional supplements can reduce V O,drift and improve the time course of recovery of neuromuscular function.

Therapeutic antibodies are highly versatile macromolecules that can be engineered to bind and inhibit a target with high specificity. Unfortunately, the cell membrane is impenetrable to antibody reagents, thus limiting their use almost entirely to extracellular targets. Expanding the application of therapeutic antibodies to intracellular targets is an exciting concept that could have a huge impact on how intracellular protein-protein interactions involved in diseases can be modulated. The modification of therapeutic antibodies with Cell Penetrating Peptides (CPPs) can enable cellular penetration, however, no general approach to modifying antibodies with CPPs has been developed that allows for systematic optimisation of both the in vivo and cell penetrating properties. In this study, neutralising single-chain variable fragment antibodies (scFvs) have been isolated from naïve scFv libraries using antibody phage display that are specific to the model intracellular targets Bcl-2 and Bcl-xL. Lead scFvs showed potent inhibition of these proteins in an in vitro assay with IC50 values of <10 nM being calculated, which is superior to the small molecule Bcl-2/xL inhibitor ABT-737. The lead anti-Bcl-xL scFv was conjugated to the CPPs octa-arginine, HIV Tat49-57 or Antp52-58. These peptides were synthesised to possess either an N-isobutyryl cysteinyl or N-maleimidopropionyl moiety and a C-terminal lysine residue, allowing for site-specific conjugation to an unpaired cysteine residue introduced to the scFv construct and regioselective introduction of 5-carboxyfluorescein to the CPP, respectively. Live-cell confocal microscopy showed that the scFv-octa-arginine conjugate possessed superior cell entry capabilities compared to the scFv-Tat49-57 and scFv-Antp52-58 conjugates. Further studies using a panel of cancer cell lines are required to determine if the anti-Bcl-xL scFv-octa-arginine conjugate can induce apoptosis through inhibition of cellular Bcl-xL. Additonally, a novel approach to controlling the cell penetrating properties of the CPP octa-arginine has been developed. It was demonstrated that carbamate protection of octa-arginine’s guanidine functionality effectively inhibited its cell entry capabilities. Moreover, esterase-labile acyloxymethyl carbonyl (AM) protecting groups were utilised to protect the guanidine functionality of octa-arginine, inhibiting its cell entry capabilities. In a HPLC based assay it was demonstrated that the AM protected octa-arginine was deprotected by pig liver esterase, suggesting that in vivo deprotection could be achieved by serum esterases. The controlled unmasking of octa-arginine is predicted to increase its circulation time and reduce non-specific tissue uptake in vivo, potentially making this CPP more suitable for in vivo applications. The methodologies utilised for the preparation of scFv-CPP conjugates and developed for the controlled unmasking of octa-arginine in this study will allow for optimisation of the cell penetrating and in vivo properties of this promising class of macromolecular therapeutic, thus providing a gateway to unlocking the immense potential of therapeutic intracellular antibodies.  .

Human axillary odour is formed by the action of Corynebacteria or Stephyloccui bacteria on odourless axilla sections. Several groups have identified axillary odorants, including 3-methyl-2-hexanoic acid (3M2H) and 3-hydroxy-3-methyl-hexenoic acid (HMHA), and how they are pre-formed and bound to amino acid conjugates. However, there is currently a lack of LC-MS methodologies and no reported NMR methods, that are required to further identify the non-volatile constituents, which would provide further information to allow understanding of the underlying physiological biochemistry of malodour. This work has incorporated a three-pronged approach. Firstly, a global strategy, through the use of NMR and LC-MS, provided a complementary unbiased overview of the metabolite composition. Metabolites were identified based on acquired standards, accurate mass and through the use of in-house or online databases. Furthermore, spectra of biological samples are inherently complex, thus, requiring a multivariate data analysis (MVDA) approach to extract the latent chemical information in the data. Secondly, semi-targeted LC-MS/MS methodologies has been used to identify metabolites with a common structural core (i.e. odour precursors) and provide structural information for the reliable identification of known and unknown metabolites. Finally, a targeted LC-MSIMS method provided an increase in specificity and sensitivity to accurately quantify known metabolites of interest (odour precursors). Initially, all methodologies were developed through the use of either an artificial sweat matrix (global strategy) or through the use of synthetic standards (semi-targeted or targeted strategy). The sample complexity was then increased by applying the methodologies to an ASG5 apocrine cell line, in order to provide further knowledge into apocrine cell metabolism and to identify whether there could be any potential male or female differences due to differences in circulating hormones. Changes in the cell metabolism were identified, and both the NMR and LC-MS data could differentiate between control, tamoxifen- and β-estradiol-treated. However, it is difficult to attribute these changes to specific pathways, as these hormones or the vehicle used (ethanol) are likely to produce a ripple effect across the cell's metabolism. Nonetheless, NMR spectroscopy quantified 25 metabolites with lactate being the most abundant at 19.1 mM, while HILIC-MS could detect a range of lipids, nucleotides, amino acids, fatty acids and vitamins. The methodologies were then applied to human apocrine sweat collected from six volunteers across five days. NMR spectroscopy was able to identify 25 and quantify 19 metabolites, with lactate being the most abundant at 13.2 mM. LC-MS/MS readily identified 12 amino acid conjugates with HMHA being the most abundant. Furthermore, a possible 20 unidentified conjugates were detected (LC-MSIMS semi-targeted methodologies) as well as putatively identifying 473 metabolites (LC-MS global methodologies). MVDA techniques such as principal component analysis (PCA) illustrated that intra-individual variation was greater than inter-individual variation, as well as secretions from both the left and right arm being consistent with one another. Moreover, MVDA illustrated the complementary nature of both NMR and MS, as the data acquired with the two types of instrumentation showed the same trends, even though these trends were based on different subsets of metabolites. The work presented herein, has successfully used a number of analytical technologies to investigate metabolite content of human apocrine sweat. It has been shown that a number of complementary techniques and multivariate analysis can provide a valuable insight into the underlying physiology of malodour.

Adipose tissue development is regulated by a complex interaction between the local actions of insulin, glucocorticoids and insulin like growth factors (IGFs). A series of experiments was undertaken in which the normal development of individual adipose tissue depots and their development following periconceptional under- and overnutrition, formula feeding and juvenile obesity was investigated in sheep. Expression and abundance of glucocorticoid receptor (GR), 11β-hydroxysteroid dehydrogenases (11β-HSDs), insulin receptor, p85 subunit of phosphatidylinositol 3-kinase (p85), glucose transporter 4 (Glut4), insulin like growth factor (IGF) 1 and 2 and their receptors (IGF-R) were measured as markers of sensitivity to glucocorticoids, insulin and IGFs in individual adipose tissue depots. It was found that during early postnatal life omental adipose tissue grows faster than other depots. In all investigated groups there were marked differences in the expression of all investigated genes between adipose tissue depots. No effect was found of periconceptional nutrition on expression of the investigated genes. Weight of the mother prior to conception was negatively associated with omental GR and 11β-HSD1. Free fatty acid levels at 4 months of age were related to omental and subcutaneous 11β-HSD1 expression. Perirenal expression of IGF1R at 4 months was negatively correlated with perirenal and subcutaneous adipose tissue mass. IGF1R expression correlated with IR and GR expression. Formula feeding resulted in reduced expression of Glut4 and increased 11β-HSD1 expression. A combination of formula feeding and juvenile obesity resulted in a redistribution of adipose tissue in favour of the perirenal depot. Obesity per se resulted in a reduction of the expression of all genes and proteins examined. We have shown significant differences in markers of tissue sensitivity to the actions of insulin, glucocorticoids and insulin like growth factors between different adipose tissue depots in the body, highlighting the importance of examining those depots individually in future studies.

The work in this thesis involves two separate projects. The first project involves the behavioural measurement of auditory thresholds in the ferret (Mustela Putorius). A new behavioural paradigm using a sound localisation task was developed which produces reliable psychophysical detection thresholds in animals. Initial attempts to use the task failed and after further investigation improvements were made. These changes produced a task that successfully produced reliably low thresholds. Different methods of testing, and the number of experimental trials required, here then explored systemically. The refined data collection method was then used to investigate frequency resolution in the ferret. These data demonstrated that the method was suitable for measuring perceptual frequency selectivity. It revealed that the auditory filters of ferrets are broader than several other species. In some cases this was also broader than neural estimates would suggest. The second project involved the measurement of neural data in the Guinea Pig (Cavia porecellus). More specifically the project aimed to test the ability of the primary auditory cortex (AI) to integrate high frequency spatial cues. Two experiments were required to elucidate these data. The first experiment demonstrated a relationship between frequency and space, though these data proved noisy. A second experiment was conducted, focussing on improving the quality of the data this allowed for a more quantitative approach to be applied. The results highlighted that though AI neurons are responsive over a broad frequency range, inhibitory binaural interactions integrate spatial information over a smaller range. Binaural interactions were only strong when sounds in either ear were closely matched in frequency. In contrast, excitatory binaural interactions did not generally depend on the interaural frequency difference. These findings place important constraints on the across frequency integration of binaural level cues.

The auditory nerve conveys spectral information, reflecting the location of maximum vibration along the frequency-tuned basilar membrane, and also information reflecting the timing of peaks in the vibrations at each location. Debate continues as to whether pitch is extracted based on the available temporal or spectral representations of tonal stimuli, or both. The aim of the current work was to determine the roles of temporal and spectral harmonicity cues for pitch, under important conditions for understanding speech in multi-talker environments. Two such conditions are the temporal integration of pitch and pitch-based segregation of sound sources. Pitch information in running speech changes over time. Therefore, pitch-extraction mechanisms must be able to follow these changes to enhance intelligibility, particularly when listening in modulated backgrounds such as competing speech. However, the temporal resolution of pitch has received little attention. In the first three chapters, the roles of temporal and spectral cues on the temporal resolution of pitch extraction were determined by measuring pitch-domain temporal modulation transfer functions and gap-detection thresholds. Temporal resolution was shown to be unaffected by the availability of spectral cues, and similarly unaffected by the overall pitch strength of the stimulus. However, the system was much more sluggish in response to changes in pitch information in stimuli presented in high-frequency regions compared to low-frequency regions. This processing strategy may reflect the progressive loss of accurate temporal information towards higher frequencies imposed by transduction processes in the auditory periphery. To understand speech in noise, the ability of the auditory system to integrate pitch information over long periods is equally important as its ability to detect rapid changes in pitch. In Chapter 4, discrimination thresholds for pitch value and pitch strength were measured in the presence and absence of spectral cues as a function of stimulus duration. The assumption was that discrimination thresholds would reach asymptote at the stimulus duration corresponding to the length of the pitch integration window. However, the pitch-strength discrimination data revealed integration was only limited by the stimulus duration, suggesting that this task may reflect the rate of decrease in the variance of internal pitch-value and pitch-strength estimates with increasing stimulus duration, but not the total integration capacity of the system. In multi-talker environments, listeners have to process multiple simultaneous tonal sound sources. The fifth study showed that temporal interactions between simultaneous tonal stimuli could aid detection in the absence of spectral cues. In contrast, harmonic resolvability is thought to be a prerequisite for pitch-based simultaneous grouping. However, data from a second experiment showed that listeners were able to perceptually segregate tonal sounds in the absence of spectral cues.

The work presented in this thesis consists of 4 manuscripts, focussed on characterising the distribution of mutant huntingtin protein in transgenic and knock-in mouse models of Huntington’s disease. The mouse lines showed a different expression level of mutant huntingtin across the different time points. In the R6/1 mice, the inclusions were present and widespread from 3.5 weeks of age. In the YAC128 mice, inclusions were not present until 15 months of age, but then developed rapidly throughout the brain. In the HdhQ92 and HdhQ150 mice, intra nuclear inclusions (NIIs) were apparent at 10 and 5 months of age, respectively, and spread anterior to posterior and ventral-dorsal directions. In this thesis, the study has shown no increase in GFAP immunoactivity in the striatum of each mice line. However we detected a small increase in GFAP immunoactivity in the cortex of transgenic mouse models. With electron microscopy, we observed ultrastructural pathology with vacuolization, uneven cell membrane and degenerated mitochondria in these mouse lines along side with the presence of inclusions. Each mouse line showed different levels of degeneration such as YAC128 and HdhQ92 mice exhibited apoptitic neurons, whereas HdhQ150 mice has shown signs of necrosis. The results demonstrate that each of the mouse lines studied has a unique pattern of development of neuropathology. Inclusion formations may not be pathogenic per se, but may be representative of the dysfunctional neuronal populations that underpin the functional disturbances found in each of these mouse lines. Electron microscopy shows different cell death morphology in these mouse lines.

Osteoarthritis (OA) is a highly prevalent, debilitating disease affecting many joints including the knee. Despite the involvement of several tissues, it is believed that the articular cartilage is the primary site of pathogenesis in humans. Within this study, a new scoring system of OA was devised, incorporating the articular cartilage and underlying bone, aimed at providing a more comprehensive means of grading the severity of tissue damage. We examined changes progressively from mild to severe and were able to deduce from the scoring system that bone changes may precede those of the overlying cartilage. Immunohistochemistry was used to assess stem cell marker expression, proliferation and progressive changes within the extracellular matrix of sectioned osteochondral plugs, however no distinct pattern of change could be extrapolated, highlighting the variable nature of this taxing disease. Previous studies have demonstrated the presence of a sub-population of chondroprogenitor cells present in normal hyaline cartilage. We demonstrated in this study that a similar group of cells reside in osteoarthritic articular cartilage. We were able to isolate and expand clonally derived primary cell lines to beyond 50 population doublings whilst maintaining a chondrogenic phenotype, and demonstrated the tri-lineage potential of these cells. That said, a significant amount of variation was observed and it was, therefore, postulated that there may be a smaller cohort of viable cells within this sub-population isolated from osteoarthritic cartilage. A preliminary study was also carried out comparing chondroprogenitors from normal articular cartilage to those isolated from OA tissue. Heterogeneity was again encountered, suggesting that there was a group of OA chondroprogenitors with similar characteristics to the normal cells, which differed from the other less metabolically active cells. This finding was agreeable with the aforementioned postulation. Data from our preliminary integration study was promising as we demonstrated the potential for using these chondroprogenitor cells in combination with other cells whilst achieving successful integration. However, further work is necessary to distinguish between the cell lines with the potential for integration from those that lacked this ability, thereby eliminating the heterogeneity. The presence of viable chondroprogenitor cells in OA tissue challenges the dogma that the tissue is irrecoverable, and opens the scope for regenerative medicine using resident progenitor cells. This is an exciting prospect that could significantly contribute to articular cartilage repair.