Cellular communication requires the transport of chemical messengers between intracellular compartments and from cell to cell. The regulated exocytosis of a secretory vesicle at the plasma membrane involves the merger of two bilayers, with markedly different lipid composition, within a millisecond time scale. The spatial and temporal control of the protein and lipid complement at these fusion sites is essential. A highly conserved family of proteins are known to drive this fusion event; SNAP-25 and syntaxin-1 (t-SNAREs) associate at the plasma membrane in a 1:1 stoichiometry to provide a binding site for the vesicle-membrane protein synaptobrevin (v-SNARE). The formation of this complex and subsequent fusion requires accessory proteins for efficient calcium-triggered exocytosis; which of these proteins facilitate the initial attachment of vesicle to the plasma membrane prior to fusion is still under debate. Specific sites for vesicle fusion have been proposed and the organisation of lipids and proteins at these fusion sites has been extensively investigated with limited spatial and temporal resolution; however the presence of raft-forming lipids at these sites as well as the arrangement of SNARE proteins at the molecular level is still under contention. The data presented within this thesis aims to elucidate the protein and lipid environment at the fusion site using super-resolution microscopy and advanced vesicle tracking. Under diffraction-limited microscopy the t-SNAREs are visualised as 200 nm homogenous clusters; however I have used single molecule localisation microscopy to reveal a more complex heterogeneous molecular arrangement. Quantification of lipid order exclusively at the plasma membrane provided insight into the influence of cholesterol-induced lipid arrangement on SNAP-25 localisation. In addition the t-SNARE interaction was investigated using TCSPC-FLIM identifying two lipid-order-dependent conformations in distinct clusters at the plasma membrane. Extensive vesicle tracking at optimum sampling rates demonstrated the ‘sampling’ behaviour of LDCVs and allowed characterisation of vesicle fusion sites. In summary I find that vesicles exhibit preference for residence and probably fusion at regions of plasma membrane with a low t-SNARE density; these proteins appear to exert control over exocytosis by adopting alternative conformations that are under cholesterol-induced regulation.

Cystic fibrosis (CF) lung disease is characterised by early airways infection and inflammation, chronic suppuration, frequent infective exacerbations and an increased influx of acute, and chronic inflammatory cells. The inflammatory process involves activation of many cell types including neutrophils, macrophages and epithelial cells, and leads ultimately to the development of progressive respiratory failure and death. Accurate assessment of the inflammatory process is a crucial part of disease monitoring and should allow appropriate evaluation of therapeutic interventions so as to maximize control of the respiratory sequelae of the disorder. Lung function markers such as FEV1 are insensitive and indirect. Direct but invasive methods such as fibreoptic bronchoscopy and biopsy are limited in application, repeatability and safety. Non-invasive methods of assessment are, therefore, attractive. Exhaled Breath Gases, Exhaled Breath Condensate and Induced Sputum provide potential for such measures. These techniques are safe, simple, repeatable and could assess all airways and can be used in children as young as 6 years. We hypothesised that biomarkers of inflammation in Cystic Fibrosis Lung Disease are measurable in samples collected noninvasively, and can be developed into clinically useful assays. These assays would have the ability to reflect the level of inflammation in the CF lungs as well as holding the potential to act as surrogate markers of CFTR function. Methods Non-invasive markers of inflammation in Cystic Fibrosis lung disease Methods. Exhaled breath gases, exhaled breath condensate, bronchoalveolar lavage fluid and induced sputum were investigated using a number of analysis techniques to identify the markers which best discriminated CF from non CF subjects. Analysis techniques used were electrochemical cells, chemiluminescene, ELISA, EIA, ion selective probes and mass spectrometry. Results Markers found to discriminate CF from non CF subjects were EBC pH and ammonium, and 38 proteomic markers were found in induced sputum. 21 proteomic markers were found in bronchoalveolar lavage fluid. One biomarker has been identified with confidence, Calgranulin A. Discussion A large component of the work of this thesis was focussed on exhaled breath condensate. Two markers, pH and Ammonium were different between the CF and control groups. The measurement of EBC pH and ammonium as markers of inflammation should be used in future gene therapy trials as they are cheap, quick and simple to perform Using clean techniques free from contamination, no proteins are repeatedly detectable in EBC using highly sensitive SELDI techniques. This technique reflects the highest sensitivity of any available proteomics instrument and therefore until new technologies become available, it would be incorrect to assay any proteins in EBC. The induced sputum proteomics study identified 38 independent markers of CF lung inflammation Therefore, sampling by collection of induced sputum should be used in gene therapy trials. The endpoints should be assessed by a combination of SELDI as an endpoint and by ELISA where this is available. The marker Calgranulin is likely to report on neutrophil recruitment to the lung. It is anticipated that this will be a sensitive marker of inflammation in the lung and it also has the potential to report on successful of gene transfer as it is raised in heterozygote carriers as well as homozygotes with CF. Therefore, the non-invasive technique induced sputum coupled to proteomic analysis would have the ability to reflect the level of inflammation in CF subjects and may also report on CFTR function.

Cancer-induced bone pain (CIBP), due to bony metastases, is a major clinical problem, significantly reducing quality of life in cancer patients. Current therapies often provide inadequate analgesia or unacceptable side effects. The aim of this thesis was to characterise behaviours of a preclinical model of CIBP and test novel analgesic interventions in this model. A secondary aim was to investigate the involvement of the N-methyl-D-Aspartate (NMDA) receptors and TRP channels (TRPM8, TRPV1 and TRPV4) in CIBP. Investigation of CIBP in a preclinical model may lead to better pain management in CIBP patients. The results presented here demonstrate that this model of CIBP develops behaviours that may be indicative of mechanical allodynia, thermal sensitivity, movement-evoked pain, ongoing pain and spontaneous pain. This suggests that this model reflects the clinical condition of CIBP, where patients suffer from constant background pain with spontaneous and movement-related breakthrough pain. In this study it was found that radiotherapy significantly attenuated movement-evoked pain and thermal sensitivity to 20°C and 40°C. XRT also significantly reduced anxiety and risk assessment behaviours (grooming behaviour and number of protected stretch attends) compared to untreated CIBP. Duloxetine attenuated CIBP-induced mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain, whereas S,S-reboxetine attenuated thermal sensitivity to 40°C but did not effect CIBP-induced mechanical allodynia or movement-evoked pain. In addition, CB 65 attenuated movement-evoked pain and thermal sensitivity to 40°C. A single dose of gabapentin did not attenuate CIBP-induced mechanical allodynia, thermal sensitivity to 40°C or movement-evoked pain. These studies confirm that the CIBP model shows characteristics and pharmacological sensitivities consistent with known and predicted mechanisms and validate it as a useful model for assessing potential new treatments proposed for use in patients. Behavioural results suggest that NMDA receptors containing the NR2A subunit are involved in CIBP-induced movement-evoked pain. This suggests that NR2A antagonists may be useful for treating CIBP-induced movement-evoked pain. Additionally, results show that there is increased expression of NR2A in the laminae I, II and III in the dorsal horn of the spinal cord. XRT treated animals also showed increased expression of NR2A in laminae I and II. The selective involvement of NR2A in CIBP is different to other chronic pain states, for example, neuropathic pain states that appear to involve the NR2B subunit. The TRPV1 antagonist AMG 9810 did not attenuate mechanical allodynia, thermal sensitivity to 40°C or movement-evoked pain. Interestingly, the TRPM8 agonist icilin attenuated movement-evoked pain, which suggests that icilin might be useful in the treatment of movement-evoked pain. The TRPV4 antagonist RN 1734 attenuated mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain in CIBP. This suggests RN 1734 may be useful in the treatment of mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain in CIBP. Results show that the expression of TRPV4 is increased in DRG ipsilateral to the cancerbearing tibia. In conclusion, these results show that the preclinical model of CIBP investigated in this thesis is suitable for testing novel analgesic interventions. This thesis identified some useful targets for the analgesic treatment of CIBP and results suggest that many different mechanisms contribute to CIBP. A point to consider is that any robust effective treatment may need to target all (or at least several) of these mechanisms.

Electrospun biomaterial scaffolds can be engineered to support the neural differentiation of induced pluripotent stem cells. As electrospinning produces scaffolds consisting of nano or microfibers, these topographical features can be used as cues to direct stem cell differentiation. These nano and microscale scaffolds can also be used to deliver chemical cues, such as small molecules and growth factors, to direct the differentiation of induced pluripotent stem cells into neural phenotypes. Induced pluripotent stem cells can become any cell type found in the body, making them a powerful tool for engineering tissues. Therefore, a combination of an engineered biomaterial scaffold with induced pluripotent stem cells is a promising approach for neural tissue engineering applications. As detailed in this thesis, electrospun scaffolds support the neuronal differentiation of induced pluripotent stem cells through delivering the appropriate chemical cues and also presenting physical cues, specifically topography to enhance neuronal regeneration. This thesis seeks to evaluate the following topics: multifunctional electrospun scaffolds for promoting neuronal differentiation of induced pluripotent stem cells, neuronal differentiation of human induced pluripotent stem cells seeded on electrospun scaffolds with varied topographies, and controlled release of glial cell-derived neurotrophic factor from random and aligned electrospun nanofibers. / Graduate / nkhadem@uvic.ca

It is well documented that engaging in resistance exercise can lead to further improvements in endurance performance. Whilst, not fully understood, it is speculated that increased motor unit recruitment, improved muscle coordination and enhanced utilisation of stored elastic energy after resistance-based exercise improves exercise economy. Nevertheless, while prolonged exposure to resistance training improves endurance performance in the long-term, a consequence of such training when unaccustomed is the appearance of exercise-induced muscle damage (EIMD). Exercise-induced muscle damage is well known to affect athletic performance requiring muscular strength and power; however, its effects on markers of endurance exercise are unclear. Therefore, the aim of this thesis was to investigate the effects of EIMD on endurance performance, with an emphasis on the physiological (oxygen uptake; , minute ventilation; ), metabolic (blood lactate; [La]), perceptual (rating of perceived exertion; RPE) and kinematic (stride length; SL, stride frequency; SF) responses during sub-maximal endurance exercise.

The notion of airplane stability and control being a balancing act between stability and control has been around as long as aeronautics. The Wright brothers’ first successful flights were born of the debate, and were successful at least in part because they spent considerable time teaching themselves how to control their otherwise unstable airplane. This thesis covers four aspects of handling for large transport aircraft: large size and the accompanying low frequency dynamics, the way in which lifting surfaces and control system elements are modelled in flight dynamics analyses, the cockpit feel characteristics and details of how pilots interact with them, and the dynamic instability associated with Pilot Induced Oscillations. The dynamics associated with large transport aircraft are reviewed from the perspective of pilot-in-the-loop handling qualities, including the effects of relaxing static stability in pursuit of performance. Areas in which current design requirements are incomplete are highlighted. Issues with modelling of dynamic elements which are between the pilot’s fingers and the airplane response are illuminated and recommendations are made. Cockpit feel characteristics are examined in detail, in particular, the nonlinear elements of friction and breakout forces. Three piloted simulation experiments are described and the results reviewed. Each was very different in nature, and all were designed to evaluate linear and nonlinear elements of the cockpit feel characteristics from the pilot’s point of view. These included understanding the pilot’s ability to precisely control the manipulator itself, the pilot’s ability to command the flight path, and neuro-muscular modelling to gain a deeper understanding of the range of characteristics pilots can adapt to and why. Based on the data collected and analyzed, conclusions are drawn and recommendations are made. Finally, a novel and unique PIO prediction criterion is developed, which is based on control-theoretic constructs. This criterion identifies unique signatures in the dynamic response of the airplane to predict the onset of instability.

published_or_final_version / Speech and Hearing Sciences / Master / Master of Science in Audiology

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The focus of this dissertation is the introduction of surface-induced dissociation (SID) into a commercially available quadrupole time-of-flight mass spectrometer as an alternative ion fragmentation method. The performance of the SID device was characterized and its applications were demonstrated by dissociating peptides, proteins, inorganic salt clusters and non-covalent protein complexes. The SID setup allowed direct comparison of SID with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of Q-TOF instrumentation, including extended mass range, high sensitivity and resolution. With the SID setup installed, no significant reduction of the ion transmission was evident. SID fragmentation patterns of peptides are, in general, similar to CID, with slight differences in the relative intensities of immonium ions, backbone cleavage b- versus y- type ions, and y- versus y-NH3 ions. This suggests enhanced accessibility to high energy/secondary fragmentation channels with SID. SID studies on cesium iodide clusters (CsI) also revealed that SID deposits more internal energy.The utility of mass spectrometric methods to probe the gas phase cyclization process was studied with [D-Ala2]-Leucine Enkephalin amide. This peptide showed prominent formation of the [M-NH3]+ ion which is believed to be the linear b5 ion with a C-terminal oxazolone structure. Other fragments in the spectra indicate that the linear b5 ion undergoes cyclization, subsequent ring opening and further dissociation to rearranged fragments that cannot be explained by the initial sequence. The similarities between the cyclic and b5-ion from the linear peptide indicated the formation of a heterogeneous ion population and this is further supported by gas-phase H/D exchange experiments. An ion funnel interface to improve ion transmission at high pressures was tested in a custom built quadrupole-surface-quadrupole instrument. The ion transmission efficiency for selected bio-molecules such as YGGFLR, insulin chain-B, ubiquitin and cytochrome c showed to approach almost 90%, with the funnel interface installed. The ion transmission efficiency was effected by several factors including: the size of the analyte, the DC gradient, the RF frequency, and the RF amplitude. The higher fragmentation efficiencies for SID in the presence of the funnel interface indicated higher internal energy deposition for the funnel interface.

Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.