Newly developed hydrogels, produced by cross linking purified poly ($\gamma$-glutamic acid) ($\gamma$PGA) with dihaloalkane compounds were utilized for slow-release of porcine somatotropin (pST). The polymer was produced by fermentation of Bacillus licheniformis. It was found that the rate of the crosslinking reaction and hydrogel yield were affected by several parameters, such as temperature, initial polymer and base concentrations as well as the type of crosslinkers. Reaction occurred rapidly at a polymer concentration of 40-80 mg/ml and temperature of 60-80$\sp\circ$C without hydrolysis of newly formed networks and polymer backbone. The optimal concentration for the base (NaHCO$\sb3$) was 1-2 mole per mole of carboxylic residues. Analysis of the hydrolysis of the hydrogels as a function of pH indicated that the hydrolysis occurred very slowly at neutral pH, but rapidly in both acidic and alkaline solution. The ester bonds were more sensitive to hydrolysis than the peptide bonds. The biodegradability of the hydrogels and polymer was further examined through enzymatic degradation by three enzymes (Cathepsin B, Pronase E and Trypsin), which were able to gradually cleave both ester and peptide bonds. Swelling of the hydrogel showed dramatic changes when experimental conditions such as the molecular weight of $\gamma$-PGA, nature of the crosslinkers and solutions used for swelling (ionic strength, organic solvent) were varied. The swelling degree was positively correlated with initial molecular weight of $\gamma$-PGA, but inversely correlated with the chain length of the crosslinkers and the crosslinking degree. Increasing the organic solvent or ionic concentration in solutions decreased the gel swelling, which was in agreement with general reports in literature. Porcine somatotropin (pST) is a very unstable hormone in aqueous environment. It was found in this research that the peptide underwent aggregation and decomposition at extreme pH, but was relatively stable in neutral pH. Aggregation was positively correlated with pST and ionic concentrations. The hormone monomer was stabilized to a certain degree in glucose solutions and at low concentration of urea. A quantitative ELISA method was established using anti-pST monoclonal and multiclonal antibodies. The method was simple and specific. The release profiles of $\gamma$-PGA based hydrogels for p-nitroaniline and BSA were studied in vitro. Hydrogels formed with longer chain crosslinkers released materials much slower than shorter chain dihaloalkanes, but unevenly release was observed for macromolecules. The hydrogels formed by dihalopentane was applied for slow-release of pST in vitro and in vivo. Although the aggregation and decomposition of pST were expected in the system, the hydrogel was able to release the hormone for a period of 20-30 days in vitro and in an in vivo rat experiment. In live piglets, the release system increased growth rate by 17% during 8-week experiment. This research demonstrated the potential application of $\gamma$PGA based hydrogels in slow-release systems for bioactive materials, especially macromolecules, such as peptides and proteins.

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<p>Exercise has long been recognized to be an important natural stimulus causing bronchoconstriction in most patients with symptomatic asthma, particularly in children and young adults. In the laboratory, bronchoconstriction caused by exercise is documented by examining a reduction in airway calibre after exercise which is measured as a fall in the forced expired volume in one second (FEV₁). Exercise bronchoconstriction is generally short lived, wearing off within 60 minutes.</p> <p>More than 50% of asthmatic subjects, experience progressively less and less airway narrowing with repeated exercise challenges on the same day. This progressive decrease in exercise bronchoconstriction is considered to be a potentially important airway inhibitory protective effect and is termed exercise refractoriness. O'Byrne and Jones (O'Byrne and Jones, 1986), first demonstrated the likely involvement of inhibitory prostaglandins in exercise refractoriness, when they demonstrated that indomethacin inhibited the effect in asthmatic subjects.</p> <p>The precise mechanism underlying exercise bronchoconstriction is unclear. However, the initiating stimulus may involve either alterations in osmolarity of the epithelial-lining fluid or changes in airway temperature resulting from the inhalation of inadequately conditioned air during exercise or both. These changes cause the release of bronchoconstrictor mediators and the development of bronchoconstriction. The work described in this thesis was undertaken to establish the role of leukotriene (LT) D₄ in the pathogenesis of exercise bronchoconstriction and of histamine-, and LTD₄ induced, inhibitory prostaglandin release in causing exercise refractoriness. The body of research described in this thesis details new findings in relation to LTD₄ and exercise responses in asthmatic subjects. It shows that tachyphylaxis, a potential airway protective mechanism, occurs to repeated challenges with LTD₄ inhalations in asthmatic subjects and that flurbiprofen, a cyclooxygenase inhibitor, blocks this effect implicating inhibitory prostaglandin release in the mechanism. Using a specific LTD₄ receptor antagonist, it shows for the first time, that LTD₄, an important mediator in asthma, is involved in exercise bronchoconstriction in asthmatic subjects. In addition, it demonstrates also that complete cross refractoriness/tachyphylaxis occurs following exercise and LTD₄ stimulation an effect which is also inhibited by flurbiprofen. However, complete cross refractoriness/tachyphylaxis does not occur following exercise and histamine.</p> <p>The conclusion from the work in this thesis is that LTD₄, rather than histamine, plays the more important role in exercise bronchoconstriction and that LTD₄ induced inhibitory prostaglandin release is the cause of exercise refractoriness in asthmatic subjects.</p> / Doctor of Philosophy (PhD)

<p>The purpose of this thesis is to investigate the partitioning of work performed by the expiratory muscles between controlling operating lung volume and increasing expiratory flow, as well as the importance of the expiratory work in determining maximal ventilation during exercise. Healthy subjects were studied at rest and six levels of exercise, including maximum exercise (WRmax). Esophageal and gastric pressure (Pes and Pga) were measured using standard balloon catheter systems. Lung volume was measured using inductance plethysmography, calibrated at each exercise work rate with inert gas dilution and spirometric volumes. The static effects of lung and chest wall recoil on esophageal pressure (Pes-stat and Pel,w respectively) were measured at rest (Pel,w was estimated for some subjects). Work of breathing was measured for both inspiratory and expiratory muscle activity and further partitioned into elastic and resistive work. During the transition from rest to mild exercise (50W), end-expiratory lung volume fell from 53.0 ± 3.7 to 47.6 ± 3.6 (M ± SE) percent of total lung capacity, there was a further significant decrease with 100W exercise to 44.0 ± 3.8 %TLC, at maximal exercise, end-expiratory volume was 40.3 ± 2.5 %TLC. During exercise, expiratory flow was greater than that which could be achieved passively (i.e. due to the combined elastic recoil of the lung and chest wall). At maximal exercise, approximately half of the measured expiratory flow was due to expiratory muscle activity. The extent to which expiratory flow was dependent on expiratory muscle activity was greatest at low lung volumes, where the combined elastic recoil of the lung and chest wall was relatively small. Expiratory esophageal pressure progressively increased during the course of exercise. This increase in pressure was associated with significant increases in expiratory flow at all work rates, including maximal exercise. Expiratory pressure remained effective throughout the majority of expiration (i.e. it did not exceed the point at which starling type resistance prevents further increases in expiratory flow). Transient ineffective pressure toward end expiration may have occurred during maximal exercise. The total work of breathing at a given level of ventilation was in agreement with studies reported in the literature. At rest all work was performed by inspiratory muscles. During mild exercise (50W), expiratory muscle work was significant, accounting for 10.5 ± 2.1 % of the total work of breathing (p < 0.05). During mild to moderate exercise (50-150W) the expiratory work used to lower end-expiratory volume below functional residual capacity exceeded that used for increasing expiratory flow (p < 0.05). At maximal exercise however, the work performed increasing expiratory flow accounted for 71 ± 9.2 % of the total expiratory work and was significantly greater than the work used to lower end-expiratory volume (p < 0.05). Ventilation during maximal exercise could not be achieved without expiratory muscle activity, illustrating the importance of these muscles in lowering end-expiratory lung volume and increasing expiratory flow. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>Asthma is a chronic respiratory disease, characterized by variable airflow obstruction, airway hyperresponsiveness and airway inflammation. Prior to starting this thesis, information on the role of inflammatory cells and cytokines in asthma was limited. The aim of this thesis was to determine the roles of eosinophils, an inflammatory cell believed to be important in asthma, and GM-CSF and IL-3, cytokines shown to regulate eosinophils in vitro, in mild and allergen-induced asthma. Fiberoptic bronchoscopy was performed on each subject and blood, bronchoalveolar lavage and bronchial biopsy samples were obtained. In comparison to non-asthmatics, eosinophil number and activity and GM-CSF levels were increased in asthmatics. GM-CSF levels correlated with eosinophil number and activity and airway responsiveness correlated with eosinophil number and activity and GM-CSF levels. IL-3 was detected in both asthmatics and non-asthmatics, with no difference apparent between the groups. These findings indicate that eosinophils, potentially regulated in vivo by GM-CSF, contribute to airway inflammation in mild asthma. In comparison to the control diluent inhalation, allergen inhalation caused increases in eosinophil number and activity and GM-CSF levels. IL-3 levels did not change after allergen. Eosinophil number and activity correlated with GM-CSF and the severity of the late asthmatic response correlated with the number and activity of eosinophils. These findings indicate that an increase in eosinophil number and activity, possibly due to GM-CSF, contributes to allergen-induced asthma. This thesis has demonstrated cellular and cytokine involvement in mild and allergen-induced asthma. For the first time, the presence of GM-CSF and IL-3 cytokines, at the protein level, have been demonstrated in the airways of subjects with mild and allergen-induced asthma. Eosinophils, potentially regulated by GM-CSF, appear to play an important role in the disordered airway function associated with asthma.</p> / Doctor of Philosophy (PhD)