<p>The pathogenesis of airway hyperresponsiveness, a characteristic feature of asthma, is uncertain. Current evidence suggests a pathogenic role for immature (progenitors) and mature inflammatory cells, particularly eosinophils. The aim of this thesis was to examine the role of eosinophils and bone marrow progenitors in the development of allergen-induced airway hyperresponsiveness in dogs. Random source mongrel dogs demonstrating skin test reactivity to Ascaris suum allergen were studied. Acetylcholine airway responsiveness was measured before and 24 hours after inhalation of Ascaris allergen. For the second and third studies, dogs were pre-treated with inhaled budesonide twice daily for one week prior to allergen challenge. Airway eosinophils were enumerated from bronchoalveolar lavage samples and eosinophil activation was assessed by measurement of eosinophil peroxidase levels in bronchoalveolar lavage fluid. Bone marrow was obtained 24 hours after allergen inhalation with progenitors counted after 8 days in culture. In the first study, dogs that developed airway hyperresponsiveness were found to have a greater number of and more activated airway eosinophils before allergen inhalation than dogs that did not develop airway hyperresponsiveness. In the second study, pre-treatment with inhaled budesonide reduced the number of eosinophils present in the airways before allergen inhalation. This reduction was associated with an attenuation of allergen-induced airway hyperresponsiveness. In the third study, allergen inhalation increased bone marrow progenitor production in dogs that developed allergen-induced airway hyperresponsiveness. Furthermore, the increases in progenitors and airway hyperresponsiveness were reduced by pre-treatment with inhaled budesonide. These results suggest that pre-existing airway eosinophilia influences the development of airway hyperresponsiveness after allergen inhalation. In addition, the results from this thesis provide the first direct evidence that allergen inhalation can increase bone marrow progenitor production and suggest that such increases may contribute to the development of airway hyperresponsiveness in asthma.</p> / Doctor of Philosophy (PhD)

<p>Recommendations regarding fluid replenishment for children who exercise are scarce and based on adult data. The objectives of this thesis were to evaluate and identify factors that would help optimize fluid replenishment for children who exercise in a warm environment. Total sweat electrolyte losses were compared among maturational groups (males and females) exercising in the heat (chapters 2 and 3). Total sweat Na⁺ and Cl⁻ losses, even when corrected for body weight, were greater in young men and women compared to their prepubescent and pubescent counterparts. Children and adults lost similar amounts of K⁺, lactate, and ammonia per kg body weight. Within the same maturational group, there were no gender differences in any of these electrolyte losses. Chapter 4 summarizes the effect of hypohydration (induced by exercise in the heat) on thirst, drink preferences, and the subsequent voluntary rehydration. Thirst and drink preferences were assessed while children dehydrated up to 0.8% of their initial body weight in four separate trials (one flavored drink in each). Thirst intensity increased during dehydration. Grape was the preferred drink during the entire dehydration phase, but its desirability did not increase as much as it did with the orange, apple and water drinks. This is possibly due to a ceiling effect. During recovery, most subjects rehydrated with all drinks, exceeding baseline levels by 0.40% (apple) to 0.76% (grape). The magnitude of overhydration, however, was greater with grape and orange than with water and apple. Chapter 5 summarizes the effect of ingesting various electrolyte drinks on thermoregulation and performance of children who were kept euhydrated while exercising in the heat. Four grape-flavored drinks were tested. One drink was water and the other three had 6% carbohydrate (4% sucrose, 2% fructose) with different [Na⁺] (0, 8.8, 18.5 mmol∙1⁻¹). Rectal temperature, heart rate and performance were similar among trials. A negative Na⁺ deficit occurred with all drinks but in a greater extent with Na⁺-free drinks. Na⁺ intake did not modify plasma [Na⁺] or osmolality. Drink composition had no effect on thirst and stomach fullness sensations, nor did it affect voluntary intake during recovery. The main conclusions of this thesis were: (1) Adults lost more Na⁺ and Cl⁻ from sweat than children. (2) Children's thirst intensity and drink preferences increased in response to minimal dehydration levels. (3) Grape was the most desirable drink. (4) Children spontaneously overhydrated following hypohydration induced by exercise. (5) Compared to water, CHO-electrolyte drinks had no effect on thermoregulation, performance, and perceptual responses of children who were kept euhydrated while exercising in the heat.</p> / Doctor of Philosophy (PhD)

<p>The purpose of this thesis was to quantify spinal deformity (nature and extent), pulmonary impairment (mechanical and gas exchanging properties), disability (working capacity) and handicap (exertional symptoms) in a large group of subjects with idiopathic scoliosis, so that the relationships between these elements could be determined. It was anticipated that these relationships would be more variable (weaker) than is commonly implied. A further aim therefore, was to identify additional factors which contribute to pulmonary impairment, disability and exertional symptoms in these subjects. The influences of respiratory muscle, peripheral muscle and cardiac factors on these relationships were considered to be potentially important.</p> <p>Seventy-nine subjects (M:F 13:66; age 21 (SD 10.1)) with mild-moderate idiopathic thoracic scoliosis (Cobb angle 45° (SD 18.4)) were studied.</p> <p>Pulmonary impairment in the group as a whole was moderate, with the vital capacity being reduced to 79% of predicted (SD 13.6). Angle of scoliosis was one of four features of the spinal deformity which contributed to pulmonary impairment; longer curves (number of vertebrae), higher curve position and loss of the normal thoracic kyphosis also had an additive influence.</p> <p>Disability was significant, with exercise capacity being reduced to 86% (SD 17.4) of predicted; nature and extent of spinal deformity was unrelated to disability. Similarly, there was no direct relationship between pulmonary impairment and the extent of disability.</p> <p>Application of psychophysical symptom rating scale showed that both the intensity of breathlessness and leg effort were increased during exercise, but most subjects stopped exercising due to leg effort rather than breathlessness. This finding focused attention on the relationship of disability to peripheral muscle factors.</p> <p>Leg muscle volume and lean body mass were closely related to maximum working capacity. The relationship of leg muscle volume to maximum oxygen consumption was similar to that previously reported in normal subjects; this suggests that reduced muscle bulk, rather than qualitative muscular differences, is a particularly important contributor to disability. Once between subject differences in muscularity were taken into account, an additional influence of pulmonary impairment on disability was evident.</p> <p>A higher heart rate response during exercise was also found in more disabled subjects, suggesting that cardiac performance may also influence work capacity in this group. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

Hypertension is a disease characterized by increased activity of the Renin Angiotensin System (RAAS) and immune related vascular dysfunction. Angiotensin II (Ang II) is the final effector molecule of the RAAS and has numerous biologic activities that perpetuates vascular remodeling and inflammation. Ang II signaling of inflammatory cells may be due the presence of RAAS components on T lymphocytes. Many studies have shown the importance of pro-inflammatory T cell phenotypes, through cytokine analysis, in hypertensive models. However, the specific characterization of the RAAS on these phenotypes has yet to be determined. We sought to establish the expression of RAAS components on naïve T cell subsets and compare that to changes in expression that may be seen with AngII treatment and anti-CD3/28 stimulation. Here we find that AngII and anti-CD3/28 treatments significantly increase the expression of RAAS components on T cell populations.

Lysyl oxidase (LOX) is the enzyme that mediates cross-linking between collagen and elastin molecules during cardiac remodeling, LOX expression and activity is upregulated in response to the mechanical stresses that occur during hypertension. The aim of this study was to investigate the role of lysl oxidase (LOX) changes in cardiac structure and mechanical function during angiotensin II (AngII) induced hypertension. C57 male mice were given the LOX suicide substrate: β-aminoproprionitrile (BAPN) in their drinking water (300mg/kg/d). On day 14 of BAPN treatment an osmotic pump of AngII (490ng/kg/hr) was implanted. Weekly echo measurements were gathered. 28 days after pump implantation cardiac tissue was harvested for various assays including, LOX enzymatic activity, hydroxyproline quantification, and histological analysis. AngII treated groups showed an increase LOX protein expression, LOX activity, collagen cross-linking, and total collagen content while ECHO results showed an up-regulation aortic velocity time integral (AoVTI) and LV mass and down regulation of E/E-A VTI. When BAPN was co administered with AngII there was an attenuation seen in all these areas. While AngII+BAPN treated mice showed a return of these parameters to normal control levels. These results provide evidence that Angiotensin II-Induced hypertension causes the overexpression of LOX. LOX's overstimulation has a major influence in the cardiac structure and function. Conversely both the structural and mechanical changes can be extenuated with administration of the LOX suicide substrate BAPN.

Clostridium difficile Is the major cause of nosocomial diarrhoea in the UK and is associated with high morbidity and mortality rates. There has been a large increase in cases of C. difficile associated disease (CDAD) in the last decade and It is thought that the emergence of the hypervirulent strain (ribotype 027) has contributed towards this rise. A major factor in the control and prevention of the disease is adequate cleaning of the clinical environment and disinfection, usually with chlorine based agents. However, the spores of C. difficile are highly resistant to many disinfectants.

This research project is concerned with the design, synthesis and development of new phosphodiesterase 5 (PDE5) inhibitors with improved selectivities and lower toxicities. Two series of a 5 member and a 6 member ring fused heterocyclic compounds were designed, and synthesized. By alteration of starting materials and fragments, two virtual libraries, each is consisted of close to hundred compounds, were obtained successfully. The screening of sexual stimulation activity with rabbits demonstrated both groups of compounds were able to stimulate rabbit penile erection significantly. The following toxicity studies revealed 2-(substituted-sulfonylphenyl)-imidazo [1,5-a]-1,3,5-triazine-4-(3H)-one group possessed an unacceptable toxicity with oral LD50 about 200mg/kg; while 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group showed an acceptable toxicity with oral LD50 over 2000mg/kg. The continued bioactivity studies showed yonkenafil, the representative of 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group, has a better selectivity towards PDE5 and PDE6 than sildenafil and a better overall profile of sexual stimulation on animals than sildenafil. Chronic toxicity studies of yonkenafil further confirmed yonkenafil did not cause any serious side effect and damage on animal models and most actions were explainable. Based on evidences of the above studies, yonkenafil were recommended to enter clinical trials by the regulation authority of China, SFDA. Currently yonkenafil has been through the Phase I clinical trials and ready to progress into Phase II. Hopefully, yonkenafil will provide an alternative to the ED patients in the future.

This research primarily focused on identifying the formulation parameters which control the efficacy of liposomes as delivery systems to enhance the delivery of poorly soluble drugs. Preliminary studies focused on the drug loading of ibuprofen within vesicle systems. Initially both liposomal and niosomal formulations were screened for their drug-loading capacity: liposomal systems were shown to offer significantly higher ibuprofen loading and thereafter lipid based systems were further investigated. Given the key role cholesterol is known to play within the stability of bilayer vesicles. the optimum cholesterol content in terms of drug loading and release of poorly soluble drugs was then investigated. From these studies a concentration of 11 total molar % of cholesterol was used as a benchmark for all further formulations. Investigating the effect of liposomc composition on several low solubility drugs, drug loading was shown to be enhanced by adopting longer chain length lipids. cationic lipids and. decreasing drug molecular weight. Drug release was increased by using cationic lipids and lower molecular weight of drug; conversely, a reduction was noted when employing longer chain lipids thus supporting the rational of longer chain lipids producing more stable liposomes, a theory also supported by results obtained via Langmuir studies· although it was revealed that stability is also dependent on geometric features associated with the lipid chain moiety. Interestingly, reduction in drug loading appeared to be induced when symmetrical phospholipids were substituted for lipids constituting asymmetrical alkyl chain groups thus further highlighting the importance of lipid geometry. Combining a symmetrical lipid with an asymmetrical derivative enhanced encapsulation of a hydrophobic drug while reducing that of another suggesting the importance of drug characteristics. Phosphatidylcholine liposornes could successfully be prepared (and visualised using transmission electron microscopy) from fatty alcohols therefore offering an alternative liposomal stabiliser to cholesterol. Results obtained revealed that liposomes containing tetradecanol within their formulation shares similar vesicle size, drug encapsulation, surface charge. and toxicity profiles as liposomes formulated with cholesterol, however the tetradecanol preparation appeared to release considerably more drug during stability studies. Langmuir monolayer studies revealed that the condensing influence by tetradecanol is less than compared with cholesterol suggesting that this reduced intercalation by the former could explain why the tetradecanol formulation released more drug compared with cholesterol formulations. Environmental scanning electron microscopy (ESEM) was used to analyse the morphology and stability of liposomes. These investigations indicated that the presence of drugs within the liposomal bilayer were able to enhance the stability of the bilayers against collapse under reduced hydration conditions. In addition the presence of charged lipids within the formulation under reduced hydration conditions compared with its neutral counterpart. However the applicability of using ESEM as a new method to investigate liposome stability appears less valid than first hoped since the results are often open to varied interpretation and do not provide a robust set of data to support conclusions in some cases.

Salt formation has extensively been studied as a strategy to improve drug solubility but it has not been explored as a strategy to improve mechanical properties. A better understanding of which factors of the solid state can have an influence in the mechanical properties of pharmaceutical powders can help to optimise and reduce cost of tablet manufacturing. The aim of this study was to form different series of amine salts of flurbiprofen, gemfibrozil and diclofenac and to establish predictive relationships between architectural characteristics and physicochemical and mechanical properties of the salts. For this purpose, three different carboxylic acid drugs were selected: flurbiprofen, gemfibrozil and diclofenac, similar in size but varying in flexibility and shape and three different series of counterions were also chosen: one with increasing bulk and no hydroxyl groups to limit the hydrogen bonding potential; a second one with increasing number of hydroxyl groups and finally a third series, related to the latter in number of hydroxyl groups but with different molecular shape and flexibility. Physico-chemical characterization was performed (DSC, TGA, solubility, intrinsic dissolution rate, particle size, true density) and mechanical properties measured using a compaction replicator. Strained molecular conformations produce weaker compacts as they have higher energy than preferred conformations that usually lie close to energy minimums and oppose plastic deformation. It was observed that slip planes, which correspond to regions of weakest interaction between the planes, were associated with improved plasticity and stronger compacts. Apart from hydrogen bonds, profuse van der Waals forces can result in ineffective slip planes. Salts displaying two-dimensional densely hydrogen bonded layers produced stronger compacts than salts showing one-dimensional networks of non-bonded columns, probably by reducing the attachment energy between layers. When hydrogen bonds are created intramolecularly, it is possible that the mechanical properties are compromised as they do not contribute so much to create twodimensional densely bonded layers and they can force molecules into strained conformations. Some types of hydrogen bonding network may be associated with improved mechanical properties, such as type II, or R (10) 3 4 using graph-set notation, versus type III, or R (12) 4 8 , columns. This work clearly demonstrates the potential of investigating crystal structure-mechanical property relationship in pharmaceutical materials.

The work presented in this thesis falls into three main categories: The design and synthesis of potential anti-tuberculosis drugs targeting a mycobacterial esterase and the enzyme dUTPase; synthesis and anti-microbial SAR studies on a set of carboxamidrazones; synthesis and anti-microbial SAR studies on a set of thiosem icarbazones.