Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Background: There has been a dramatic increase in the use of dietary creatine supplementation among sports men and women, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but knowledge of its myocardial effects is limited. Objectives: To investigate the effects of dietary creatine supplementation with and without exercise on 1) basal cardiac function, 2) susceptibility to ischaemia/reperfusion injury and 3) myocardial protein expression and phosphorylation and 4) mitochondrial oxidative function. Methods: Male Wistar rats were randomly divided into control or creatine supplemented groups. Half of each group was exercise trained by swimming for a period of 8 weeks, 5 days per week. At the end of the 8 weeks the open field test was performed and blood corticosterone levels were measured by RIA to determine whether the swim training protocol had any effects on stress levels of the rats. Afterwards hearts were excised and either freeze-clamped for biochemical and molecular analysis or perfused on the isolated heart perfusion system to assess function and tolerance to ischaemia and reperfusion. Five series of experiments were performed: (i) Mechanical function was documented before and after 20 minutes global ischaemia using the work heart model, (ii) A H2O filled balloon connected to a pressure transducer was inserted into the left ventricle to measure LVDP and ischaemic contracture in the Langendorff model, (iii) The left coronary artery was ligated for 35 minutes and infarct size determined after 30 minutes of reperfusion by conventional TTC staining methods. (iv) Mitochondrial oxidative capacity was quantified. (v) High pressure liquid chromatography (HPLC) and Western Blot analysis were performed on blood and heart tissue for determination of high energy phosphates and protein expression and phosphorylation. Results: Neither the behavioural studies nor the corticosterone levels showed any evidence of stress in the groups investigated. Hearts from creatine supplemented sedentary (33.5 ± 4.5%), creatine supplemented exercised rats (18.22 ± 6.2%) as well as control exercised rats (26.1 ± 5.9%) had poorer aortic output recoveries than the sedentary control group (55.9 ± 4.35% p < 0.01) and there was also greater ischaemic contracture in the creatine supplemented exercised group compared to the sedentary control group (10.4 ± 4.23 mmHg vs 31.63 ± 4.74 mmHg). There were no differences in either infarct size or in mitochondrial oxygen consumption between the groups. HPLC analysis revealed elevated phosphocreatine content (44.51 ±14.65 vs 8.19 ±4.93 nmol/gram wet weight, p < 0.05) as well as elevated ATP levels (781.1 ±58.82 vs 482.1 ±75.86 nmol/gram wet weight, p<0.05) in blood from creatine supplemented vs control sedentary rats. These high energy phosphate elevations were not evident in heart tissue and creatine tranporter expression was not altered by creatine supplementation. GLUT4 and phosphorylated AMPK and PKB/Akt were all significantly higher in the creatine supplemented exercised hearts compared to the control sedentary hearts. Conclusion: This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise trained animals by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation. / AFRIKAANSE OPSOMMING: Agtergrond: Die gebruik van kreatien as dieetaanvulling het in die afgelope aantal jaar dramaties toegeneem onder sportlui, sowel as mediese praktisyns wat dit as ‘n terapeutiese middel vir die behandeling van spier- en neurologiese siektes aanwend. Die effekte van kreatien op skeletspier is reeds deeglik ondersoek, maar inligting aangaande die miokardiale effekte van die preperaat is beperk. Doelwitte: Om die effekte van kreatien dieetaanvulling met of sonder oefening ten opsigte van die volgende aspekte te ondersoek: 1) basislyn miokardiale funksie, 2) vatbaarheid vir iskemie/herperfusie besering, 3) proteïenuitdrukking en -fosforilering in die miokardium en 4) mitochondriale oksidatiewe funksie. Metodes: Manlike Wistar rotte is ewekansig in kontrole of kreatien aanvullings groepe verdeel. Helfte van elke groep is aan oefening in die vorm van swemsessies, vir ‘n periode van 8 weke, 5 dae per week blootgestel. Gedrags- en biochemiese toetse is aangewend om die moontlike effek van die swemprotokol op die rotte se stres vlakke te bepaal. In hierdie verband is die oop area toets gebruik, asook bloed kortikosteroon vlakke gemeet deur radioaktiewe immuunessais. Harte is daarna uit die rotte gedissekteer en gevriesklamp vir biochemiese en molekulêre analise, of geperfuseer op die geïsoleerde werkhart perfusiesisteem om sodoende funksie en weerstand teen iskemie en herperfusie beskadeging te bepaal. Vyf eksperimentele reekse is uitgevoer: (i) Meganiese funksie is noteer voor en na 20 minute globale isgemie in die werkhart model; (ii) ‘n Water gevulde plastiek ballon, gekoppel aan ‘n druk omsetter, is in die linker ventrikel geplaas om sodoende linker ventrikulêre ontwikkelde druk (LVDP), asook iskemiese kontraktuur te meet; (iii) Linker koronêre arterie afbinding is vir ‘n periode van 35 minute toegepas en die infarktgrootte bepaal na 30 minute herperfusie deur gebruik te maak van standaard kleuringsmetodes; (iv) Mitochondriale oksidatiewe kapasiteit is gemeet; (v) Hoë druk vloeistof chromatografie (HPLC) en Western Blot analises is uitgevoer op bloed en hartweefsel vir die bepaling van hoë energie fosfate (HEFe), sowel as proteïenuitdrukking en -fosforilering. Resultate: Beide gedragsstudies en kortikosteroonvlakke het geen teken van stres in die betrokke groepe getoon nie. Die groep blootgestel aan kreatienaanvulling en oefening se harte het na iskemie funksioneel swakker herstel as harte van die onaktiewe kontrole groep (18.22±6.2% vs 55.9±4.35%; p<0.01), asook ‘n groter ikgemiese kontraktuur in vergelyking met die onaktiewe kontrole groep ontwikkel (31.63±4.74 mmHg vs 10.4±4.23 mmHg). Daar was geen verskille in infarktgrootte of mitochondriale suurstofverbruik tussen die verskillende groepe waargeneem nie. HPLC analise het verhoogde fosfokreatien (44.51±14.65 vs 8.19±4.93 nmol/gram nat gewig, p<0.05) en adenosientrifosfaat (ATP) bloedvlakke (781.1±58.82 vs 482.1±75.86 nmol/gram nat gewig, p<0.05) in kreatien aanvullings vergelyk met die kontrole groepe getoon. Daar was egter geen meetbare veranderings in HEF vlakke in hartweefsel nie. Gepaardgaande hiermee het kreatienaanvulling geen effek gehad op die uitdrukking va die kreatien transporter nie. In vergelyking met onaktiewe kontrole harte was GLUT4, en fosforileerde AMPK en PKB/ Akt beduidend hoër in harte van geoefende rotte met kreatienaangevulling. Gevolgtrekking: Hierdie data dui daarop dat kreatienaanvulling geen effek op basislyn miokardiale funksie het nie. Kreatienaanvulling het egter die miokardium se weerstand teen iskemiese skade verlaag in harte van rotte blootgestel aan oefening: iskemiese kontraktuur is verhoog en aorta-uitset tydens herperfusie is verlaag. Die presiese meganismes hierby betrokke is egter onbekend en vereis dus verdere studie. / Division of Medical Physiology (University of Stellenbosch), The National Research Foundation and the Harry Crossley Fund for financial support.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.

Hyperthermia can occur in lungs and airways during both physiological and pathophysiological conditions. A previous study carried out in our laboratory showed that hyperthermia activates and sensitizes vagal bronchopulmonary Cfiber afferents, whether this effect is through a direct action of hyperthermia on sensory nerves is not known. This dissertation study was aimed to investigate the thermal-sensitivity of pulmonary sensory neurons, and the roles of thermalsensitive transient receptor potential vanilloid (TRPV) channels. Whole-cell patch-clamp recordings of neurons isolated from nodose/jugular ganglia were applied in the study. Results of this study showed that hyperthermia directly activates pulmonary sensory neurons, and this effect is partially mediated through the TRPV subtype 1 (TRPV1) channel as well as other thermal-sensitive TRPV (2–4) channels. In addition, hyperthermia exerts potentiating effects on responses of pulmonary sensory neurons to TRPV1 activators, but not to non- TRPV1 activators. Furthermore, results obtained in the study of TRPV1-null mice revealed that TRPV1 plays a dominant role in mediating the potentiating effect of hyperthermia on pulmonary sensory neurons, but is only partially involved in the direct activation of these sensory neurons by increasing temperature. These results suggested that the thermal-sensitivity of pulmonary sensory neurons is dependent upon the function of the TRPV1 channel, and TRPV1-mediated sensitization of these sensory neurons may contribute to airway hyperreactivity and augmented reflex responses under hyperthermic conditions.

Since apoE allele status is the predominant Alzheimers disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNPs) in brain apoE receptors represent excellent candidates for association with AD. Therefore, three low density lipoprotein receptor (LDLR) SNPs were evaluated by TaqMan allelic discrimination assays for association with AD and I found that certain haplotypes alter the odds of AD. A SNP within LDLR exon 12, rs688, was identified in silico as neutralizing a putative exon splicing enhancer (ESE). Since LDLR is a major apoE receptor in the brain, I hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, I analyzed splicing patterns in human hippocampus samples and established that this SNP was associated with significantly decreased LDLR exon 12 splicing efficiency when the minor allele T is present in vivo. Lastly, I evaluated whether rs688 associates with AD by genotyping DNA from the Religious Orders Study (ROS) series. The rs688T/T genotype was associated with increased AD odds in males, but not in females, in a dataset consisting of 1,457 men and 2,055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males (recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13- 1.97, uncorrected p=0.005), but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion.

Acid Sphingomyelinase (ASMase) activity has been proposed to mediate LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS-induced TNFα secretion. ASMasedeficient (asm-/-) mice and isolated peritoneal macrophages produce several fold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS. The mechanism for these differences however is not transcriptional but post-translational. The TNFα converting enzyme (TACE) catalyzes the maturation of the 26kD precursor (proTNFα) to the active 17kD form (sTNFα). In mouse peritoneal macrophages, the activity of TACE rather than the rate of TNFα mRNA synthesis was the rate-limiting factor regulating TNFα production. Substantial portion of the translated proTNFα was not processed to sTNFα; instead it was rapidly internalized and degraded in the lysosomes. TACE activity was 2 to 3 fold higher in asm-/- macrophages as compared to asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and SMase. In asm-/- but not in asm+/+macrophages, indirect immunofluorescence experiments revealed distinct TNFα-positive structures in close vicinity of the plasma membrane. Asm-/- cells also had higher number of EEA1-positive early endosomes. Co-localization experiments that involved inhibitors of TACE and/or lysosomal proteolysis suggest that in asm-/-cells a significant portion of proTNFα is sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis it is recycled to the plasma membrane and processed to sTNFα.

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that sense extracellular signal and activate intracellular signaling pathways. The serotonin 5HT2A receptor (or 2AR) is one of the GPCRs coupled to Gq proteins, activating PLC and hydrolyzing PIP2. This hydrolysis causes a diffusion of bound PIP2 away from the channel binding site resulting in G protein-gated inwardly rectifying K+ channel (GIRK) inhibition and a downstream stimulation of Ca2+ release from endoplasmic reticulum stores. Previous experiments have demonstrated that the serotonin 5HTA receptor is a target of serotonergic psychedelic drugs, such as LSD, and partially mediates the action of many atypical antipsychotic drugs. However, the portion responsible for the functional activity and response of these drugs is unknown. The purpose of this study was to functionally characterize four deconstructed analogs of risperidone, an atypical antipsychotic agent, using two assays: by application to 5HT2A receptors in Xenopus oocytes and by calcium epifluorescence imaging in a HEK293 cell line stably expressing 2AR. Our experiments revealed that two analogs, RHV-006 and RHV-008, are partial agonists by themselves and greatly antagonize the effects of serotonin. RHV-006 and RHV-008 contain the piperidine and benzisoxizole ring systems of risperidone. RHV-023 and RHV-026, on the other hand, are more efficacious agonists than RHV-006 and RHV-008 but display a non-antagonistic effect with serotonin. RHV-023 and RHV-026 contain both the piperidine and benzisoxizole ring systems in addition to part of the diazabicyclo ring, thus containing more of risperidone’s structure than RHV-006 and RHV-008.

Type 2 diabetes mellitus is a growing problem across the world and has significant pathological changes associated with it, including diabetic cardiomyopathy, wherein cardiac function is reduced. MicroRNA-21 has been shown to play a role in both the heart and diabetes so it was thought that knocking out miR-21 could have a protective effect on oxidative phosphorylation function in diabetic mice. Subsarcolemmal and interfibrillar mitochondria were isolated from adult male WT, miR-21 KO, db/db, and double knockout mice (db/db and miR-21 KO cross) and evaluated for function. Knocking out miR-21 in diabetic mice showed a restorative effect in Complex I and Complex II function even though it increased ROS production in Complex I and did not show a significant change in MPTP opening. Knocking out miR-21 could potentially restore oxidative phosphorylation function in diabetic patients but at the expense of producing more ROS.

Lentiviral gene therapy has held great promise for treating a wide range of neurological disorders due to its ability to stably integrate into the genome of nondividing cells like neurons, in addition to dividing cells. The nervous system is a complex and highly heterogeneous system, and while a therapeutic intervention may have beneficial effects in one population of cells it may have severe side effects in another. For this reason, specific targeting of lentiviral vectors is crucial for their ultimate utility for research and clinical research use. Two different approaches for focusing the targeting of lentiviral vectors were employed in these studies. The first method involved assessing the effects of vector production strategies on the resulting virus’s tropism both in vivo and in vitro. The changes in vector transduction were determined via flow cytometry on cells in culture and immunohistochemistry following brain injections. Results from these experiments suggest that while the production conditions do impact the vectors efficacy, there is not a distinct effect on their tropism. A unique characteristic of retroviral and lentiviral vectors is their capacity for being pseudotyped, conferring a new tropism on the vector. Native tropisms are generally not specific beyond very broad cell types, which may not be sufficient for all applications. In this case, chimeric targeting molecules can provide an even more refined targeting profile compared to native pseudotypes. The second approach utilizes novel chimeric glycoproteins made from nerve growth factor and the vesicular stomatitis virus glycoprotein. These chimeras are designed to pseudotype lentiviral vectors to target nociceptive sensory neurons for a variety of disorders. While these chimeras were successfully produced as protein, they were misfolded and sequestered in the endoplasmic reticulum and therefore unavailable to produce lentivirus. While neither strategy was completely successful, they do provide interesting information for the design and creation of lentiviral vectors. This research shows that small differences in the steps followed as part of a lentivirus production protocol can greatly impact the resulting vectors efficacy. It also shows that while VSV has been used to create chimeric glycoproteins, not all targeting molecules are suitable for this purpose.

Asthmatics most "at risk of death" from their asthma are those that have severe asthma with a history of severe life threatening attacks (SLTAs). Some asthmatics are poor perceivers of the severity of their asthma. It is not known whether these two groups form a homogeneous population. The aim of this thesis was to establish whether this was the case by comparing the sensitivity of asthmatics classified as severe with a history of severe life threatening attacks, asthmatics without a history of SLTAs, mild and moderate asthmatics, and matched controls to externally added resistive loads during inspiration. It also aimed to establish whether poor sensitivity to externally added resistive loads during inspiration was associated with diminished amplitude or increased latency of scalp potentials evoked by inspiratory occlusion. Two studies were conducted to meet the first aim. The first study tested the applicability of the equal variance normal-normal model of signal detection theory as a descriptor of subjects' performance when discriminating between different levels of externally added resistive loads during inspiration. Twelve subjects rated their capacity to distinguish between pairs of resistive loads ranging from 2-33cmH2O/L/s presented during a single inspiration. Data were collected for 100, 200 and 300 single breath trials. The results showed that the model fitted the data well; that 200 trials provided performance indices that were sufficiently precise to distinguish between relatively low, average and high levels of sensitivity; and that, for the modality tested, respiratory sensation complied with Weber’s Law. The second study applied the validated procedure to test the ability of 25 asthmatics classified either as severe, or mild or moderate, and 25 matched controls to distinguish between pairs of external resistive loads ranging from 2-33cmH2O/L/s presented during a single inspiration. Analysis of the data (ANOVA) showed that there were no significant differences in the ability of subjects to distinguish between resistive loads regardless of whether they were severe asthmatics with or without a history of SLTAs, mild or moderate asthmatics, or matched controls. It was concluded that asthmatics with poor sensitivity to the respiratory sensation tested and asthmatics most "at risk of death" from their asthma were not a homogeneous group, but poor sensitivity coupled with severe asthma, and a history of SLTAs most likely increased the risk of death to that patient. Four studies were conducted to meet the second aim. The first three studies were concerned with determining the reproducibilty of the scalp potential evoked by inspiratory occlusion (designated respiratory event related evoked potentials, RREP) and establishing a reliable recording protocol for the RREP that was relatively free of artefact. The results showed that the RREP was relatively stable over time and the best recording sites were cephalic electrode pairs C3-Cz and C4-Cz (10-20 International System). The fourth study examined the relationship between the ability of 25 asthmatic subjects and their matched controls to distinguish between levels of resistive loads added to inspiration and the latencies and amplitudes of the first positive and first negative components of the RREP. Correlation analysis showed that there was no relationship between sensitivity to added resistive loads during inspiration and RREP component parameters. It was concluded that diminution or absence of the early components of the RREP was not indicative of impaired perception of respiratory sensation though some issues regarding the appropriateness of the analysis still require resolution. / Whole document restricted, but available by request, use the feedback form to request access.

A thin cardiac muscle preparation of rat (ventricular trabeculae) was used in the two studies described in this Thesis. First, I investigated the mechanisms by which volatile anaesthetics decrease cardiac contractility using trabeculae loaded with a fluorescent Ca2+ indicator. Second, I employed confocal laser scanning microscopy to determine the three-dimensional microstructure of perimysial collagen fibres over the range of sarcomere lengths (1.9 - 2.3 µm) in which passive force increases steeply. The volatile anaesthetics halothane (0.23 – 3 %) and isoflurane (0.48 - 4%) produced dose-dependent decreases in the amplitudes of the intracellular Ca2+ transients and twitch force. They also produced changes in cellular autofluorescence, consistent with an increase in the concentration of the reduced forms of nicotinamide adenine nucleotides and flavoproteins. When the fluorescent Ca2+ indicator signals were corrected for changes in cellular autofluorescence, the volatile anaesthetics did not significantly change diastolic [Ca2+]. In the presence of halothane or isoflurane, restoration of Ca2+ transients to control levels, achieved by elevation of external [Ca2+], did not restore peak force. Moreover, maximal Ca2+-activated force, elicited using ryanodine tetani, was reduced by halothane and isoflurane. Thus, the negative inotropic actions of halothane and isoflurane in cardiac muscle reflect both reduced availability of Ca2+ and decreased responsiveness of the contractile system to Ca2+. Reconstructed three-dimensional images showed that perimysial collagen fibres are wavy (as distinct from coiled) cords which straighten considerably as sarcomere length is increased from ~1.85 µm (near-resting length) to ~2.3 µm. These observations are consistent with the notion that the straightening of these fibres is responsible for limiting cardiac sarcomere length to ~2.3 µm.