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The objective of this research is to investigate both mathematically and numerically the effects of vascular geometry upon the cellular dynamics in the endothelium and its consequence in the localisation of atherosclerosis. It is widely accepted that the formation of atherosclerotic plaques preferentially occurs at specific locations in the vasculature, such as arterial branches and bends. It has also been observed that, at the sites of plaque formation, the physiological functions of the vascular endothelium are impaired due to a defect in the production mechanisms of or diminished activities of endothelial nitric oxide (NO). From these observations, a correlation between the vascular geometry, which is effected via local haemodynamic forces, and local bioavailability of endothelial NO has been postulated. The research areas that have been involved in the investigation of atherosclerosis's localisation in the past, haemodynamics, medicine, calcium dynamics, NO kinetics and endothelial cell biology, have been studied individually, and there appears to be no integrated model to date that allows investigation of coupled haemodynamic and cellular mechanism applied in physiologically realistic model geometries. An integrated numerical model that includes these mechanisms will be developed in this research, which will lead to a further, more comprehensive understanding of the pathogenesis of atherosclerosis.

This manuscript presents an investigation and application of the medical radiographic technique of Dynamic Contrast-enhanced Computed Tomography with an emphasis on its application to the measurement of tissue perfusion using the techniques of CT Perfusion. CT Perfusion was used in association with Fluoro- Deoxy Glucose Positron Emission Tomography (FDG PET) to investigate altered blood flow due to the angiogenic effects of tumour in the clinical setting of medical imaging for cancer diagnosis and staging. CT perfusion, CT enhancement and Doppler ultrasound studies were compared in a series of patient studies performed for the assessment of metastatic liver disease. There was good correlation between all techniques for the arterial phase but not between Doppler measurements of the portal phase and any CT measurement. A new method was developed for quantifying CT perfusion and enhancement values, the Standardised Perfusion Value (SPV) and the Standardised Enhancement Value (SEV). The SPV was shown to correlate with FDG uptake in a series of 16 patient studies of lung nodules, an unexpected and potentially important finding that if confirmed in a larger study may provide an additional diagnostic role for CT in the assessment of lung nodules. Investigation of a commercially available package for the determination of CT Perfusion, CT Perfusion GE Medical Systems, was undertaken in a small series of brain studies for assessment of acute stroke. This data set showed the technique to positively identify patients with non-hemorrhagic stroke in the presence of a normal conventional CT, to select those cases where thrombolysis is appropriate, and to provide an indication for prognosis. An investigation of the accuracy and cost-effectiveness of FDG PET in solitary pulmonary nodules using Australian data was carried out. FDG PET was found to be accurate, cost saving and cost effective for the characterisation of indeterminate solitary pulmonary nodules in Australia. This work was expanded to include the impact of quantitative contrast enhancement CT (QECT) on the cost-effectiveness of FDG PET. The addition of QECT is a cost effective approach, however whether QECT is used alone or in combination with FDG PET will depend on local availability of PET, the cost of PET with respect to surgery and the prior probability of malignancy. A published review of CT perfusion, clinical applications and techniques, is included in the body of the work. Dynamic contrast-enhanced CT and FDG PET were used to investigate blood flow, expressed as SPV, and metabolic relationships in non-small cell lung cancers (NSCLC) of varying size and stage. A significant correlation between SPV and FDG uptake was only found for tumours smaller than 4.5 cm2. Blood flow-metabolic relationships are not consistent in NSCLC but depend on tumour size and stage. Dynamic contrast-enhanced CT as an adjunct to an FDG study undertaken using integrated PET-CT offers an efficient way to augment the assessment of tumour biology with possible future application as part of clinical care. In summary the work has developed a method for standardizing the results of dynamic contrast-enhanced CT and investigated its potential when applied with FDG PET to improve the diagnosis and staging of cancers.

Cette thèse, intitulée « L’exploration fonctionnelle de repos et à l’exercice dans l'hypertension pulmonaire », est le résultat de différents travaux de physiologie clinique effectués dans le cadre des maladies vasculaires pulmonaires (MVP).La première partie de cette thèse est consacrée à l’étude de la diffusion des gaz dans l’hypertension artérielle pulmonaire (HTAP), l’hypertension pulmonaire thromboembolique chronique (HTP-TEC) et la maladie veino-occlusive pulmonaire (MVOP). Nous nous sommes particulièrement intéressé à l’étude de la double diffusion du monoxyde de carbone (DLCO) et d’azote (DLNO), permettant notamment la mesure du volume capillaire (Vc) et de la diffusion membranaire (Dm). Nous avons mis en évidence une diminution significative de ces paramètres dans les trois maladies et plus particulièrement dans la MVOP. Cette pathologie a la particularité de présenter un rapport DLNO/DLCO significativement plus élevé, évocateur de l’hémangiomatose capillaire associée à l’atteinte veinulaire. De plus, nous avons mis en évidence que le volume capillaire et la diffusion membranaire sont corrélés aux paramètres de capacités à l’exercice, dont la consommation en oxygène (VO2), dans un groupe de patients avec HTAP indemnes de toute comorbidité. Enfin, nous avons mis en évidence une corrélation significative entre la Dm et la survie dans l’HTAP.La deuxième partie de cette thèse fait référence a l’étude du cathétérisme cardiaque droit durant l’exercice. Dans un premier travail, nous avons collaboré à la proposition d’une nouvelle définition de l’hypertension pulmonaire à l’exercice (HTPe). Dans une cohorte rétrospective de patients investigués au Centre National de référence de l’Hypertension Pulmonaire, nous avons montré que l’association de résistances pulmonaires totales supérieures à 3 UW au pic de l’effort avec une pression artérielle pulmonaire moyenne (PAPm) supérieure à 30 mmHg présentent des valeurs de sensibilités et de spécificités élevées pour le diagnostic de patients avec MVP ou cardiopathie gauche sans hypertension pulmonaire de repos. Nous avons confronté ces résultats à une cohorte historique de sujets sains et trouvé des valeurs similaires. Dans un autre travail, nous avons analysé la concordance entre trois différentes définitions utilisées jusqu’à présent dans la littérature concernant l’HTPe, en montrant qu’elles ne sont pas équivalentes. Enfin, nous avons collaboré à une étude hémodynamique des sujets présentant une PAPm au repos comprise en 21 et 24 mmHg. Nous avons démontré que ces sujets présentent une hémodynamique d’effort anormale et qu’il existe une association entre la PAPm de repos et la fréquence de l’ HTPe.Dans la troisième partie de la thèse, nous avons contribué à l’étude de la distensibilité vasculaire dans les MVP. Dans une première étude rétrospective, nous avons collaboré à la validation du modèle alpha de distensibilité vasculaire. Nous avons démontré dans une cohorte de sujets avec maladies vasculaires pulmonaires que le coefficient alpha permet de détecter précocement des anomalies de la circulation pulmonaire chez des sujets présentant une PAPm de repos inférieure à 25 mmHg. De plus, dans une étude prospective, nous avons également étudié une nouvelle technique diagnostic : le signal Doppler pulmonaire, basé sur la pulsatilité de la paroi vasculaire, et donc reflétant la distensibilité des vaisseaux. Nous avons démontré que la performance diagnostique de cette technique était tout à fait acceptable en comparaison avec le cathétérisme cardiaque droit.Enfin, dans la dernière partie, nous avons exploré les différences de réponses à l’effort entre un groupe de patients avec HTAP et un groupe de patients avec une forme distale d’HTP-TEC. Nous avons observé une réponse ventilatoire différente, notamment avec une inefficience ventilatoire plus importante liée à un effet espace mort, expliquant une partie de la limitation fonctionnelle. / This thesis, entitled « Rest and exercice functional investigation in pulmonary hypertension » is the sum of several physiological and clinical works in the field of pulmonary vascular diseases (PVD).The first part of this thesis is focused on the study of gas diffusion in pulmonary arterial hypertension (PAH), chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary veno-occlusive disease (PVOD). We have a special interest in the study of combined nitric oxide diffusion (DLNO) and carbon monoxide diffusion (DLCO) technic, which allows the measurement of capillary blood volume (Vc) and membrane diffusion (Dm). We have demonstrated a significant decrease of those parameters in the three forms of PH, specifically emphasized in PVOD. Furthermore, DLNO/DLCO ratio was remarkably increased in PVOD, suggesting a component of capillary hemangiomatosis associated to venular remodeling. Moreover, we have demonstrated that Vc and Dm were correlated to exercice capacity, such as peak VO2, in a group of PAH patients without confunding factors. Finally, we have found a significant correlation between Dm and survival in PAH.The second part of this thesis was about right heart catheterization during exercice. In this work, we have collaborated to the proposition of a new definition of exercice pulmonary hypertension (ePH). In a retrospective cohort of patients investigated in the French National Center for Pulmonary Hypertension, we have shown that total pulmonary resistance above 3 WU during exercise associated with a mean pulmonary arterial pressure (mPAP) above 30 mmHg displayed high sensitiviy and specificity for diagnosis of patients with PVD or left heart disease without resting pulmonary hypertension. We have compared these results with an historical cohort of healthy subjects and found similar results. In an other work, we have analyzed the concordance between three different definitions of ePH recently used in the litterature. We have demonstrated that they were not equivalent. Finally, we have collaborated to an haemodynamic study of subjects with resting mPAP between 21 and 24 mmHg. We have demonstrated that these subjects displayed abnormal haemodynamics during exercice. We have also demonstated that a progressive rise of resting mPAP was associated with an increase of the frequence of ePH.The third part of this manuscript is consacrated to the study of pulmonary vascular ditensibility in PVD. In a retrospective study, we have collaborated to the validation of the alpha model which allow the estimation of a distensibility coefficient. We have demonstrated in a cohort of patients with PVD that alpha allowed the early detection of abnormal pulmonary circulation in subjects with normal mPAP. In a prospective study, we have used a new technic called the lung Doppler signal (LDS) to diagnose PH, which investigates the pulsatile distensibility of pulmonary vascular walls. We have demonstated that LDS diagnostic performance was acceptable in comparison with the gold standard right heart catheterization.Finally, in the last part of the thesis, we have investigated differences of exercice profile between patients with PAH and distal CTEPH. We have demonstated an abnormal ventilatory pattern, with hyperventilation and marked ventilatory inefficiency in distal CTEPH. This is related to the increased physiological deadspace, which explained a part of fonctional limitation in distal CTEPH.

This thesis details a 1-d model of a lymphatic vessel, developed from a model by Reddy. Some additions to the modelling techniques were found to be necessary to prevent numerical phenomena not found in experiment. Furthermore the details of the wall and valve were important to the mechanics of the system. This developed model presents flow characteristics which are not represented in the existing lumped parameter or 1-d models of the lymphatic system. Additional terms allow more realistic representation of some modes of flow such as those occurring during collapse. The model was validated using Poiseuille flow calculations and experimental work. Features found in experiment were reproduced in the model. Such as the shark tooth shape of the radius time graph. A study of the sensitivity of the model to experimental parameters was performed. Features that increased flow included: increased compliance of the vessel, a larger diameter, amplitude of contraction or frequency, or a faster contraction wave. A lumped parameter model, relating the radius directly to the pressure, was investigated but this did not reproduce flow features such as the shark tooth shaped radius with time relationship or the radius peak at the beginning of a contraction or passive relaxation of the vessel. In the 1-d model the time constant of this passive relaxation increased with the magnitude of contraction. This value may have physiological relevance.

This study examined the effects of rapid and uncompensable core temperature (Tc) acquisition on cerebral oxygenation and haemodynamics, cerebral neural function, decision-making, and rate and magnitude salivary cortisol appearance. Fourteen male subjects (mean age, 33.6 ± 12.1 years) performed an incremental treadmill exercise test to a termination criterion in a control session (CON) and an experimental session (PPE). The incremental treadmill exercise test protocol included an initial 5-minute stage at 3.5 mph and a 0% grade, the second stage was 5-minutes at 3.5 mph at 4% grade, the third stage was 50-minutes at 3.5 mph and an 8% grade, and the final stage was 1-hour at 3.5 mph and a 12% grade. The Instrumentation included a near-infrared spectroscopy (NIRS) monitor, MUSE EEG monitoring system, Equivital integrated physiological monitoring system, Tc capsules, and salivary cortisol oral swabs and ELISA kit for salivary analysis. Important physiological results were significant differences in the physiological strain index (PSI) at all common points of measurement. Important cerebral oxygenation and haemodynamics results were a plateau in left-side prefrontal cortex (PFC) HbO2 and tHb at roughly Tc 38°C in both CON and PPE, 80% of TTT in CON, and 60% of TTT in PPE. Additionally, there was higher left-side PFC activation during PPE as indicated by a significant decrease in TSI % from start to end of exercise and double the decrease in TSI % per minute in PPE when compared to CON. There were no significant differences during the CON session. An analysis of frontal theta EEG power results showed a significant decrease when comparing pre- and post-exercise values during a Go/No-go test in PPE (F(1,13) = 6.069, p ≤ 0.05)). There was also a significant difference when evaluating incorrect responses between pre- and post-exercise values in PPE (F(1,13) = 12.785, p ≤ 0.01)); these differences were not observed during CON. There was also a difference in the rate of cortisol appearance (CON = 0.002 µg dL-1 min-1; PPE = 0.018 µg dL-1 min-1). In the PPE condition, mean cortisol values between start of exercise and the measurement point associated with Tc 38°C and between the start and end of exercise during PPE were significantly different (F(1,13) = 22.71, p ≤ 0.01). Lastly, there was a significant difference between magnitude of cortisol values at the termination between CON and PPE. These data suggest that rapid and uncompensable Tc acquisition during PPE caused an altered cerebral oxygenation and haemodynamic response in the left-side PFC when compared to CON. The left PFC could be working harder to prevent fatigue in PPE. This could have implications for cognitive processes during and/or following exercise in the heat while wearing PPE. These data also suggest rapid and uncompensable Tc acquisition results in decreased cognitive control. This could have implications for individuals whose occupation requires PPE and critical decision making while experiencing rapid Tc heat storage. Lastly, these results show a difference between PPE and CON in regards to rate and magnitude of salivary cortisol appearance, potentially affecting individuals chronically exposed to acute heat stress. Increased acute cortisol concentration decreases anabolic response, cognitive performance, and mood states. The chronic effects of increased cortisol concentration are many: largely related to atherosclerosis development and subsequent cardiovascular disease. Additional issues include anthropometric, endocrine, metabolic, and haemodynamic disturbances. This study makes a strong argument for the rate of thermal acquisition factor. CON and PPE differences in PSI at all measurement points provides justification and support for the changes in other variables. Rapid and uncompensable Tc acquisition needs to be taken into account, as it potentially puts the lives of employees who wear PPE and those around them at risk. / Graduate

This study compared uncomplicated canine babesiosis (CB) with various grades of experimentally induced normovolaemic anaemia (EA) and the physiological state (controls) in the dog using Doppler variables of the abdominal aorta and splanchnic vessels. There were 14 cases of uncomplicated CB, and each EA and control group had 11 Beagles. There were significant increases in the abdominal aorta, cranial mesenteric artery, coeliac artery and main portal vein blood flow velocities, and in portal blood flow during EA when compared with the physiological state. There were significant reductions in resistance indices of the same vessels, and those of the hilar splenic artery. Changes were most notable during severe EA and less consistent during lower grades of anaemia. Significant changes in renal haemodynamics were found only during severe acute EA. In contrast to other abdominal vessels, left renal artery pulsatility and resistive indices increased significantly during EA while those of the interlobar artery remained unchanged. There was a significant increase in peak systolic velocity and significant decrease in end diastolic velocity. Renal artery time-averaged mean velocity (TAVmean) (P < 0.008) and end diastolic velocity (P = 0.041) were significantly lower than the corresponding variables of the aorta, cranial mesenteric and coeliac arteries during the EA but not the physiological state. The TAVmean ratio was significantly (P< 0.014) lower during EA when compared to the physiological state, and significantly (P< 0.004) lower than the corresponding variables of cranial mesenteric or coeliac artery during the EA but not the physiological state. There was a striking similarity between CB and EA regarding haemodynamic change patterns of Doppler variables in all vessels. In spite of this, renal resistive indices during CB were significantly higher than during EA and the physiological state. The similarity between CB and EA haemodynamic patterns is attributed to anaemia while significant differences between them may be attributable to pathophysiological factors peculiar to CB. This observation supports the view that CB impairs renal circulation through certain mechanisms such as capillary blockage with sequestered red blood cells. Doppler ultrasonography is a useful technique for clinical investigation of haemodynamics in CB and related diseases. / Thesis (PhD (Veterinary Science))--University of Pretoria, 2005. / Companion Animal Clinical Studies / unrestricted

Limb haemodynamic responses during heat-stress and the importance of local vs. central temperature-sensitive mechanisms towards their regulation remain poorly understood, both at a whole-limb level and within individual tissues (i.e. skeletal muscle and skin). The aims of this thesis were to 1) investigate the haemodynamic responses at rest to direct thermal challenges both at a local level and during progressive elevations in systemic heat stress, 2) to ascertain the contribution of local vs. systemic mechanisms towards this regulation, and 3) to investigate the same responses during single-legged small-muscle mass exercise to near maximal levels. Results from Chapters 4 and 5 characterised the haemodynamic responses during isolated cooling and heating of the arm and leg, and provided evidence of alterations in both skin and skeletal muscle blood flow controlled solely through local temperature-sensitive mechanisms. While local cooling led to modest decreases in limb blood flow due to decreases in mean blood velocity alone, increases during heating occurred as a result of an increased antegrade flow, a diminished retrograde flow, and a reduction in the potentially pro-atherogenic oscillatory shear index. In Chapter 6, whole-body heating with isolated single leg cooling displayed the continued control of limb blood flow via local thermosensitive mechanisms alone, as cooled leg blood flow remained unchanged despite significant elevations in core temperature, cardiac output, and opposing heated leg blood flow. Furthermore, elevations in heated leg V̇O2 suggested a possible metabolic contribution to the observed skeletal muscle hyperaemic response. During incremental single-legged knee-extensor exercise to near maximal levels, blood flow was determined by a combination of metabolic workload and local tissue temperatures, regardless of whether systemic heat stress was present. Chapter 7 revealed that whilst skin and muscle blood flow in the leg continued to increase in line with local temperatures to levels of severe heat stress, rapid cooling of the leg when hyperthermic resulted in a similar reverse response in muscle tissues only, as skin blood flow remained elevated despite the abolition of high skin and subcutaneous temperatures. In addition, evidence was provided that moderate levels of whole-body heat stress provided little additional benefit to anti-atherogenic shear profiles than that experienced during isolated limb heating alone. Taken together, these findings suggest that local thermosensitive mechanisms dominate limb blood flow control during direct rapid heating in humans both at rest and during small muscle mass exercise, but that underlying central mechanisms may act to maintain flow when local temperatures are reduced in the face of high core temperatures.

Activation of A3 adenosine receptors has been shown to protect the myocardium from ischaemia reperfusion injury in a number of animal models. The PI3K - AKT and MEK1/2 - ERK1/2 cell survival pathways have been shown to play a critical role in regulating myocardial ischaemia reperfusion injury. In this study we investigated whether the A3 adenosine receptor agonist 2-CL-IB-MECA protects the myocardium from ischaemia reperfusion injury, when administered at reperfusion or post reperfusion and whether the protection involved the PI3K – AKT or MEK 1/2 – ERK1/2 cell survival pathways. In the Langendorff model of ischaemia reperfusion injury isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. Administration of 2-CL-IB-MECA (1nM) at reperfusion significantly decreased infarct size to risk ratio compared to non-treated ischeamic reperfused control hearts. This protection was abolished in the presence of the PI3K inhibitor Wortmannin or MEK1/2 inhibitor UO126. Western blot analysis determined that administration of 2-CL-IB-MECA (1 nM) upregulated ERK1/2 phosphorylation. In the adult rat cardiac myocyte model of hypoxia/reoxygenation cells underwent 6 hours of hypoxia and 18 hours of reoxygenation. Administration of 2-CL-IB-MECA (1 nM) at the onset of reoxygenation significantly decreased cellular apoptosis and necrosis. Administration of 2-CL-IB-MECA (1nM) in the presence of the Wortmannin or UO126 significantly reversed this anti-apoptotic effect and anti-necrotic effect. Our data further showed that 2-CL-IB-MECA protects myocytes subjected to hypoxia/reoxygenation injury via decreasing cleaved-caspase 3 activity that was abolished in presence of the PI3K inhibitor but not in the presence of the MEK1/2 inhibitor UO126. Administration of 2-CL-IB-MECA (100nM) at the onset of reperfusion also significantly decreased infarct size to risk ratio in the ischaemic reperfused rat heart compared to controls that was reversed in the presence of Wortmannin or Rapamycin. This protection was associated with an increase in PI3K-AKT / p70S6K / BAD phosphorylation. 2-CL-IB-MECA (100nM) administered at reoxygenation also significantly protected adult rat cardiac myocytes from hypoxia/reoxygenation injury 28 in an anti-apoptotic and anti-necrotic manner. This anti-apoptotic/necrotic effect of 2-CL-IB-MECA was abolished in the presence Wortmannin. Furthermore, that this protection afforded by 2-CL-IB-MECA (100nM) when administered at reoxygenation was associated with a decrease in cleaved caspase 3 activity that was abolished in the presence of the Wortmannin Interestingly, postponing the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion significantly protected the isolated perfused rat heart from ischaemia reperfusion injury in a Wortmannin and UO126 sensitive manner. This protection was associated with an increase in AKT and ERK1/2 phosphorylation. Administration of the A3 agonist 2-CL-IB-MECA 15 or 30 minutes after the onset of reoxygenation significantly protected isolated adult rat cardiac myocytes subjected to 6 hours of hypoxia and 18 hours of reoxygenation from injury in an anti-apoptotic/necrotic manner. This anti-apoptotic was abolished upon PI3K inhibition with Wortmannin or MEK1/2 inhibition with UO126. The anti-necrotic effect of 2-CL-IB-MECA when administered 15 or 30 minutes post-reperfusion was not abolished in the presence of the inhibitors. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after reoxygenation was associated with a decrease in cleaved-caspase 3 activity that was abolished in the presence of Wortmannin but not in the presence of the MEK 1/2inhibitor UO126. Collectively, we have demonstrated for the first time that administration of 2-CL-IB-MECA at the onset of reperfusion protects the ischaemic reperfused rat myocardium from lethal ischaemia reperfusion injury in a PI3K and MEK1/2 sensitive manner. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion of reoxygenation also significantly protects the isolated perfused rat heart from ischaemia reperfusion injury and the adult rat cardiac myocyte from hypoxia/reoxygenation injury in an anti apoptotic / necrotic manner. Furthermore, that this protection is associated with recruitment of the PI3K-AKT and MEK1/2 – ERK1/2 cell survival pathways.

3-nitrotyrosine (3NT), a stable end product arising from the interaction of proteins and reactive nitrogen species such as peroxynitrite, is produced during periods of oxidative stress. 3NT is, therefore, of interest as a potential biomarker in a variety of disease states where oxidative stress is known to be involved in the pathology, for example intermittent claudication. The aim of this thesis was to develop sensitive and specific immunoassays to assess the levels of 3NT in plasma samples from claudicants and to investigate the protein nitration profile. Clinical data and plasma samples were collected from claudicant (n=33) and control (n=6) subjects. Analysis of data confirmed the difficulty of using parameters such as ankle brachial index (ABI) in diagnosis, supporting the need for investigations into potential biomarkers. Development of indirect and competitive ELISAs using electrochemically nitrated bovine serum albumin as the standard revealed that the detection of 3NT was dependent on the antibody being able to access the 3NT-residues within the protein. Various denaturing conditions and different types of microtitre plate were utilised during development. Initially the presence of 3NT in claudicant or control whole plasma samples could only be detected using dot blot immunodetection. Affinity purification techniques for the fractionation of the plasma proteins were therefore applied. Subsequently, 3NT-containing plasma proteins were found to be present in all of the claudicant and control samples using the developed competitive ELISA. Proteomic analysis of the 3NT-affinity purified samples, using MALDI-MS and LC-ESI-MS/MS, confirmed the presence of human serum albumin, serotransferrin and apolipoprotein A1 and A2 precursors within those protein bands staining immunopositive for 3NT on SDS-PAGE gels. The identification of apolipoprotein A1 within 3NT-immunopositive bands confirms previous reports suggesting the oxidative modification of HDL may contribute to the link between inflammation and the pathology of atherosclerosis.