Thesis (M.I.P.H.)--School of Public Health, University of Sydney, 2004. / "This treatise is submitted in partial satisfaction of the requirements for the Degree of Master of International Public Health (Hons), University of Sydney". Bibliography: leaves 10-15.

Thesis (M.I.P.H.)--University of Sydney, 2004. / Title from title screen (viewed 14 May 2008). Submitted in fulfilment of the requirements for the degree of Master of International Public Health (Hons) to the School of Public Health. Includes bibliographical references. Also available in print form.

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The lymphatic system is responsible for several vital roles in human body, one of which is maintaining fluid and protein balance. There is no central pump in the lymphatic system and the transport of fluid against gravity and adverse pressure gradient is maintained by the extrinsic and intrinsic pumping mechanisms. Any disruption of the lymphatic system due to trauma or injury can lead to edema. There is no cure for lymphedema partly because the knowledge of the function of the lymphatic system is lacking. Thus, a well-developed model of the lymphatic system is crucial to improve our understanding of its function. Here we used a lumped parameter approach to model a chain of lymphangions in series. Equations of conservation of mass, conservation of momentum, and vessel wall force balance were solved for each lymphangion computationally. Due to the lack of knowledge of the parameters describing the system in the literature, more accurate measurements of these parameters should be pursued to advance the model. Because of the difficulty of the isolated vessel and in-situ experiments, we performed a parameter sensitivity analysis to determine the parameters that affect the system most strongly. Our results showed that more accurate estimations of active contractile force and physiologic features of lymphangions, such as length/diameter ratios, should be pursued in future experiments. Also further experiments are required to refine the valve behavior and valve parameters.

<p>Thrombosis is a common complication of chemotherapy for breast cancer patients. However, the specific mechanisms by which chemotherapy agents modulate these hemostatic pathways are not well understood. In this thesis, we investigated the mechanism(s) by which chemotherapy agents can upregulate procoagulant pathways (tissue factor (TF), phosphatidylserine exposure, and cell-free DNA (CFDNA) release) and impair the protein C (PC) anticoagulant pathway. We examined the effects of chemotherapy agents doxorubicin, epirubicin and the cyclophosphamide metabolite acrolein on cell surface procoagulant activity. We found that treatment of endothelial cells with the chemotherapy drugs increased phosphatidylserine exposure and TF activity on treated endothelial cells, blood monocytes and/or smooth muscle cells. This corresponded to an increase in thrombin generation on chemotherapy-treated cells exposed to recalcified, defibrinated plasma. We also found that found that doxorubicin and epirubicin can increase CFDNA release from breast cancer chemotherapy patients and healthy mice, which corresponds to an increase in thrombin-antithrombin levels. Treatment of venous whole blood and isolated neutrophils with doxorubicin and epirubicin increased CFDNA release. We found that exposure of recalcified plasma to CFDNA isolated from epirubicin-treated whole blood increased thrombin generation by activating the contact pathway. We investigated the effects of chemotherapy on the PC anticoagulant pathway. We found that acrolein decreased EPCR while increasing thrombomodulin expression on treated endothelial cells. A corresponding decrease in activated PC generation was measured on acrolein-treated endothelial cells exposed to recalcified, defibrinated plasma. Healthy mice treated with acrolein and cyclophosphamide increased PC antigen levels, but no measurable increase in plasma APC levels. Breast cancer chemotherapy drugs elevate thrombin generation by activating coagulation through the TF and contact pathways, and by promoting phosphatidylserine exposure, as well as by impairing PC activation EPCR expression. These studies provide insight into the mechanisms of breast cancer chemotherapy-induced hypercoagulation.</p> / Doctor of Philosophy (Medical Science)

The venom of the Eastern coral snake Micrurus fulvius can cause respiratory paralysis in the bitten patient, which is attributable to -neurotoxins (-NTx). The aim of this work was to study the biodistribution and lymphatic tracking by molecular imaging of the main -NTx of M. fulvius venom. -NTx was bioconjugated with the chelator diethylenetriaminepenta-acetic acid (DTPA) and radiolabeled with the radionuclide Gallium-67. Radiolabeling efficiency was 60%-78%; radiochemical purity 92%; and stability at 48 h 85%. The median lethal dose (LD50) and PLA(2) activity of bioconjugated -NTx decreased 3 and 2.5 times, respectively, in comparison with native -NTx. The immune recognition by polyclonal antibodies decreased 10 times. Biodistribution of -NTx-DTPA-Ga-67 in rats showed increased uptake in popliteal, lumbar nodes and kidneys that was not observed with Ga-67-free. Accumulation in organs at 24 h was less than 1%, except for kidneys, where the average was 3.7%. The inoculation site works as a depot, since 10% of the initial dose of -NTx-DTPA-Ga-67 remains there for up to 48 h. This work clearly demonstrates the lymphatic system participation in the biodistribution of -NTx-DTPA-Ga-67. Our approach could be applied to analyze the role of the lymphatic system in snakebite for a better understanding of envenoming.

Plasma volume (PV) rises by up to 50% in normal pregnancy, a phenomenon associated with a favourable pregnancy outcome. A previous study of pregnant women with sickle cell (haemoglobin SS) disorder found that PV paradoxically contracts in late pregnancy. A cross-sectional study was performed to determine PV (Evans blue method) and volume regulatory hormones and electrolytes in pregnant women with haemoglobin (Hb) SS and in non-pregnant and Hb AA controls. PV rose in pregnant HbAA and was significantly correlated with plasma angiotensinogen. Non-pregnant Hb SS women had supranormal PV measurements and reduced glomerular filtration rate (GFR). Their PV did not rise in pregnancy and was not correlated with angiotensinogen. Their plasma renin concentration also failed to rise significantly by 36 weeks gestation and was significantly less than in Hb AA pregnancy although aldosterone concentration was raised as expected. A general vasoconstriction in pregnancy can cause inactivation of the renin-angiotensin system and could explain this, with aldosterone being elevated by non Angiotensin II dependent stimulation such as plasma potassium, which was significantly higher in the pregnant Hb SS women. Further studies demonstrating a deficiency of vasodilator substances in pregnant Hb SS women will strengthen this hypothesis.

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.

Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43(-/-) mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43(∆LEC)) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.

Intraocular pressure (IOP) is the most important risk factor for glaucoma development and progression. Most anti-glaucoma treatments aim to lower IOP by enhancing aqueous humor drainage from the eye. Aqueous humor drainage occurs via well-characterized trabecular meshwork (TM) and uveoscleral (UVS) pathways, and the recently described ciliary lymphatics. The relative contribution of the lymphatic pathway to aqueous drainage is not known. We developed a sheep model to quantitatively assess lymphatic drainage along with TM and UVS outflows. Following intracameral injection of 125I-bovine serum albumin (BSA), lymph and blood samples were continuously collected. Lymphatic and TM drainage were quantitatively assessed by measuring 125I-BSA recovery. This quantitative sheep model enables assessment of relative contributions of lymphatic drainage (1.64% ± 0.89%), TM (68.86% ± 9.27%) and UVS outflows (19.87% ± 5.59%), and may help to better understand the effects of glaucoma agents on outflow pathways.