Doctor of Philosophy (PhD) / Over 85% of Australia’s population live in urban areas and many turtle populations occur on Australia’s east coast where urban development is particularly concentrated. In the state of NSW, over half of the freshwater coastal wetlands have been highly modified or completely destroyed, and urban freshwater creeks often have only a narrow strip of weedy bushland left along their banks. Even though habitat degradation may result in declines in density and distribution of turtle populations, there are few data on Australian freshwater turtles in urban areas. In addition to extreme habitat alteration, urban waterways are innundated with anthropogenic contaminants from sources including wet weather surface runoff and industrial and sewage discharges. Pollutants can impact all systems of the body with potentially severe effects on reproduction and survival that can result in deterioration of animal populations. Turtles are particularly susceptible to anthropogenic contaminants due to their intimate contact with the aquatic environment, an often high trophic level, their ability to accumulate toxins, and their longevity. For almost all contaminants, the degree of accumulation in and effect on reptile species is unknown. Sublethal effects in field situations are particularly poorly studied and have never been documented in pleurodiran turtles. As a pioneering work in Australian reptile ecotoxicology, this thesis takes a broad approach, but focuses primarily on immunotoxicity and reproductive toxicity – two areas that greatly impact the size and continuance of animal populations. The aim of the thesis is to provide baseline data on haematology, cellular immunology and tissue metal concentrations for freshwater turtles in Sydney – data which were lacking for all Australian turtle species prior to this study. After initial assessment of the distribution and density of freshwater turtles in Sydney, the study examines the potential for Sydney’s turtles as sentinel species for measuring the effects of pollution on haematology, cellular immunity, and parasite loads; and considers the relationships between urban metal pollution and reproductive variables. The relative suitability of non-lethally sampled tissues (blood, carapace, egg) for use in biomonitoring is also assessed. Three species of Australian freshwater turtles were found in the Sydney region, with Chelodina longicollis occurring naturally in the area, and populations of Emydura macquarii and Elseya latisternum likely to have originated from translocated individuals. The North American turtle Trachemys scripta elegans was not encountered during this study despite concerns that it was establishing in the Sydney area. Chelodina longicollis populations were widespread, although poor recruitmment was indicated by low capture rates and comparatively low percentage of juveniles at some sites. Not so widespread, Emydura macquarii was present in much larger numbers than C. longicollis and with a high juvenile component in some areas of southeastern Sydney. I provide information on erythrocyte and leucocyte parameters in C. longicollis over a range of sites, pollution conditions, and seasons. In C. longicollis, numbers of lymphocytes, heterophils and eosinophils varied over sites, but not due to pollution from sewage treatment plant outfalls. There was significant temporal variation in erythrocyte, lymphocyte, eosinophil, heterophil, and basophil number, the heterophil:lymphocyte ratio, and haematocrit, but not consistently among sites. Future studies should ensure simultaneous sampling across sites for comparative purposes. Similarly, turtle populations downstream of sewage treatment plant outfalls showed no consistent difference in number, body condition, blood haemogregarine load, or leech (haemogregarine vector) load from upstream populations. Leech (Helobdella papillornata, with some Placobdella sp.) load and haemogregarine numbers increase dramatically once C. longicollis reach a carapace of 110 mm. The number of leeches on turtles varied across season, year, and site. Turtles with large numbers of leeches had reduced haematocrit, but the presence of leeches had no other correlations with haematological parameters. Haemogregarine numbers did not change across season or year, and were not correlated with haematological variables. The hypothesis that pollutants lead to an increase in normal blood protozoa due to reduced immunity thus was not supported. The concentration of metals in C. longicollis and E. macquarii carapace and in lagoon sediments varied significantly over four urban and four national park sites, but not based on this split. Pollution in periurban areas, such as illegal dumping of toxic wastes and atmospheric deposition of pollutants, means that each site must be classified separately as to degree of metal pollution. There was little or no affect of species, size, sex, or gravidity on metal concentrations in the carapace of adult turtles. Emydura macquarii had higher concentrations of blood Fe than C. longicollis from a different site, but this is possibly due to an increase in haemoglobin resulting from the site’s low aquatic oxygen concentration rather than any increased environmental exposure. Chelid turtles in Sydney do not show much promise as a biomonitoring tool. Carapace analysis is largely discounted as a potential tool for metal biomonitoring due to poor correlations between potentially toxic metals in non-lethally samplable tissues (carapace, claw) and internal organs (liver, kidney) or bone (femur). However, carapace metal concentrations still potentially reflect long-term metal presence or different dietary exposures as evidenced by the significant variation in concentrations over sites. A rare correlation was found for concentrations of aquatic Pb and carapace Pb, and a correlation was also found for concentrations of blood Pb and carapace Pb in E. macquarii. Thus any potential for tissue biomonitoring seems to lie with this highly ecotoxicologically relevant metal. Although two other ecotoxicologically relevent metals, Cu and Se, were significantly higher in egg contents of C. longicollis compared to E. macquarii, these elements are also essential and a lack of baseline values means it is not known if this simply reflects natural taxonomic variation. Ni, a metal of toxicological concern in sea turtles, was not present in egg contents, and only variably present in eggshell. The absence of Pb from eggs, despite its presence in many maternal tissues, suggests that selective metal uptake into eggs may be protective of toxic elements, rather than eggs serving as a maternal method of toxic metal elimination as has been previously suggested. The paucity of toxic metal detection in eggs renders them unlikely tissues for biomonitoring. The maternal tissue or tissues or environmental source from which egg metals originate remains obscure, although a significant negative effect of maternal carapace concentrations of Ca and Mg on eggshell thickness in E. macquarii indicates that there may be mobilisation of Ca and Mg from the carapace for eggshell formation. The only metal whose eggshell concentration correlated with eggshell thickness was Mg, indicating that ecotoxic metals previously associated with eggshell thinning are not problematic in the Sydney chelids. As with North American turtles living at polluted sites, none of the chelid hatchlings were found to have any overt abnormalities. Hatching success was poor and hatching mass low for eggs of both C. longicollis and E. macquarii, although results from natural nests are required to determine whether or not this was an outcome of hormonally-induced oviposition and artificial incubation. It is difficult to interpret metal concentrations found in the soft tissues, calcified tissues, and eggs of chelonians due to the paucity of comparative data, and much more research is required on tissue metal concentrations before patterns will emerge. This especially applies to pleurodires for which no previous information is available. From comparisons with the limited data available for other freshwater turtles, marine turtles, and other aquatic reptiles, it does not appear that Sydney’s turtle populations have unusually high metal concentrations in tissues. Exclusion of toxic metals such as Pb from the egg may also be protective to the developing embryo. An ability to live in polluted habitats, while limiting the accumulation of toxic contaminants, may be one key to their persistence in urban waterways from which other freshwater fauna have disappeared. Reproductive impacts such as low embryo survival and small hatchling weights require more rigorous examination, but may have less effect on these animals which have such naturally high egg and hatchling mortality. Although it was generally hard to demonstrate biochemical, physiological or population impacts of contaminants, C. longicollis from a site with severe sewage pollution did display unusual alterations in a number of haematological variables, body condition, and carapace bone structure. Despite this, the population was large and had a comparatively high ratio of juveniles. Additionally, the adverse haematological alterations appeared reversible. Thus, successful populations in Sydney probably are more dependent on basic ecological needs being met, than on low levels of environmental contaminants. The ongoing persistence of chelid populations in Sydney is likely to be dependent to some extent on their opportunistic diets, which generally make animals less vulnerable to habitat modification and the reduction in prey item diversity following pollution (Mason 1996, Allanson & Georges 1999), with a further benefit possibly bestowed at some sites on E. macquarii by its omnivory.

The thesis is comprised of a comparative study of iron metabolism with particular reference to iron deficiency in the Indian and the African; Europeans were included when naterlal was available. Fifty four patients with iron deficiency anaemia were studied. There ware 43 Indians as compared with 11 Africans although the overall proportion of Indians to Africans admitted to the same ward was 1 : 4. Amongst the Indians the commonest cause of anaemia from blood loss was peptic ulceration (6 patients); while 3 had cirrhosis of the liver, one had hookworm anaemia and one was a case of ulcerative colitis. Gynaecological lesions were found in 2 patients, one had a proliferative endometrium and the other had endometrial polypi. Idiopathic iron deficiency anaemia was found in 60.5% of the Indian patients (both male and female). Amongst the Africans on the other hand, there were 2 cases of hookworm anaemia and 2 of cirrhosis of the liver while peptic ulceration was suspected in one patient who alao had amoebic dysentery and urinary hllharsiesis. None of the Africans had Idiopathic iron deficiency anaemia with the possible exception of one who had 4 Caesarean sections in rapid succession. There were 175 Indians, 175 Africans and 139 Europeans who were studied haematologlcally during pregnancy. The Incidence of iron deficiency anaemia among them was 26.7%, 2% and 4% respectively. The third aspect of the thesis is confined to an analysis of necropsy materiel for iron stores. Two hundred Africans and 58 Indians were studied. It was found that the incidence of *siderosis" in the African was high. There was a significant difference in the iron concentrations in the stores of the 2 racial groups. It was concluded that iron deficiency anaemia is common in the Indian. Diet appears to play an important role in its production. / Thesis (M.D.)-University of Natal, Durban, 1963.

This study focuses on women with both pre-eclampsia and Human Immunodeficiency Virus (HIV). Pre-eclampsia is a pregnancy-specific syndrome that occurs after 20 weeks gestation. Thrombocytopenia is the most common haematological abnormality in pre-eclampsia. Further, studies suggest that the immunological mechanism plays some role in the aetiology of pre-eclampsia. The immunological hallmark of HIV infection is a progressive decline in the number of CD4 T lymphocytes and significant haematological abnormalities are also common in HIV-infected individuals i.e. anaemia, thrombocytopenia and leukopenia. The study population comprised of two groups i.e., pre-eclamptic HIV-positive African women and preeclamptic HIV-negative African women as the control group. Samples were analysed for haematological parameters (full blood count) and immunological markers (flow cytometry). There was no statistical significance in the following parameters: RBC, Hb, haematocrit, MCV, MCH, MCHC, platelets, MPV, WBC, lymphocytes, neutrophils, eosinophils, monocytes, basophils and CD8. There was a statistical difference in the CD3 and CD4 counts between both the groups. However, the CD3 and CD4 counts were within the normal range in the HIV-negative pre-eclamptic group and even though CD3 decreased, it was still within the normal range in the HIV-positive pre-eclamptic group, with CD4 decreasing below the normal range in the HIV-positive pre-eclamptic group. This suggests that immune mechanisms involving CD estimations do not play a role in pre-eclampsia since the decrease in the counts can be solely attributed to HIV infection. Results obtained in this study do not show any severe haematological or immunological abnormalities when women have both pre-eclampsia and HIV infection. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2007.

Abstract Both qualitative and quantitative limitations of two-dimensional (2DE) imaging have lead to the emergence of the three-dimensional echo (3DE) technique. Until recently the development of 3DE technology has been slow due to inferior image quality and longer processing and acquisition times compared with 2DE. However, in recent times 3DE technology has advanced to “real-time” (RT3D) and has become feasible as a standard clinical tool. The overall hypothesis of the studies undertaken in this thesis is that the increased accuracy of left ventricular (LV) assessment using RT3D justifies its use in clinical decision-making based on both single and sequential measurements in routine echocardiographic practice. It shows the increased accuracy and feasibility of 3DE using online and offline techniques and opacification, and evaluates the prognostic implications of LV measurements globally and regionally using 3DE compared with 2DE. Further, it assesses the accuracy of LV assessment using 3DE and demonstrates its superior accuracy to 2DE in cross-sectional studies. The thesis initially reviews the use of physiologic parameters used for clinical decision making and looks at the principles of incremental value in diagnostic testing. The assessment of ventricular structure and function is discussed as well as the current problems with clinical echocardiography, which have lead to the evolution and development of 3DE. The second chapter describes the methodologies used in this thesis, with particular emphasis on new technologies used in the studies. Chapter 3 looks at the role of experience as a barrier to incorporation of 3DE into standard practice. We tested attendees before and after an intensive interactive training course to evaluate the learning curve. An interactive teaching course with rehearsal and direct mentoring appears to overcome this limitation and may improve the uptake of the 3DE technique. Chapters 4 to 6 validate the use of 3DE by comparing it to 2DE and magnetic resonance imaging (MRI) with respect to online and offline techniques, the use of left ventricular opacification (LVO) and the serial follow-up of patients. The availability of automated on-line software may increase the feasibility of real-time 3DE for LV volume calculation in clinical practice. Chapter 4 looks at the comparison of off- and on-line approaches with magnetic resonance imaging. Online measurement of LV volumes was found to be feasible and more accurate than with 2DE, however the offline approach was more accurate - although more time-consuming. Chapter 5 involves two studies which sought to validate 3DE in a multicentre setting. They examine the variability of LV measurements across multiple sites and to validate 3DE against MRI. The first study found that the semi-automated measurement of 3DE reduces the variation of LV volume between centers. The second study provides information on the role of different potential sources of error and provides guidelines for future users on how to minimize these errors as well as how to interpret their findings. The next chapter further validates 3DE with use of LVO as a technique to improve the accuracy of LV volume measurements. We sought to examine the accuracy of non-contrast and contrast enhanced 2DE and 3DE for calculation of LV volumes and ejection fraction (EF), relative to cardiac magnetic resonance imaging. 2DE with LVO was analogous to 3DE in accurate categorization of LV function. However, 3DE with LVO was feasible and superior to other non-contrast and contrast enhanced techniques in patients with previous infarction. Chapter 7 examines the technical details of regional volume measurements. The use of a fixed external frame of reference in analyses of regional wall motion in the apical four-chamber view is prone to a systematic error and the use of a floating-axis analysis corrects for this, but this is based on landmarks by MRI (apex, annulus) and center of mass by 3DE. The axis is likely to be influenced by reverse remodeling after intervention and therefore we sought to evaluate 3DE regional volume assessment vs. cardiac MRI over follow-up. The use of a center-line based on center of mass with RT3D may contribute to problems with use of this modality to track regional volumes over time. Although global 3DE volumes compare well with MRI volumes, new developments in image quality and automated software will be needed before changes in regional volumes can be reliably followed with 3DE. 2DE ESE is limited by sampling in standard imaging planes and suboptimal acquisition may cause poor matching of pre- and post-stress images. 3DE may avoid these orientation problems and contraction front mapping (CFM) may also provide a method for defining the temporal homogeneity of contraction. Chapter 8 examines the incremental benefit of 3DE CFM to 2DE and 3DE, using coronary angiography as the reference standard. It found that analysis of the temporal distribution of contraction may be an alternate means than 3DE wall motion assessment for identification of ischemia for ESE. Another significant validation needed for 3DE is not only for the LV but for the right ventricle (RV). The non-geometric nature of the RV makes it difficult to measure. Chapter 9 shows that 3DE is superior to 2DE for follow-up of RV function by validation vs. cardiac MRI. It was found that 3DE was indeed more accurate than 2DE approaches and reduces test-retest variation of RV volumes and EF measurements in follow-up RV assessment. Chapter 10 investigates the accuracy and reproducibility of the use of 3DE for serial follow-up of LV measurements. Echocardiographic follow-up of LV volumes is difficult because of the test-retest variation of 2DE. In this follow-up study, sequential 3DE measurements of change of volumes and EF were similar to those obtained using MRI, but 2DE overestimated change in EDV. As MRI is expensive, of limited availability and cannot be performed on patients with implanted devices, 3DE appears to be an effective long-term follow-up imaging tool for when sequential measurement of LV volumes is sought to guide management decisions. Chapter 11 investigates whether 3DE is more predictive of outcome than 2DE. In this outcome study, 3DE measurements of ESV and EF showed a stronger prediction of outcomes than 2DE. 3DE now appears to be the measurement of choice for when LV volumes and EF are sought to guide management decisions. In conclusion, the work reported in this thesis demonstrates that 3DE increases the accuracy of LV assessment and justifies its use in clinical decision-making based on both single and sequential measurements in routine echocardiographic practice.

Antarctic fish from the suborder Notothenioidei inhabit what is perhaps the most thermally stable ocean environment on Earth. Evolutionary theory suggests that by specialising for this environment, Antarctic fish have traded-off their ability to respond to variations in temperature, and like their environment, have become extremely stenothermal. However, previous research has revealed that the Antarctic notothenioid fish Pagothenia borchgrevinki is not as thermally limited as evolutionary theory might predict, and is capable of acclimation to 4 ℃ during a one month period. The purpose of the current research was to investigate the physiological mechanisms that underpin this remarkable acclimatory ability. P. borchgrevinki were acclimated for one month to 4 ℃ and changes in oxygen consumption, prolonged swimming ability, cardiovascular function, enzyme activity and haematology were measured. Significant changes in resting oxygen consumption rate and prolonged swimming ability occurred during the acclimation period, and these changes were mediated by adjustments of enzyme activity and specific aspects of the haematology. By monitoring resting oxygen consumption and prolonged swimming ability over a much longer, six month, acclimation period it was confirmed that the adjustments evident during one month at 4 ℃ were sustainable in the long-term, and were not short-term compensatory mechanisms. Interestingly, fish infected with x-cell gill disease did not possess the same ability to acclimate as was demonstrated by healthy P. borchgrevinki. P. borchgrevinki are unusual among the notothenioids, possessing an active, pelagic lifestyle which differs from the sedentary, benthic lifestyle of most other species within the suborder. Therefore, it was hypothesised that the acclimatory ability demonstrated by this species may also be unusual among the notothenioids. To test this hypothesis, the acclimation ability of three sedentary, benthic notothenioids (Trematomus bernacchii, T. hansoni and T. pennellii) was investigated. Results confirmed the hypothesis, with all three species demonstrating very poor survival at 4 ℃ and absolutely no capacity for acclimation. Such results present a disturbing scenario for the future of Antarctic notothenioid fish in Earth?s rapidly warming climate, and highlights the need for continued research combined with immediate action to combat the warming which currently threatens Antarctic marine biodiversity.

Doctor of Philosophy (PhD) / Over 85% of Australia’s population live in urban areas and many turtle populations occur on Australia’s east coast where urban development is particularly concentrated. In the state of NSW, over half of the freshwater coastal wetlands have been highly modified or completely destroyed, and urban freshwater creeks often have only a narrow strip of weedy bushland left along their banks. Even though habitat degradation may result in declines in density and distribution of turtle populations, there are few data on Australian freshwater turtles in urban areas. In addition to extreme habitat alteration, urban waterways are innundated with anthropogenic contaminants from sources including wet weather surface runoff and industrial and sewage discharges. Pollutants can impact all systems of the body with potentially severe effects on reproduction and survival that can result in deterioration of animal populations. Turtles are particularly susceptible to anthropogenic contaminants due to their intimate contact with the aquatic environment, an often high trophic level, their ability to accumulate toxins, and their longevity. For almost all contaminants, the degree of accumulation in and effect on reptile species is unknown. Sublethal effects in field situations are particularly poorly studied and have never been documented in pleurodiran turtles. As a pioneering work in Australian reptile ecotoxicology, this thesis takes a broad approach, but focuses primarily on immunotoxicity and reproductive toxicity – two areas that greatly impact the size and continuance of animal populations. The aim of the thesis is to provide baseline data on haematology, cellular immunology and tissue metal concentrations for freshwater turtles in Sydney – data which were lacking for all Australian turtle species prior to this study. After initial assessment of the distribution and density of freshwater turtles in Sydney, the study examines the potential for Sydney’s turtles as sentinel species for measuring the effects of pollution on haematology, cellular immunity, and parasite loads; and considers the relationships between urban metal pollution and reproductive variables. The relative suitability of non-lethally sampled tissues (blood, carapace, egg) for use in biomonitoring is also assessed. Three species of Australian freshwater turtles were found in the Sydney region, with Chelodina longicollis occurring naturally in the area, and populations of Emydura macquarii and Elseya latisternum likely to have originated from translocated individuals. The North American turtle Trachemys scripta elegans was not encountered during this study despite concerns that it was establishing in the Sydney area. Chelodina longicollis populations were widespread, although poor recruitmment was indicated by low capture rates and comparatively low percentage of juveniles at some sites. Not so widespread, Emydura macquarii was present in much larger numbers than C. longicollis and with a high juvenile component in some areas of southeastern Sydney. I provide information on erythrocyte and leucocyte parameters in C. longicollis over a range of sites, pollution conditions, and seasons. In C. longicollis, numbers of lymphocytes, heterophils and eosinophils varied over sites, but not due to pollution from sewage treatment plant outfalls. There was significant temporal variation in erythrocyte, lymphocyte, eosinophil, heterophil, and basophil number, the heterophil:lymphocyte ratio, and haematocrit, but not consistently among sites. Future studies should ensure simultaneous sampling across sites for comparative purposes. Similarly, turtle populations downstream of sewage treatment plant outfalls showed no consistent difference in number, body condition, blood haemogregarine load, or leech (haemogregarine vector) load from upstream populations. Leech (Helobdella papillornata, with some Placobdella sp.) load and haemogregarine numbers increase dramatically once C. longicollis reach a carapace of 110 mm. The number of leeches on turtles varied across season, year, and site. Turtles with large numbers of leeches had reduced haematocrit, but the presence of leeches had no other correlations with haematological parameters. Haemogregarine numbers did not change across season or year, and were not correlated with haematological variables. The hypothesis that pollutants lead to an increase in normal blood protozoa due to reduced immunity thus was not supported. The concentration of metals in C. longicollis and E. macquarii carapace and in lagoon sediments varied significantly over four urban and four national park sites, but not based on this split. Pollution in periurban areas, such as illegal dumping of toxic wastes and atmospheric deposition of pollutants, means that each site must be classified separately as to degree of metal pollution. There was little or no affect of species, size, sex, or gravidity on metal concentrations in the carapace of adult turtles. Emydura macquarii had higher concentrations of blood Fe than C. longicollis from a different site, but this is possibly due to an increase in haemoglobin resulting from the site’s low aquatic oxygen concentration rather than any increased environmental exposure. Chelid turtles in Sydney do not show much promise as a biomonitoring tool. Carapace analysis is largely discounted as a potential tool for metal biomonitoring due to poor correlations between potentially toxic metals in non-lethally samplable tissues (carapace, claw) and internal organs (liver, kidney) or bone (femur). However, carapace metal concentrations still potentially reflect long-term metal presence or different dietary exposures as evidenced by the significant variation in concentrations over sites. A rare correlation was found for concentrations of aquatic Pb and carapace Pb, and a correlation was also found for concentrations of blood Pb and carapace Pb in E. macquarii. Thus any potential for tissue biomonitoring seems to lie with this highly ecotoxicologically relevant metal. Although two other ecotoxicologically relevent metals, Cu and Se, were significantly higher in egg contents of C. longicollis compared to E. macquarii, these elements are also essential and a lack of baseline values means it is not known if this simply reflects natural taxonomic variation. Ni, a metal of toxicological concern in sea turtles, was not present in egg contents, and only variably present in eggshell. The absence of Pb from eggs, despite its presence in many maternal tissues, suggests that selective metal uptake into eggs may be protective of toxic elements, rather than eggs serving as a maternal method of toxic metal elimination as has been previously suggested. The paucity of toxic metal detection in eggs renders them unlikely tissues for biomonitoring. The maternal tissue or tissues or environmental source from which egg metals originate remains obscure, although a significant negative effect of maternal carapace concentrations of Ca and Mg on eggshell thickness in E. macquarii indicates that there may be mobilisation of Ca and Mg from the carapace for eggshell formation. The only metal whose eggshell concentration correlated with eggshell thickness was Mg, indicating that ecotoxic metals previously associated with eggshell thinning are not problematic in the Sydney chelids. As with North American turtles living at polluted sites, none of the chelid hatchlings were found to have any overt abnormalities. Hatching success was poor and hatching mass low for eggs of both C. longicollis and E. macquarii, although results from natural nests are required to determine whether or not this was an outcome of hormonally-induced oviposition and artificial incubation. It is difficult to interpret metal concentrations found in the soft tissues, calcified tissues, and eggs of chelonians due to the paucity of comparative data, and much more research is required on tissue metal concentrations before patterns will emerge. This especially applies to pleurodires for which no previous information is available. From comparisons with the limited data available for other freshwater turtles, marine turtles, and other aquatic reptiles, it does not appear that Sydney’s turtle populations have unusually high metal concentrations in tissues. Exclusion of toxic metals such as Pb from the egg may also be protective to the developing embryo. An ability to live in polluted habitats, while limiting the accumulation of toxic contaminants, may be one key to their persistence in urban waterways from which other freshwater fauna have disappeared. Reproductive impacts such as low embryo survival and small hatchling weights require more rigorous examination, but may have less effect on these animals which have such naturally high egg and hatchling mortality. Although it was generally hard to demonstrate biochemical, physiological or population impacts of contaminants, C. longicollis from a site with severe sewage pollution did display unusual alterations in a number of haematological variables, body condition, and carapace bone structure. Despite this, the population was large and had a comparatively high ratio of juveniles. Additionally, the adverse haematological alterations appeared reversible. Thus, successful populations in Sydney probably are more dependent on basic ecological needs being met, than on low levels of environmental contaminants. The ongoing persistence of chelid populations in Sydney is likely to be dependent to some extent on their opportunistic diets, which generally make animals less vulnerable to habitat modification and the reduction in prey item diversity following pollution (Mason 1996, Allanson & Georges 1999), with a further benefit possibly bestowed at some sites on E. macquarii by its omnivory.

The objective of the work presented in this thesis was to explore the role of potential alternatives to platelet transfusions and specifically to investigate whether desmopressin could be used for treatment of thrombocytopenia or platelet dysfunction. Patients with thrombocytopenia or platelet dysfunction are often treated with platelet transfusions to treat or prevent bleeding. However the evidence for the efficacy of platelet transfusion is limited and there is some evidence of harm. I have focused on thrombocytopenic patients with haematological malignancies or critically ill patients, who are amongst the groups most commonly treated with platelet transfusions. The aims of this research were to determine: 1. If levels of Von Willebrand factor (VWF) or other measures of haemostasis are predictive of bleeding in severe thrombocytopenia; 2. Whether VWF compensates for thrombocytopenia in vitro; 3. The evidence for the efficacy of desmopressin in all patients undergoing surgery or invasive procedures; 4. The evidence for desmopressin for platelet dysfunction or thrombocytopenia; 5. If it is feasible to use desmopressin to treat critically ill thrombocytopenic patients in a clinical trial. To identify derangements of haemostasis that may signify candidates for alternatives to platelet transfusions, I analysed blood samples from an observational trial of fifty patients with haematological malignancies and profound thrombocytopenia due to intensive chemotherapy. I used a panel of tests to investigate measures of primary haemostasis, thrombin generation, cross-linked fibrin formation and fibrinolysis. Using multivariable logistic regression, I found no consistent correlation between any measures of haemostasis and the risk of clinically significant bleeding. VWF antigen levels were the best predictor of clinically significant bleeding on the same day (odds ratio 0.31, 95% confidence interval 0.10 to 0.98, p=0.047) but were not predictive of severe bleeding over the 24 hours after the test (odds ratio 0.48, 95% confidence interval 0.10 to 2.34, p=0.36). In a separate set of experiments, I evaluated thrombus formation under flow in thrombocytopenia. This technique was sensitive to the platelet count . Addition of exogenous VWF to thrombocytopenic blood resulted in improvement in thrombus formation, suggesting that agents that affect or influence VWF pathways might have a role. Desmopressin can be used to increase VWF levels, so leading on from my laboratory experiments; I used systematic reviews and meta-analyses to assess whether desmopressin could be used in unselected patients to reduce bleeding peri-operatively. I identified 62 randomised controlled trials. Overall there was no evidence of benefit for administering desmopressin to unselected patients. However further analysis of eleven randomised controlled trials that focused on patients with platelet dysfunction found that desmopressin resulted in transfusion of fewer units of red cells (equivalent to a 25% reduction compared to control), less blood loss (equivalent to a 23% reduction compared to control) and a lower risk of requiring a re-operation due to bleeding (Peto odds ratio 0.39, 95% confidence interval 0.18 to 0.84). There was no evidence for an increase in thrombotic events. There was no randomised controlled trial evidence for perioperative desmopressin for patients with thrombocytopenia. These specific research gaps were addressed by designing new clinical trials. I have commenced a randomised controlled feasibility trial of desmopressin versus placebo for critically ill patients with thrombocytopenia undergoing invasive procedures. This trial is ongoing and is the first randomised trial evaluating peri-procedural desmopressin in thrombocytopenia. The programme of work arising from this research has the potential to benefit a large number of patients by preventing bleeding and reducing exposure to allogeneic blood components such as platelets. The results presented in this thesis are exploratory but are an important step on a path towards larger trials using desmopressin as an alternative, or adjunct to platelet transfusion.

Currently cellular therapies, such as hematopoietic stem cell transplantation (HSCT), are produced at a small scale on a case-by-case basis, usually in a clinical or near-clinical setting. Meeting the demand for future cellular therapies will require a robust and scalable manufacturing process that is either designed around or controls the variation associated with biological starting materials. Understanding variation requires both a measure of the allowable variation (that does not negatively affect patient outcome) and the achievable variation (with current technology). The prevalence of HSCT makes it an ideal case study to prepare for more complex biological manufacturing with more challenging regulatory classifications.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anaemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterised by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. The cellular and molecular pathology of FA is poorly understood, resulting in a severe lack of effective treatment options. In this thesis, I describe the first viable vertebrate model of RAD51 loss. Phenotypic characterisation of zebrafish rad51 loss-of-function mutants showed that they develop key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. Taking advantage of the unique properties of the zebrafish model, I show that some of these symptoms stem from both decreased proliferation, as well as increased apoptosis of embryonic haematopoietic stem and progenitor cells. Co-mutation of p$L was able to rescue the haematopoietic defects seen in the single mutants, but led to tumour development, underscoring the role of rad51 as a tumour suppressor. I further demonstrate that prolonged inflammatory stress can exacerbate the haematological impairment, leading to an additional decrease in kidney marrow cell numbers. In contrast, prolonged aldehyde-derived stress did not induce symptoms in the mutant fish. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signalling during embryogenesis leads to the haematological defects seen later in life in FA. It also strengthens the evidence for the involvement of haematopoietic stress, such as inflammation, in the development of bone marrow failure. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

The aim of the present study was to assess an influence of two different therapeutical concentrations of peracetic acid on selected haematological and biochemical parameters in grass carp (Ctenopharyngodon idella). Fish were radomly distributed to aquaria and exposed to concentrations of 0 mg.l-1 PAA (control group), 1.0 mg.l-1 PAA (P1 group), 3.0 mg.l-1 PAA (P2 group). Almost total mortality of fish was observed in the concentration 3.0 mg.l-1 PAA during the treatment comparing with the P1 group and untreated control where no mortality was observed. After the end of the experimental exposure of fish to peracetic acid, the sampling of blood has been realised. The samples of the blood were examined in order to determine haematological and biochemical parameters. Consequently, there were no significant differences (p<0.05) in a haematological profile of fish exposed to concentration of 1.0 mg.l-1 PAA. Goblet cells count and size have risen, that caused exposure of fish to peracetic acid. In the biochemical profile of fish, significant changes (p<0.01) in three parameters were found after exposure of fish to peracetic acid in concentration 1.0 mg.l-1. Changed parameters were: aspartate aminotransferase, creatine kinase and lactate dehydrogenase. The changes were moderate and it can be supposed that these changes are reversible. No significant change (p<0.05) in haematological parameters points out to the minimum negative influence of recommended therapeutical concntration (1.0 mg.l-1 PAA) to the health of C. idella.