Impacts of a fuel oil spill on seagrass meadows in a subtropical port, Gladstone, Australia - The value of long-term marine habitat monitoring in high risk areas.

We used an established seagrass monitoring programme to examine the short and longer-term impacts of an oil spill event on intertidal seagrass meadows. Results for potentially impacted seagrass areas were compared with existing monitoring data and with control seagrass meadows located outside of the oil spill area. Seagrass meadows were not significantly affected by the oil spill. Declines in seagrass biomass and area 1 month post-spill were consistent between control and impact meadows. Eight months post-spill, seagrass density and area increased to be within historical ranges. The declines in seagrass meadows were likely attributable to natural seasonal variation and a combination of climatic and anthropogenic impacts. The lack of impact from the oil spill was due to several mitigating factors rather than a lack of toxic effects to seagrasses. The study demonstrates the value of long-term monitoring of critical habitats in high risk areas to effectively assess impacts.

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

Risk factors associated with corneal nerve alteration in type 1 diabetes in the absence of neuropathy: A longitudinal in vivo corneal confocal microscopy study

Purpose The aim of this study was to determine alterations to the corneal subbasal nerve plexus (SNP) over four years using in vivo corneal confocal microscopy (IVCM) in participants with type 1 diabetes and to identify significant risk factors associated with these alterations. Methods A cohort of 108 individuals with type 1 diabetes and no evidence of peripheral neuropathy at enrollment underwent laser-scanning IVCM, ocular screening, and health and metabolic assessment at baseline and the examinations continued for four subsequent annual visits. At each annual visit, eight central corneal images of the SNP were selected and analyzed to quantify corneal nerve fiber density (CNFD), branch density (CNBD) and fiber length (CNFL). Linear mixed model approaches were fitted to examine the relationship between risk factors and corneal nerve parameters. Results A total of 96 participants completed the final visit and 91 participants completed all visits. No significant relationships were found between corneal nerve parameters and time, sex, duration of diabetes, smoking, alcohol consumption, blood pressure or BMI. However, CNFD was negatively associated with HbA1c (β=-0.76, P<0.01) and age (β=-0.13, P<0.01) and positively related to high density lipids (HDL) (β=2.01, P=0.03). Higher HbA1c (β=-1.58, P=0.04) and age (β=-0.23, P<0.01) also negatively impacted CNBD. CNFL was only affected by higher age (β=-0.06, P<0.01). Conclusions Glycemic control, HDL and age have significant effects on SNP structure. These findings highlight the importance of diabetic management to prevent corneal nerve damage as well as the capability of IVCM for monitoring subclinical alterations in the corneal SNP in diabetes.

Anisochrony of O-methylene protons of ethyl ester functions and structural factors in the connected chiral entities

(i) Incistrans pairs of cyclic 1,3-dicarboxylic acid ethyl esters thecis-foms exhibit higher O-methylene proton (HA, HB) anisochrony than thetrans-forms; (ii) anisochrony, easily observed in certain decalin-10-carboxylic ethyl esters, ‘disappears’ on one of the rings attaining the possibility of transforming into a ‘twist’ form; (iii) in certain pairs of chiralsecethyl esters and theirtert-methylated analogues anisochrony is higher in the latter, contrary to expectation, while, in certain others, the reverse is observed. Attempted explanations are based on assessments whether H A and H B are or are not in highly different magnetic environments in confomers regarded as preferred. This subsumes the possibility thatXYZC-CO2H A H B Me chiral ethyl acetates differ fromXYZC-CH A H B Me ethanes because intervention by the carboxyl group insulates the prochiral centre and allows anisotropic effects to gain somewhat in importance among mechanisms that discriminate between H A and H B so long as rotamerpopulation inequalities persist. Background information on why rotamer-population inequalities will always persist and on a heuristic that attempts to generalize the effects ofXYZ inXYZC - CU AUB V is provided. Possible effects when connectivity exists between a pair amongX, Y, Z or when specific interactions occur betweenV andX, Y orZ are considered. An interpretation in terms of ‘increasing conformational mobility’ has been suggested for the observed increase in the rate of temperature-dependence of O-methylene anisochrony down a series of chiral ethyl esters.

First Report of a Toxic Nodularia spumigena (Nostocales/Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8-47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 mu g/kg, range 32.3-56.8 mu g/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

The effects of environmental factors on biomass and microcystin production by the freshwater cyanobacterial genera Microcystis and Anabaena

Several cyanobacterial genera produce the hepatotoxins, microcystins. Microcystins are produced only in cells that have microcystin synthetase gene (mcy) clusters, which encode enzyme complexes involved in microcystin biosynthesis. Microcystin-producing and nonmicrocystin-producing genotypes of single cyanobacterial genus may occur simultaneously in situ. Previously, the effects of environmental factors on the growth and microcystin production of cyanobacteria have mainly been studied by means of isolated cyanobacteria cultures in the laboratory. Studies in the field have been difficult, owing to the lack of methods to identify and quantify the different genotypes. In this study, genus-specific microcystin synthetase E (mcyE) gene primers were designed and a method to identify and quantify the mcyE copy numbers was developed and used in situ. Microcystis and Anabaena mcyE genes were observed in two Finnish lakes. Microcystis appeared to be the most abundant microcystin producer in Lake Tuusulanjärvi and in one basin of Lake Hiidenvesi. Because the most potent microcystin-producing genus of a lake can be identified, it will be possible in the future to design genus-targeted strategies for lake restoration. Effects of P and N concentrations on the biomass of microcystin-producing and nonmicrocystin-producing Microcystis strains and an Anabaena strain were studied in cultures. P and N concentrations and their combined effect increased cyanobacterial biomass of all Microcystis strains. The biomass of microcystin-producing Microcystis was higher than that of nonmicrocystin-producing strains at high nutrient concentrations. The P concentration increased Anabaena biomass, but the effect of N concentration was statistically insignificant for growth yield, probably due to the ability of the genus to fix molecular N2. P and N concentrations and combined nutrients caused an increase in cellular microcystin concentrations of the Microcystis strain cultivated in chemostat cultures. Cyanobacteria are able to hydrolyse nutrients from organic matter through extracellular enzyme activities. Leucine aminopeptidase (LAP) activity was observed in an axenic N2-fixing Anabaena strain grown in batch cultures. The P concentration caused a statistically significant increase in LAP activity, whereas the effect of N concentration was insignificant. The highest LAP activities were observed in the most eutrophic basins of Lake Hiidenvesi. LAP activity probably originated mostly from attached heterotrophic bacteria and less from cyanobacteria.

Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Study of the structure and function of the sheath of tropically adapted bulls and risk factors for preputial eversion and preputial prolapse

Preputial prolapse is an obvious condition affecting bulls from many breeds. Unfortunately, the losses in production and welfare concerns associated with preputial prolapse can remain undetected for long periods of time in the extensive beef areas of northern Australia where the bulls are not inspected regularly. Thus, there is a critical need to identify the structural factors predisposing to preputial prolapse in young bulls so that they can be culled early. Despite there being no firm scientific evidence of an association between preputial eversion and preputial prolapse, it seems logical that the increased exposure of the sensitive prepuce as a consequence of preputial eversion may increase the risk of bulls developing preputial pathology, in particular preputial prolapse. This may be particularly relevant in Bos indicus bulls as they have a more pendulous sheath and thus eversion of the prepuce may be associated with a greater risk of injury to the prepuce compared to that in Bos taurus bulls. Further, studies of preputial eversion in Bos taurus bulls have concluded that there is an association between polledness and increased prevalence and severity (length of everted prepuce and duration of eversion) of preputial eversion due primarily to the absence or poor development of the caudal preputial muscles. No similar definitive work in Bos indicus bulls has been conducted and thus anatomical studies reported in this thesis were conducted to determine if a similar association occurred in Bos indicus bulls. A survey of a sample of large beef breeding herds in northern Australia found that preputial prolapse is a significant problem in Bos indicus and Bos indicus derived bulls and affected both young and older bulls. The importance of preputial prolapse confirmed the value of further research into the causes of this problem. A series of anatomical studies confirmed that preputial eversion in Bos indicus derived bulls was not more prevalent in polled bulls than horned bulls and was not associated with deficiency of the caudal preputial muscles as was established in Bos taurus bulls. An anatomical study of Bos indicus derived bulls with preputial prolapse found that preputial prolapse occurred in horned bulls of varying ages and these bulls did not have any evidence of deficiency in the caudal preputial muscles. However, preputial prolapse was observed in young polled bulls that had poorly developed or absent caudal preputial muscles. It was concluded that deficiency of the caudal preputial muscles in polled Bos indicus derived bulls may predispose to preputial prolapse at an early age, but no predisposing anatomical factors were found for horned Bos indicus derived bulls. In these studies, preputial eversion and preputial prolapse were found in horned Bos indicus derived bulls that did not have any preputial muscle deficiency and it was noted that preputial eversion was not related to the length of the prepuce. Further studies confirmed that preputial eversion was linearly and consistently associated with position of the glans penis within the sheath in Bos indicus derived bulls, and movement of the glans penis towards the preputial orifice consistently resulted in preputial eversion in these bulls. A method to objectively measure the relationship between movement of the glans penis within the sheath and preputial eversion was developed. Studies in humans have linked function of some abdominal muscles to function of the pelvic organs. This relationship was investigated in Bos indicus derived bulls to determine whether the function of specific abdominal muscles affected position of the penis in the sheath. Using the method developed to objectively measure the relationship between penis movement and preputial eversion, the abdominal muscles that potentially were associated with movement of the glans penis or preputial eversion were examined but no significant relationships were observed. In the anatomical study of Bos indicus derived bulls not affected with preputial prolapse a more pendulous sheath was associated with increased prevalence of preputial eversion. This relationship was confirmed for horned and polled bulls in the penis movement studies. Bos indicus derived bulls with more pendulous sheaths evert their prepuces more than bulls with less pendulous sheaths thus increasing the risk of damage to the prepuce either from the environment, other bulls, or from them inadvertently stepping on the everted prepuce when they get to their feet. Culling Bos indicus derived bulls with more pendulous sheaths should reduce the incidence of preputial eversion and possibly preputial prolapse. The anatomical study of Bos indicus derived bulls that did not have preputial prolapse demonstrates that there are herds of bulls where the polled bulls do not have any evidence of deficiency of the caudal preputial iv muscles. There is a need to develop a practical and cost effective test to identify polled Bos indicus bulls that have a deficiency in their caudal preputial muscles.