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No description available.

No description available.

Cilia are finger-like protrusions that function in the detection and processing of a wide variety of signals. In vertebrates, they are intimately involved in early embryonic patterning, the differentiation and function of sensory neurons, morphogenesis and physiology of duct epithelia, cell motility and metabolism. Lipid composition of the ciliary membrane and its contribution to cilia function is one of the least studied areas of cilia biology. Here I use a collection of transgenic lines to determine the phosphoinositide content of the ciliary membrane and to test the role of phosphoinositides in cilia morphogenesis and function. My studies reveal that specific types of phosphoinositides are found in the ciliary membrane and that their content plays an important role in cilia morphology and function. Moreover, I generated a zebrafish model of a human ciliopathy to test whether some aspects of its mutant phenotype can be alleviated by manipulating the phosphoinositide content in the ciliary membrane. Finally, my studies developed a method that is generally applicable to the manipulation of ciliary content of other PIs in a living vertebrate using other enzymes.

Changes in bone characteristics during soft tissue putrefaction were investigated over 140 days, equating to between 638 and 1450 cumulative cooling degree days (CCDD) depending on ambient temperature using a porcine experimental model in surface and burial depositions. The hypothesis that changes observed in bone characteristics during soft tissue putrefaction could be utilised for possible forensic applications was proved. Human bones were tested for comparison. The techniques used were colorimetric analysis of staining, measurement of micro-crack lengths (in the order of 0.1 to 1.0 mm) on fractured bone surfaces under scanning electron microscopy, inductively coupled plasma optical emission spectroscopy elemental profiling, thermogravimetric analysis (TGA), zoological mass spectrometry profiling non-collagenous peptide content, and Vickers hardness testing. The findings pertaining to the experimental porcine bone samples were as follows. Stain colour did not equalise between periosteal and fractured cortical bone surfaces. The fracture is widely considered perimortem if said surfaces are homogeneous in colour and postmortem if different. Observed inconsistences in colour change limit the potential of this technique as a potential forensic test of postmortem interval (PMI). After 28 CCDD, shorter intersecting micro-cracks changed to longer linear micro-cracks tracking lamellae. A longitudinal to tangential Vickers hardness (HV) ratio of 1.5 to 1 associated with minimal decomposition indicated 250 CCDD or less elapsed. The same ratio associated with marked decomposition indicated 1450 CCDD or more elapsed. A ratio of less than 1:1 indicated 250 to 1450 CCDD. Decreases in iron, sodium and potassium concentrations associated with tissue fluids can determine if bone is in the early stages of decomposition. TGA correlation of water loss between 22 and 100˚C with observed changes in micro-crack lengths, HV, and elemental profiles suggested progressive dehydration as the underlying common factor. These techniques demonstrated some potential to be developed as forensic tests of PMI. As no correlation with PMI was evident with proteomic profiling of non-collagenous peptides, no such potential was demonstrated.

An important group of inherited metabolic disorders in man produce abnormal excretion of organic acids in the urine. Diagnosis is usually based on identification of the abnormal metabolites, working back from there to characterise the defect at enzymatic level. The chirality of these metabolites may be significant in that the different enantiomers of a substance usually have different metabolic origins. Thus, knowledge of the chirality of a metabolite aids in the understanding of the mechanism of the disorder. The chirality of a number of urinary metabolites in inherited metabolic disease was examined using gas chromatography-mass spectrometry, most of the analyses being performed using single ion monitoring. This analytical method requires the use of chiral reference compounds of known configuration. Chiral lactic and glyceric acids are available commercially, but a range of chiral beta-hydroxy acids were prepared by the reduction of the corresponding beta-ketocarboxylic acid esters with fermenting baker's yeast. After hydrolysis of the esters, separation of the enantiomers is based on their reaction with a suitable chiral reagent to form a volatile mixture of the diastereoisomers which are resolved by gas-liquid chromatography using a capillary column with a non-chiral stationary phase. Information from the analysis of the chiral standards was used to assign the absolute configuration of the following urinary metabolites; lactic acid in an unusual case of lactic aciduria, glyceric acid in the glyceric acidurias, beta-hydroxybutyrate in ketonuria, beta-hydroxyvalerate in propionic acidaemia, 2-methyl beta-hydroxybutyrate in beta-ketothiolase deficiency and beta-hydroxyadipic acid in hydroxydicarboxylic aciduria. The biochemical significance of the chirality of each of these metabolites is discussed. The assigning of configuration to urinary beta-hydroxyvalerate in propionic acidaemia, 2-methyl beta-hydroxybutyrate in beta-ketothiolase deficiency and beta-hydroxyadipic acid in hydroxydicarboxylic aciduria represents original work and has helped to elucidate the metabolic origins of these compounds.

The aim of this thesis was to develop online biosensing systems for dialysate tissue metabo- lite detection in real time, to provide an insight into the health of tissue in various in vivo applications. An autocalibration system was developed using LabSmith programmable components to improve the accuracy of results obtained over long monitoring times. A method of col- lecting dialysate into storage tubes for online analysis while retaining temporal resolution was developed and validated. Microfluidic biosensor systems were developed for online measurement of glucose and lactate. One approach employed the use of biosensors, using a combined needle electrode with enzyme encapsulated in a hydrogel layer. The dynamic range of the biosensors was extended by adding an outer polyurethane layer. An alternative approach used automated syringe pumps and valves to develop a microfluidic system for in-flow enzyme addition to the dialysate stream. The existing rsMD system was applied for detection of tissue ischaemia during and after free flap surgery, by measuring dialysate glucose and lactate levels in real time. The system was able to detect flap failure, both during surgery and afterwards in the intensive therapy unit (ITU), much earlier than traditional methods. The rsMD system was adapted to enable monitoring of lactate levels in two dialysate streams and was applied for monitoring isolated porcine kidneys during two methods of cold preservation and subsequent re-warming. Significant differences in the lactate concentrations were observed between the two techniques. The system was extended for use with human transplant kidneys and with both porcine and human pancreases. A novel 3D printed wearable biosensor system was developed for direct integration with a clinical microdialysis probe. The system considerably improved the lag time and dispersional smearing compared with the existing rsMD system. The device was used in a proof-of-concept study with wireless potentiostats to monitor cyclists during exercise.

The axial mesoderm is a specialised population of cells lying at the midline of the embryo. It is composed of two cell populations: the anterior prechordal mesoderm (PM), bounded posteriorly by the notochord (NC). A wealth of studies have shown that both PM and NC are key organising centers that pattern and regionalise the overlying neuroectoderm into fore-, mid-, hindbrain and spinal cord. However, it is unclear how the axial mesoderm becomes regionalised into PM and NC with a sharp boundary established between the two domains. Here I use the chick embryo to address this question. One of the reasons that studies into the development of axial mesoderm have been hampered is due to the lack of an exclusive marker of the PM. Here, I show that Tbx18 is a novel and exclusive marker of the PM and is expressed once the axial mesoderm has fully extended. Much emphasis has been placed in the literature upon the importance of Nodal signalling in axial mesoderm formation, however, little is known about its role in the fully extended axial mesoderm. Here, I show that Nodal initiates Tbx18 expression in the fully extended axial mesoderm, i.e. acting to further specify PM. My studies reveal, further, that Nodal signalling is inhibited by the paraxial mesoderm and retinoic acid. Together, the antagonistic signals appear to establish the posterior limit of the PM and the anterior limit of the NC. Finally, I find that Tbx18 sharpens the PM-NC boundary by downregulating the NC marker 3B9 and establishing a third subpopulation of Shh- axial mesoderm that lies at the PM-NC interface. I discuss the potential significance of this third axial mesoderm population.

Developmental mechanisms provide insight into potential therapies for auditory regeneration. Gata3 is one of the earliest expressed transcription factors during auditory development and it is essential for the development of the auditory sensory epithelium. Haploinsufficiency in man manifests as hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. In a heterozygous mouse model of this condition, hearing loss has an early onset that is apparently associated with functional defects in outer hair cells but the cause of hearing loss is unknown. Our aims were to identify the earliest electrophysiological deficits in hair cells to explain the observed hearing loss in HDR syndrome and to characterize the developmental function of Gata3 in the auditory sensory epithelium. We then proposed to identify factors that might upregulate Gata3 and restore hearing. Electrophysiological recordings of embryonic, neonatal and young adult outer hair cells showed that they differentiate normally in heterozygous mice, although some die at very early postnatal stages. However, inner hair cells suffer deficits in the function of the potassium conductances Ik;f and Ik;n at the onset of normal hearing. When Gata3 was knocked down selectively in inner hair cells from around embryonic day E16, similar deficits were observed. This is the first evidence that Gata3 has a cell autonomous function in the physiological differentiation of hair cells. Conditionally immortal, Gata3 reporter cell lines were then derived from otocysts of mice carrying the SV40 immortalising gene and a BAC construct with the Gata3 enhancer region linked to Egfp. The Gata3 reporter faithfully reproduces the expression pattern of endogenous Gata3 in the BAC transgenic mouse during normal inner ear development and it correlates with GATA3 protein in the reporter cell lines. These lines can thus be used for more detailed studies on how Gata3 regulates functional expression of potassium conductances in hair cells. More importantly, they can be used to screen for small molecules and drugs that might be able to upregulate Gata3 in vitro, which could potentially rescue the phenotype of HDR syndrome and provide an important therapeutic component for sensory regeneration.

Regeneration is the ability of organisms to restore their structures in form and function. While it is present in the complete animal kingdom, humans retain the capacity to restore some tissues and organs, but this capacity is limited in comparison with that of other vertebrates such as the zebrafish. Further, the growth of regenerative diseases and the lack of therapies to fully restore damaged organs and limbs highlight the importance of the study of regeneration. To further the understanding of the regeneration process, this study focuses on molecular and cellular dissection of the tail regeneration of zebrafish larvae. The molecular dissection found that the molecular signalling Hedgehog regulates regeneration through the Wnt and Fibroblast Growth Factor (FGF) developmental pathways as follows. The regeneration marker genes msxc, dlx5a and raldh2 are regulated by Hedgehog (Hh) through Wnt signalling, while cell proliferation is regulated by Hh through FGF signalling. Further, the role of Hh during regeneration is unique and different to its role during normal tail development, where it is not essential. Finally, it was found that the muscle differentiation marker myod is expressed sequentially after the regeneration marker genes raldh2 and dlx5a during the late stage of tail restoration. For the cellular dissection cell lineage tracings during regeneration were performed using several Cre expressing lines with tissue specific promoters, where Cre recombination is controlled by 4OH Tamoxifen administration. The lineage tracing analysis showed that both blood vessels and periderm cells participate during tail restoration and that both cell types remain lineage committed. Altogether the results presented in this thesis show that the zebrafish larva tail is a suitable model to study both molecular and cellular aspects of regeneration. The knowledge generated during this PhD research can contribute to set the basis for the development of clinical therapies to assist human regeneration.