Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra of the brain. The pathogenesis and etiology of PD are unclear. Mitochondrial dysfunction occurs in PD, causing a decrease in complex I activity in postmortem brain, and exacerbating reactive oxygen species production and ATP deficiency contributing to neuronal cell death. Mutation of leucine-rich-repeat kinase 2 (LRRK2) gene is the most common genetic factor identified in both familial and sporadic PD cases. Several mutations in LRRK2 have been linked to PD, in which R1441G is the second commonest mutation after G2019S. LRRK2 protein is ubiquitously expressed in human body, in which a portion is localized to the mitochondria. Mutations of LRRK2 directly or indirectly cause mitochondria dysfunction. Dysfunction of mitochondrial respiratory complexes has been described in skin fibroblasts and skeletal muscle of PD patients. Therefore, these clinically accessible tissues are good for monitoring disease progression. The objectives of this study were to investigate how LRRK2 R1441G mutation affects normal mitochondrial function, and whether this specific LRRK2 mutation potentiates age-dependent deterioration of mitochondrial function. To achieve these aims, colonies of skin fibroblast carrying LRRK2 R1441G mutation or wild-type LRRK2 were derived from a novel LRRK2 R1441G knock-in (KI) mouse model and its wild-type (WT) littermates. Skeletal muscles were dissected from the hind legs of WT and KI mice. The effects of aging and LRRK2 R1441G mutation on mitochondrial function were investigated in vitro using these derived skin fibroblast cultures, and ex vivo using skeletal muscle obtained from young (3-month-old) and aged (18-month-old) WT and KI mice. Reduction-oxidation activities of mitochondrial complex I and complex II in skin fibroblasts and skeletal muscle were measured spectrophotometrically. Intracellular ATP levels in skin fibroblasts were determined by bioluminescent assay. Phase-contrast microscopy showed that aging and LRRK2 R1441G mutation did not affect cell morphology of the derived skin fibroblast cultures. Complex I activity determined in skin fibroblasts and skeletal muscle derived from KI and their WT littermates revealed that, aging caused a significant increase in complex I activity in WT but not KI skin fibroblasts. Conversely, a significant decrease in complex I activity was observed in both WT and KI skeletal muscle, demonstrating an aging effect ex vivo. LRRK2 R1441G mutation did not affect complex I activity in WT and KI skin fibroblasts and skeletal muscle. Moreover, complex II activity in these two tissues was neither affected by aging nor R1441G LRRK2 mutation. Intracellular ATP levels in the skin fibroblast cultures were also unaltered by aging and LRRK2 R1441G mutation. In conclusion, my current findings indicated a significant aging effect on mitochondrial complex I activity ex vivo, supporting the role of age-dependent deterioration of complex I activity in mitochondrial dysfunction of PD. LRRK2 R1441G mutation did not affect complex I and II activities in both skin fibroblasts and skeletal muscle. Also, this mutation did not potentiate the age-dependent deterioration of complex I activities as observed in skin fibroblasts and skeletal muscle of the LRRK2 R1441G knock-in mice. / published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics / Master / Master of Philosophy

published_or_final_version / Orthopaedic Surgery / Master / Master of Philosophy

No description available.

Congenital heart disease (CHD) is the major cause of death in the first year of life, the estimated incidence being 0.5-5% of live births; therefore it is important to understand the genetic causes underlying the complex process of heart formation to help prophylaxis, diagnosis and treatment of affected patients. CHD is the commonest phenotype associated with left-right (LR) disorders. LR asymmetry is determined during embryonic development. The three major body axes ? antero-posterior, dorso-ventral and left-right ? are patterned at gastrulation. LR asymmetry is established shortly after the two other major axes are patterned. The process of LR determination can be sub-divided into four integrated steps: 1. breaking of molecular symmetry in the gastrulation organizer; 2. transfer or relay of this asymmetric information to the lateral plate mesoderm (LPM), from which most internal organs will be formed; 3. reinforcement and propagation of asymmetric cues throughout the LPM and 4. conversion of asymmetric molecular information into proper organ morphogenesis. The goal of this work is to investigate mechanisms involved at two specific points in the laterality pathway: the initial generation/maintenance of asymmetric gene expression in the LPM and the morphogenetic translation of these early events into correct heart formation in the mouse. My emphasis has been on the characterization of laterality targeted cells via careful analysis of Pitx2c expression using a Pitx2c-lacZ reporter transgene, the role of BMP signalling, via Smad1, in generation/maintenance of early asymmetric signalling in the LPM, and the later involvement of both Smad1 and Pitx2 in cardiac morphogenesis through analyses of knockout mice.

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population. The main treatments for schizophrenia are antipsychotic drugs that target dopamine receptors, yet the underlying biological mechanisms through which they alleviate the symptoms of schizophrenia remain ill defined. In this study, we used microarray analysis to profile the expression changes of thousands of genes simultaneously, following antipsychotic drug treatment of mice. Mice were treated chronically (28 days), or for a novel intermediate time-point (7 days), with one of three antipsychotic drugs: clozapine, haloperidol or olanzapine. The use of three drugs enabled us to discern antipsychotic-specific effects co-regulated by multiple drugs, rather than the side effects of individual compounds. Transcript profiling and validation by quantitative PCR of whole brain tissue revealed antipsychotic drug regulation of genes in diverse biological pathways, including: dopamine metabolism, neuropeptide and second-messenger signalling, neurogenesis, synaptic plasticity, cell adhesion, myelination, and voltage-gated ion channels. The regulation of voltage-gated channels by antipsychotic drugs has been suggested previously by electrophysiological studies, although thorough analysis has not been undertaken in vivo. Therefore, the second aim of this study was to characterise the regional mRNA and protein expression of two genes altered by multiple APDs, the voltage-gated potassium channel ??-subunit (Kcna1) and voltage-gated potassium channel interacting protein (Kchip3). Regional characterisation and expression analyses were carried out by immunohistochemistry, in situ hybridisation, and Western blot analysis of mouse brain regions of interest to schizophrenia and its treatment. Following 7-day haloperidol treatment we observed up-regulation of Kcna1 in the striatum and dentate gyrus, with increased protein in the striatum, hippocampus and midbrain; and down-regulation of Kchip3 in the striatum, with decreased protein in the cortex, hippocampus and midbrain. These studies implicate voltage-gated potassium channels in the antipsychotic drug regulation of midbrain dopaminergic neuronal activity, adult neurogenesis and/or striatothalamic GABAergic neuronal inhibition. These findings indicate that regulation of potassium channels may underlie some of the mechanisms of action of antipsychotic drugs, and that voltage-gated ion channels may provide alternative drug targets for the treatment of schizophrenia.

Copies of author's previously published articles inserted. / Bibliography: leaves 117-130. / v, 130, [51] leaves, [33] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The general aim of this thesis is to develop methods for the identification of markers for pluripotent cell subpopulations in the developing mouse embryo. A screen for mouse embryonic stem (ES) cell markers is carried out, to identify transcripts that are differentially expressed between ES cells and X cells, and to investigate pluripotent cell heterogeneity during early development. The study demonstrates the potential to identify and characterise molecular heterogeneity within the developing pluripotent cell pool in vivo, via the controlled progression and analysis of pluripotent cells in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997