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Typewritten copy / Includes bibliographical references (leaves 141-144) / [5], 144 leaves : ill. ; 27 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / An account of experimental investigations carried out from January 1952 to September 1954. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physics, 1956

Thesis (M.Res.(Med.))--University of Hong Kong, 2007.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Melbourne, Dept. of Paediatrics, The Bruce Lefroy Centre for Genetic Health Research, The Murdoch Childrens Research Institute, 2009. / Typescript. Includes bibliographical references (leaves 183-207)

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references.

Primary cilia are cellular organelles that protrude into the extracellular space, acting as antennas. They detect a wide range of chemical cues, including SHH and PDGF, as well as fluid flow, and they modulate downstream signalling systems, such as WNT and ERK. Due to this cue-sensing ability and the close association of the primary cilium with the centrosome the organelle is able to influence both cell cycle progression and cell migration. This work investigated the effect of mutations on two genes associated with primary cilia: Arl13b and Ift172. The effects of the HNN genotype of Arl13b and the WIM genotype of Ift172 on cell migration were assessed uniquely within the context of direct current electric fields. Both cell lines showed a decreased migratory response when compared to WT cells, despite no clear involvement of cilia in sensing the direction of the electric field. This corroborated with previous data of in vivo Arl13b cellular migration. Through the use of in utero electroporation the migratory deficits of IFT172 knock down were then confirmed in vivo in the developing mouse neocortex. Further in vitro investigation revealed a slower proliferation rate of HNN and WIM cells, though this was not confirmed in vivo after IFT172 knock down using a standard BrDU protocol. Nevertheless, further in vitro investigations revealed a wide variety of cell cycle and intracellular changes within both cell lines. The commonalities included lower numbers of cells in the S-phase and lower MAPK3 phosphorylation compared to WT, and differences such as GSK3β phosphorylation on Ser9. This work showed for the first time that ciliopathies affect galvanotaxis, and revealed fundamental commonalities in cell migration and proliferation between various ciliary mutations, as well as differences in specific signalling pathways. This will hopefully aid in developing future therapeutic interventions for ciliary diseases.

Lithium is given to millions of bipolar disorder or post-traumatic disorder patients. The recent studies also support a role for lithium in treating neurodegenerative disease such as Parkinson's disease and stroke. Lithium treatment leads to lithium nephropathy, which includes lithium-induced nephrogenic diabetic insipidus (NDI), lithium-induced renal cell proliferation leading to the formation of microcysts in the kidney, and lithium-induced renal fibrosis. However, there is still a gap in understanding the mechanisms and signaling pathways involved in regulating lithium-induced nephropathy. mTOR pathway activation and primary cilia are known to be associated with the abnormal renal cell proliferation and the formation of renal cysts in polycystic kidney disease, a renal disease model similar to our lithium model. The activation of hedgehog pathway is associated with the renal fibrosis observed in the unilateral ureteral obstruction and unilateral ischemia reperfusion injury models of chronic renal injury. Thus, I hypothesize that mTOR signaling pathway, primary cilia and hedgehog pathway may all contribute to lithium-induced nephropathy. To address the hypothesis that the mTOR signaling pathway may be responsible for lithium-induced renal collecting duct proliferation, mTOR pathway activation was assessed in lithium-treated mice and lithium-treated mouse inner medullary collecting duct (mIMCD3) cells. Lithium activated mTOR signaling pathway in renal collecting duct cells both in vivo and in vitro. Rapamycin, an inhibitor of mTOR, blocked lithium-induced renal cell proliferation in renal cortex and medulla in vivo and in renal collecting duct cells in vitro, supporting the hypothesis. However, rapamycin did not improve lithium-induced reduction of urine osmolality, suggesting mTOR signaling pathway may not contribute to lithium-induced NDI. To address the hypothesis that primary cilia may be necessary for lithium-induced mTOR activation and renal cell proliferation, primary cilia deficient cells were used to assess mTOR pathway activation and cell proliferation in response to lithium treatment. The absence of primary cilia abolished lithium-induced activation of mTOR pathway and cell proliferation, which supports the hypothesis. To address the hypothesis that lithium elongates primary cilia length, which is mediated by mTOR signaling pathway, primary cilia length alternation was assessed in the kidney and in mIMCD3 cells in response to lithium treatment. Lithium increased primary cilia length in renal collecting duct cells of cortex, outer medulla, and inner medulla kidney regions in vivo and in mIMCD3 cells in vitro. Rapamycin reversed lithium-induced elongation of primary cilia in renal cortical and outer medullary collecting duct cells in vivo, and blocked the increase of primary cilia length in mIMCD3 cells in vitro, which support the hypothesis. To address the hypothesis that lithium activates the hedgehog pathway in a Smoothened (smo, a key regulator of the hedgehog pathway)-dependent manner in renal collecting duct cells, mIMCD3 cells were treated with lithium or lithium/Smo inhibitor or lithium/Smo activator. Hedgehog signaling pathway is activated by lithium in mIMCD3 cells, which is partially Smo-dependent. However, the role of hedgehog signaling pathway in regulating lithium-induced fibrosis was not assessed in the study. Future studies are required to determine the role of the hedgehog pathway in the lithium model.

Drosophila melanogaster is a widely used and efficient genetic model to study nervous system development. The conservation of many genes from Drosophila to vertebrates and a short reproduction cycle makes the fruitfly a great tool for providing insight into crucial events in nervous system formation. In studying the development of the sensory nervous system, Drosophila also provides a model for understanding the formation and function of structurally diverse cilia. Cilia are hairlike organelles present throughout our bodies and responsible for many processes such as chemo, mechano, and thermosensation, fluid movement, hearing and fertility. In Drosophila the only somatic ciliated cells are the Type I sensory neurons in which a cilium forms the sensory dendrite. There are more than two diverse subtypes of the ciliated sensory neurons and the mechanism by which this diversity is achieved remains unclear. The mechanism of ciliated sensory neuron differentiation was hereby studied on an example of a differentially expressed ciliary gene - CG6129 - a Drosophila orthologue of human Rootletin, a main protein components of ciliary rootlets. CG6129 expression is specific to the ciliated cells and exhibits so called chordotonal-enriched pattern - a strong and permanent expression in the chordotonal subtype of type I neurons and weaker and transient expression in the external sensory subtype. I have shown that CG6129 knock-down causes severe disruption of the chordotonal organs function without any obvious change in the structure of the cilium, other than the lack of ciliary rootlet. The function of the external sensory subtype was only slightly affected which further highlights the difference between the two types of ciliated sensory organs. The fact that CG6129 is differentially expressed in the two subtypes of the Drosophila ciliated sensory neurons suggests that the genes involved in the formation of various cilia are differentially regulated. I have shown that CG6129 is regulated by the two well known ciliary transcription factors - RFX and fd3F (distant homologue of Foxj1). Of the two enhancers found the early-to-late enhancer is almost entirely dependent on RFX and not on fd3F while the late enhancer is dependent on both fd3F and RFX. The fact that there is some residual CG6129 expression in the absence of both RFX and fd3F suggests involvement of another regulator that may contribute to the cilia diversity. Zmynd10 is a recently characterised ciliary gene that is involved in the axonemal dynein arms assembly. Mutations in human Zmynd10 cause primary ciliary dyskinesia (PCD) and Drosophila Zmynd10 mutants have immotile cilia that lack dynein arms. Due to the presence of specific protein domains Zmynd10 has been suggested to act as a transcriptional regulator. I have shown that the transcript levels of CG6129 and other ciliary genes are reduced in the Zmynd10 mutant. This implies that Zmynd10 may regulate ciliary genes on a transcriptional or post transcriptional level and may contribute to the regulatory network governing ciliogenesis.

Cystic fibrosis (CF) is a common lethal hereditary disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR dysfunction affects multiple organ systems and most morbidity and mortality in CF results from lung disease. The CF lung appears healthy at birth, but spontaneously develops airway disease characterized by infection, inflammation, mucus plugging and airway remodeling. A CF pig model was recently generated to determine the events that initiate lung disease. CF pigs recapitulate many findings seen in humans with CF, including the spontaneous development of lung disease. I used newborn CF pigs to investigate two leading hypotheses regarding CF disease initiation: abnormal airway surface liquid (ASL) composition and defective mucociliary transport (MCT). I developed an assay to study ASL composition and found that CF ASL contained similar sodium concentrations, elevated potassium concentrations, and a decreased fraction of volatile material. I developed an assay to measure MCT in vivo. By tracking individual particles in 3-dimensions I found that newborn pigs exhibit a ventrally directed cilia orientation in the trachea. I also found that MCT is highly heterogeneous and particles traveled at different speeds within airways and between airways, challenging the classic view that airway mucus exists as continuous blanket. Comparing particle transport revealed that non-CF and CF newborn pigs exhibit similar basal particle clearance and speeds. Cholinergic stimulation induces mucus and fluid secretion. Particles became stuck in newborn CF pigs after cholinergic stimulation and stasis persisted with tissue submersion. This challenged the leading hypothesis that attributes CF airway disease pathogenesis to ASL depletion. I hypothesized that adherent mucus impairs mucociliary transport in CF airways and I developed an assay to visualize mucus stasis in submerged tracheal segments ex vivo. CF trachea stimulated in vivo exhibited highly adhesive mucus entities that emerged exclusively from submucosal gland ducts. These adherent entities impaired MCT even with extremely high ASL depths. Non-CF trachea with combinatorial disruption of HCO3- and Cl- transport reproduced the defect in CF signifying that anion transport disruption was responsible for adherent mucus. These data suggest that CFTR disruption directly produces multiple host defense defects, including defective bacterial killing and abnormally adherent mucus. Therapeutic targeting of the described defects may provide new opportunities to intervene early and improve the lives of those with CF.