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

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)

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.

Mammalian skin is a dynamic organ that constantly undergoes self-renewal during homeostasis and regenerates in response to injury. Crucial for the skin’s self-renewal and regenerative capabilities is the epidermis and its stem cell populations. Here we have interrogated the role of primary cilia and Intraflagellar Transport 20 (Ift20) in epidermal development as well as during homeostasis and wound healing in postnatal, adult skin. Using a transgenic mouse model with fluorescent markers for primary cilia and basal bodies, we characterized epidermal primary cilia during embryonic development as well as in postnatal and adult skin and find that both the Interfollicular Epidermis (IFE) and hair follicles (HFs) are highly ciliated throughout development as well as in postnatal and adult skin. Leveraging this transgenic mouse, we also developed a technique for live imaging of epidermal primary cilia in ex vivo mouse embryos and discovered that epidermal primary cilia undergo ectocytosis, a ciliary mechanism previously only observed in vitro. We also generated a mouse model for targeted ablation of Ift20 in the hair follicle stem cells (HF-SCs) of adult mice. We find that loss of Ift20 in HF-SCs inhibits ciliogenesis, as expected, but strikingly it also inhibits hair regrowth. Closer examination of these mice reveals that Ift20 is crucial in maintaining HF-SC identity. Specifically, ablation of Ift20 in HF-SCs results in loss of SOX9 expression in HF-SCs and results in ectopic expression of the IFE marker KLF5 in HF-SCs. Additionally, ectopic differentiation is observed in HF-SCs following loss of Ift20. Finally, using both in vitro and in vivo models, we also characterize the role of primary cilia and Ift20 in epidermal wound healing. We find that loss of Ift20 slows collective keratinocyte migration in vitro and also slows HF-SC migration in vivo during wound repair. Interestingly our data suggests that Ift20 regulates keratinocyte migration in a primary cilia-independent manner. Instead, we find that Ift20 mediates focal adhesion (FA) turnover during keratinocyte migration. Specifically, Ift20 together with Rab5, regulates recycling of FA integrins and loss of Ift20 inhibits proper return of integrins to the keratinocyte surface. Overall, we demonstrate that the epidermis is highly ciliated throughout development and in postnatal skin. We show that Ift20 is crucial in maintaining HF-SC identity and the telogen to anagen transition in HFs. We finally demonstrate that Ift20 regulates keratinocyte migration independent of its function in ciliogenesis and instead regulates recycling of FA integrins through a Rab5 dependent mechanism.

An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.

Current sperm processing methods used in assisted reproductive technologies can cause damage to the sperm cell. New ways that mimic the natural guidance mechanisms present in the female genital tract may offer ways to sort sperm with better fertility parameters. Sperm that respond to these cues may have better over sperm health. Human sperm exhibit positive rheotaxis by orienting and swimming against the fluid released by the female genital tract. At certain flow rates sperm can actively orient and swim against the flow. Sperm retrieved that exhibit positive rheotaxis have higher motility and better morphology than the original semen sample. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 106-108). Also available via the Internet.

Thesis (doctoral)--University of Stockholm, 1983. / Includes bibliographical references (p. 39-44 (first group)).

Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.

Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary in order to maintain neuronal cilia. Regional heterogeneity in the effect of this gene mutation on neuronal cilia suggests that the functions of some brain regions might be more compromised than others.