published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

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The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2010-11 / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Quantitative biology is an emerging field that attracts intensive research interests. Pattern formation is a widely studied topic both in biology and physics. Scientists have been trying to figure out the basic principles behind the fascinating patterns in the nature. It’s still difficult to lift the complex veil on the underling mechanisms, especially in biology, although lots of the achievements have been achieved. The new developments in synthetic biology provide a different approach to study the natural systems, test the theories, and develop new ones. Biological systems have many unique features different from physics and chemistry, such as growth and active movement. In this project, a link between cell density and cell motility is established through cell-cell signaling. The genetic engineered Escherichia coli cell regulates its motility by sensing the local cell density. The regulation of cell motility by cell density leads to sequential and periodical stripe patterns when the cells grow and expand on a semi-solid agar plate. This synthetic stripe pattern formation system is quantitative studied by quantitative measurements, mathematical modeling and theoretical analysis. To characterize the stripe pattern, two novel methods have been developed to quantify the key parameters, including cell growth, spatiotemporal cell density profile and cell density-dependent motility, besides the standard molecular biological measurements. To better understand the underlying principle of the stripe pattern formation, a quantitative model is developed based on the experiments. The detailed dynamic process is studied by computer simulation. Besides, the model predicts that the number of stripes can be tuned by varying the parameters in the system. This has been tested by quantitatively modulation of the basal expression level of a single gene in the genetic circuit. Moreover, theoretical analysis of a simplified model provides us a clear picture of the stripe formation process. The steady state traveling wave solution is obtained, which leads to an analytic ansatz that can determine the phase boundary between the stripe and the no-stripe phases. This study does not only provide a quantitative understanding about the novel mechanism of stripe pattern formation, but also sets an good example of quantitative studies in biology. The techniques, methods and knowledge gleaned here may be applied in various interdisciplinary fields. / published_or_final_version / Physics / Doctoral / Doctor of Philosophy

The aim of this dissertation is to develop theoretical models for the motion of rigid and flexible particles through very tight spaces. The geometries of conduits which will be investigated are cylindrical tubes, parallel plane walls and rectangular channels. This work is motivated by an interest in the flow and deformation of single red blood cells in very narrow capillaries, in spleen and in bone marrow. Mammalian red cells are highly flexible, but their deformations satisfy two significant constraints. They must deform at constant volume, because the contents of the cell are incompressible, and also at nearly constant surface area, because the red cell membrane strongly resists dilation. Consequently, there exists a minimal tube diameter below which passage of intact cells is not possible. A cell in a tube with this diameter has its critical shape: a cylinder with hemispherical ends. The motion of red cells is analysed using lubrication theory. When the tube diameter is slightly larger than the minimal value, the cell shape is close to its shape in the critical case. However, the rear end of the cell becomes flattened and then concave with a relatively small further increase in the diameter. The changes in cell shape and the resulting rheological parameters are analysed using matched asymptotic expansions for the high-velocity limit and using numerical solutions. A rapid decrease in the apparent viscosity of red cell suspensions with increasing tube diameter is predicted over the range of diameters considered. The red cell velocity is found to exceed the mean bulk velocity by an amount which increases with increasing tube diameter. The same type of analysis is applied to the flow and deformation of red blood cells between two parallel plates with near-minimal spacings. First, the critical shape of the particle and the minimum gap width are determined using calculus of variations. In this case, it is a disk with a rounded edge. The flow in the plasma layers between the cell and the plates is described using lubrication theory. Approximate solutions can be obtained using a locally two-dimensional analysis at each point of the rim of the cell. Cell shapes, pressure distributions, membrane stresses and particle velocities are deduced as functions of geometrical parameters. One significant finding is that the gap width between the cell and the wall decreases with distance from the axis of symmetry parallel to the flow direction. The red cell velocity may be smaller or larger than the mean fluid velocity far from the cell, depending on the spacing of the plates, with equality when the width of the red cell is about ninety percent of the spacing between plates. The same procedure is also applied to rectangular channels.

The ability of mammalian cells to locomote is important in a variety of normal and pathological processes. Previous work has suggested that low doses of x-irradiation may perturb cell motility, a finding that may have important consequences in embryogenesis, cancer metastasis, and immune response. This thesis has sought to study in more detail the effect of radiation on mammalian cell motility. Work performed in other laboratories used the colloidal gold assay and time lapse cinemicroscopy to study x-irradiation induced changes to 3T3 fibroblast motility in tissue culture. These studies were repeated here, with qualitative results similar to those reported earlier. However, these methods were not amenable to a detailed quantitative analysis. For this, spatial and temporal information on the motility and dynamic morphology of a large number of cells is required. Such a task would be impossible to perform manually, thus an automated microscope system was developed that used a computer-driven precision stage and a solid state optical sensor to track individual cells in tissue culture. Information on motility and morphology was concurrently extracted from many cells. As part of the thesis, several techniques were developed to analyze and display these data, and to correlate motility and morphology observations. These techniques were directed at preserving the actual process of 3T3 cell motility, and parameters were measured to quantify the short term persistence of cell movement (on a time scale of 0.5 to 2 hours), and the long term persistence of cells in maintaining certain characteristic behaviour (on a time scale of 3 to 12 hours). The response of 3T3 fibroblasts to x-irradiation was characterized by a number of parameters. The population average cell speed was measured following treatment, and a dose response and time response was determined in the range of 1.5 Gy. Other motility parameters indicate that the normal process of cell motility, evidenced by a series of motile segments, was disrupted by x-rays. This was thought to reflect perturbation to the control mechanisms of cell motility. The morphology of 3T3 cells stained with Coomassie blue was examined in an effort to correlate the observed motility changes with changes in the fixed cell morphology. This stain is a general structural protein stain with higher affinity toward microfilaments. High doses of x-rays were required to produce significant perturbation to cell morphology, and in the dose regime of interest, the morphology of irradiated cells was not identifiably different from control. Of note is that it was the well spread, quiescent cells that seemed least perturbed by large doses of irradiation. In summary, x-rays apparently disrupt the normal process of cell motility. Several lines of evidence suggest that actively migrating cells are the most perturbed by irradiation. This work has developed techniques to quantify cell motility in a meaningful way, and to characterize the x-ray induced perturbations. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The regulation and maintenance of normal cell movements and shape play a vital role in the normal development and health of every living thing. The characterization of 6 zebrafish Zipper Interacting Protein Kinase homolog has helped to better understand how changes in cell cytoskeletal elements can lead to changes in cell shape and movement. Zebrafish are ideal model organisms for studying ZIPK because it has been previously shown that zebrafish ZIPK has closer sequence homology to human ZIPK than rodent ZIPK, and because zebrafish embryos are ideal for studying cell shape and movement in vivo. Using Whole Mount In Situ Hybridization we found that the zebrafish ZIPK is expressed during all stages of embryonic development, but most importantly during gastrulation when cell motility and changes in cell shape can best be studied. To determine where zebrafish ZIPK is expressed at the sub-cellular level, GFP-ZIPK and Flag-ZIPK clones were created and used for transfecting into Hek293T cells and Hela Cells. From these transfections, cell counterstaining and confocal microscopy we found that ZIPK is expressed ubiquitously throughout the cell, although mainly cytoplasmic. To study the effects on cell shape various ZIPK mutants were created through site-directed mutagenesis. These mutants were made to study the effects of the kinase domain of the protein, or other functional domains within the protein. From these studies it was shown that ZIPK does affect cell shape through changes in the actomyosin cytoskeleton resulting in aberrant cytoskeletal structures. Finally, we have also shown through phosphorylation assays that ZIPK phosphorylates and thus regulates MYPT-1, a scaffolding protein of the myosin protein phosphate complex and directly phosphorylates myosin light chain, both of which play a role in changes in cell shape and movement.

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Includes list of publications by the author. Includes bibliographical references (leaves 103-123) Evaluates the nature of the interactions occurring between semen and cells of the uterus that occur following mating in pigs. Describes a novel ability of porcine seminal plasma to induce dose dependent cell-cell adhesion and mitogenesis amongst peripheral blood lymphocytes in vitro.