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In this thesis, femtosecond (fs) cellular transfection of Chinese Hamster Ovary (CHO) cells was performed using a tightly focused Gaussian beam. The beam focus was positioned on the cell membrane and three laser doses, each of 40 ms duration, were delivered allowing for the formation of a highly localized pore on the cell membrane. The membrane pore, induced as a result of a multiphoton process known as photoporation, permitted the surrounding DNA to diffuse into the cell cytoplasm. 48 hours after laser irradiation, the viable photoporated cells expressed a red fluorescent protein. The topography of a photoporated cell, targeted with tightly focused fs pulses, was also monitored as a function of the input power using Atomic Force Microscopy. Following this, I generated and implemented a “non-diffracting” quasi-Bessel beam (BB) by means of a conical shaped lens, the axicon, which successfully provided an alternative route for photoporation to the highly divergent Gaussian beam. A comparison was given between the two beam approaches for photoporation. The “non-diffracting” character of the BB resulted in the first successful attempt towards automating optical transfection. This was achieved by using an axicon and a spatial light modulator (SLM) to provide phase modulation on the annular spatial spectrum field of the BB. This approach provided control over the lateral and axial position of the beam with respect to the cell membrane, allowing for point and click photoporation. Successful photoporation of CHO cells was also demonstrated using for the first time an axicon tipped optical fibre. The applicability prospects of this method are significant, ranging from potential endoscopic embodiments of the technique to advanced studies of tissue properties in vitro and in vivo.

Colorectal cancer is thought to originate in the epithelial cells that line the colorectal crypt and, in most cases, is associated with a mutation in Wnt-signalling pathway. These mutations cause cells to alter their proliferative behaviour, make their cytoskeleton less deformable and increase their levels of cell-cell and cell-substrate adhesion. In this thesis we develop three different types of models for the proliferation and movement of epithelial cells in a colorectal crypt. We use these models to investigate how changing the cell adhesion, cytoskeleton and proliferation properties of mutant cells affects their ability to establish a mutant population within the crypt. First we develop a continuum model of two cell populations, normal and mutant, using a spatially-varying source term to model Wnt-dependent proliferation and using Darcy's law to describe cell movement down pressure gradients. We distinguish between mutant cells and normal cells by assuming the former have a spatially independent source term, representing proliferation, and a different viscosity to normal cells, to model changes in their cytoskeleton and levels of adhesion. The model is solved analytically by an asymptotic expansion of the variables and numerically using a collocation method. The results show that the ability of mutant cells to remain in the crypt depends on the position of the initial mutation and their viscosity: the further up the crypt a cell suffers a mutation the more rigid and adhesive the cell must be for a mutation to persist. We then consider a discrete cell-centre model based on the work of Meineke et al. (2001). Cell-cell interaction forces are modelled by springs and are balanced by a viscous drag term. Adaptations to Meineke et al. (2001) include unpinning of stem cells from the bottom of the crypt, dependence of cell-drag on cell size, dependence of cell-cell interaction forces on their area of contact and the inclusion of mutant cells. Using agile software engineering techniques, the software environment, CHASTE, is developed and used to solve the model numerically and to reproduce experimental findings such as crypt homoeostasis and monoclonality. The results again reveal that increasing the drag on the mutant cells increases the likelihood of a mutant population establishing itself within the crypt. The third approach is a discrete cell-vertex model. The model decouples cell-cell adhesion forces from cell deformation forces and movement is determined by a free-energy gradient balanced by a viscous drag term. Numerical simulations show that the model can generate similar results to the cell-centre model, and reveal that increased cell-cell adhesion of the mutant cells increases the likelihood of the mutant population invading the crypt. Finally the three models are compared in terms of their suitability for modelling epithelial tissue.

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The major findings of this investigation are that polyamine biosynthetic enzymes and polyamine levels are regulated by specific cellular growth factors. A serum-free defined medium was developed for the Chinese hamster ovary cell line to examine the regulation of ornithine decarboxylase (ODCase) (EC.4.1.1.17), S-adenosylmethionine decarboxylase (SAMDCase) (EC.4.1.1.50), as well as polyamine catabolism. The activity of ODCase is dependent primarily on the presence of insulin, and appears to be modulated by transferrin and ferrous sulfate, indicating that iron transport may be important in the expression of ODCase activity. The enzyme activity can also be increased by depriving the substrate ornithine, which probably acts through a putrescine mediated event. This substrate limitation leads to an intracellular decrease in putrescine and spermidine, but not spermine. The activity of SAMDCase is not influenced by alterations in the growth factors or by ornithine deprivation. Since the spermidine levels are lower as compared to cells growing in medium with serum, it appears that SAMDCase activity is not generally regulated in a negative manner by spermidine. The polyamine interconversion enzymes, such as spermidine/spermine N¹-acetyltransferase and polyamine oxidase, appear to be regulated by growth factors other than insulin, transferrin, and ferrous sulfate. Cells maintained in defined medium are much more tightly attached to the surface of the dishes in which they are growing, which may be related to the growth factors present or a lack of cellular polyamines. Vinculin, a cell surface protein associated with focal adhesion plaques, moves away from the cell surface and into the nuclear area in defined medium cells as evidenced by fluorescent antibody staining. The major conclusions of this work are that ODCase synthesis is regulated by growth factors, that enzyme activity is also regulated post-transcriptionally by substrate and end-product, and that general polyamine metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism and interconversion of polyamines.

The experiment was conducted at the University of Arizona Campus Agricultural Center to evaluate the effect of soil moisture stress on photosynthesis, transpiration, diffusive resistance, temperature differential, leaf temperature, and specific leaf weight of seven sorghum Sorghum bicolor (L.) Moench cytoplasms represented by nine lines. As soil moisture stress increased, diffusive resistance and leaf temperature increased whereas photosynthesis and transpiration decreased. Temperature differential was highest under high soil moisture stress and lowest under medium soil moisture stress. Specific leaf weight was highest under medium soil moisture stress. Three lines, AKS37, AKS38, and A2Tx398, representing two different germplasms under high soil moisture stress exhibited high photosynthesis and transpiration rates, high specific leaf weights, and low diffusive resistance. Differences in photosynthesis rates under non-soil moisture stress between A1 and A2 cytoplasmic sterility systems were significant.

No description available.

No description available.

The purpose of this study was to develop novel optical microscopy techniques in order to investigate colloidal drug particle endocytosis by mammalian cells. A total internal reflection microscope (TIRM) was initially developed for high resolution cellular imaging. TIRM is a non-fluorescent imaging technique based on the principle of ‘scattering’ of the evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass and air. The key design considerations with respect to development of a TIRM instrument are discussed. The technique is also compared and contrasted to the more commonly known non-fluorescent RICM (Reflection Interference Contrast Microscopy) technique using computer simulations. Time-lapse video TIRM is applied to imaging the interaction between A549 and 3T3 cells, and a polylysine coated substrate. Real-time label-free visualisation of 0.5 and 1 m polystyrene particle endocytosis by living cells is then demonstrated. Modifications to the TIRM system to include a dual-colour fluorescent TIRF (Total Internal Reflection Fluorescence) microscope are described in detail. Results are shown which demonstrate the ability of a combined TIRM/TIRF instrument to selectively image the basal cell membrane both label-free and fluorescently. 3T3 fibroblast cells were genetically modified using standard molecular biology protocols to express the fluorescent fusion protein EGFP-Clathrin LCa (enhanced green fluorescent protein clathrin light chain a). Finally, colloidal particle endocytosis by the genetically modified cell was imaged using the TIRM/TIRF microscope. Direct visualisation of the internalisation of 500 nm particles via clathrin coated pits in 3T3 cells was shown for the first time.

Freshwater planarian Schmidtea mediterranea is an emerging model for studying in vivo gene functions and regulation in native cell niches. The obligate asexual strain of this species reproduces by fission, in which succession of soma occurs without passing through the germline. To achieve this somatic immortality the somatic stem cells need to overcome the end replication problem. Therefore it can be hypothesised that somatic telomere maintenance in asexual S. mediterranea must possess a germ-like property, with which age-related erosions can be adequately repaired. In this PhD project, the telomere repeat unit in S. mediterranea was confirmed to be the vertebrate-like TTAGGG. Attrition of whole body telomere length was found in ageing sexual worms and also in asexual worms which had not gone through recent fission events. Opposite telomere length dynamics were observed in regenerated samples of the two strains, with erosion in the sexuals and reset in the asexuals. The telomere maintenance was found to increase during regeneration in both strains, with a higher level of increase in asexual worms. A homolog of the telomerase reverse transcriptase subunit, Smed_Tert, was identified and characterised in this organism. High level of Smed_Tert expression was seen in germ cells in mature sexual worms and adult stem cells in asexual worms. Knockdown of Smed_Tert expression by RNA interference caused progressive telomere erosion, however effects on cell proliferation and viability have not been observed in knockdown samples. Four alternate splice isoforms of Smed_Tert were identified. The enhanced telomerase activity during regeneration correlates with a proportional increase in the full-length isoform and a decrease in isoforms with a truncated TRBD domain, suggesting a dominant negative regulation of telomerase by alternative splicing. Significant increase in the expression of the full-length isoform was seen in regenerating asexual samples but not in sexual strains, which correlates with their telomere length dynamics. It is hoped that the comparative studies between the sexual and asexual strains can improve our understanding of how soma can evolve to become an effective inheritable unit.