• Magnetic force imaging and handling of cancer cells on the nanoscale

      Liu, Jinyun (University of BedfordshireUniversity of Bedfordshire, 2017-11)
      Cancer treatment has become one of the top priorities in health. Great efforts have been devoted to the diagnosis and therapy of cancers. Culturing cells with drugs is a common method used to investigate cancer therapy in experiments. However, this method has limitations in cancer treatment because of the lack of capabilities of handling cells, targeting specific cells and measuring the nanoscale changes in cell structures. Magnetic nanoparticles (MNPs) and magnetic force microscopes (MFMs) have been used to study biological samples due to their advantages in tracing, manipulating and measuring, which has motivated to research the method for implanting MNPs into cancer cells, to target the cancer cells and to measure their changes during the treatment. Research reported in this thesis focuses on magnetic force imaging and handling of targeted cancer cells on the nanoscale for possible new cancer therapies. A new differential MFM imaging method and a new compensation MFM imaging method were developed in this research to improve the MFM imaging quality. The former reverses the magnetized direction of probe from upward to downward and the latter scans the samples with three scanning directions of 0°, 45° and 90°. With these methods, the obtained MFM images achieve a high resolution, SNR, image contrast and accuracy. A pair of innovative MNPs picking up method and MNPs releasing method were developed in this research to achieve flexible MNPs picking up and releasing. The picking up method handles the magnetic tip following a helical structure as the capture path when approaching to the target MNPs. The MNPs releasing method uses a biaxiably-oriented polypropylene (BOPP) film together with a magnet allowing MNPs to separate from the MFM tip surface. With these methods, the target MNPs can be picked up by the MFM tip and released from the tip surface successfully. This research discovered, for the first time in the world to the author knowledge, the differences in morphological features (height, length, width and roughness) and mechanical properties (adhesive force and Young‟s modulus) between multinuclear and mononuclear colon cancer cells after treating the cells with fullerenol. This discovery provides guidance to the selection of cells for target treatment. The results indicate that the mononuclear SW480 cells are more sensitive to fullerenol than the multinuclear SW480 cells and the multinuclear SW480 cells exhibit a stronger drug-resistance than the mononuclear SW480 cells. A new MNPs implantation method was developed in this research, which enables the FITC-MNPs functioned tip to insert into cells so that MNPs are implanted into the target cells. Fluorescence microscope images show that the FITC-MNPs are released into the cells successfully. Cells being treated with MNPs (Cell-MNPs) manipulation III methods are explored by magnet and controllable electromagnets to manipulate the target cancer cells. The results show that the cell-MNPs have magnetic force manipulated capability and they can be manipulated to have the leftward, rightward, upward and downward flexibilities.
    • Mechanisms of androgen-induced hypertrophy: lessons from muscle cell models

      Hughes, David Conway (University of BedfordshireUniversity of Bedfordshire, 2014-04)
      Androgen endocrine physiology is responsible for many processes in the body, from bone metabolism to skeletal muscle maintenance. Testosterone is one of the most potent androgens produced, with decades of research highlighting its androgenic-anabolic effect on skeletal muscle growth. The research has been further driven through the emergence of pharmaceutical derivates (steroids) of testosterone and their use in interventions for muscle wasting associated with disease and advancing age (sarcopenia). However, the molecular regulation of testosterone and the influence it has on cellular processes in promoting muscle development, hypertrophy and satellite cell activation (proliferation, differentiation) remain poorly understood. In summary, this thesis has highlighted the impact of testosterone in rescuing an aged phenotype in myoblast cells (PD cells) displaying prior reductions in regeneration and hypertrophy vs. relevant ‘un-aged’ controls (Sharples et al., 2011, Sharples et al., 2012). Importantly, the results provide evidence towards the mechanisms for testosterone induced hypertrophy, where in the presence of testosterone and a functional androgen receptor (shown via AR antagonism/ flutamide administration) testosterone is fundamental in regulating appropriate myoD, myogenin and myostatin expression in the control of myotube differentiation and hypertrophy. Increases in androgen receptor protein levels in PD myoblasts under a testosterone stimulus highlight the intrinsic responsiveness of aged myoblasts to a hypertrophic stimulus which may somewhat explain the use of testosterone as an effective clinical treatment in muscle wasting disease. Finally, myotube hypertrophy occurred in both aged and un-aged myoblasts with testosterone administration even in the presence of an IGF-I receptor inhibitor (Picropodophyllin) suggesting a limited role of the IGF-IR and associated signaling in testosterones mediated increases in differentiation and hypertrophy. Testosterone did appear however to increase Akt phosphorylation in cells that show a prior impaired regenerative capacity (PD cells), even in the presence-IGF-IR inhibitor suggesting that testosterone can directly activate Akt independently of upstream IGF-IR a novel finding that is presented in chapter 5. This interaction maybe mediated by myostatin, where increases in myostatin has been linked with directly inhibiting Akt (Dubois et al., 2014, Léger et al., 2008, Morissette et al., 2009), and in the present study we see an increase in myostatin mRNA and corresponding decrease in phospho-Akt when AR’s (via flutamide) action is inhibited and the protein level of AR is low (chapter 5). Furthermore in chapter 6 we further showed that inhibition of PI3K/AKT using LY294002 was sufficient to remove the prior rescuing of differentiation and hypertrophy in aged cells after testosterone administration. This was coupled with reductions in myostatin and increases in mTOR with testosterone administration. Overall AR seems to be the most fundamental pathway in testosterones induction of myotube differentiation and hypertrophy with important roles for myogenin, Akt, mTOR and myostatin in mediating these processes.