Multicellular development in fungi is fundamentally different from that of animals or plants. In filamentous fungi, multicellular structures are formed by aggregation and adhesion of hyphae, followed by septation and specialisation of hyphal compartments within the aggregate. The perithecium, a flask-shaped sexual fruitbody produced by both Neurospora crassa and Sordaria macrospora, provides a model system in which to study fungal multicellular development. This study presents a detailed description of the morphological stages of perithecial morphogenesis in N. crassa and S. macrospora and its early stages, the ascogonial and protoperithecial stages, using a range of microscopical techniques. Details of the development of several mutants impaired in perithecial development are described, including: gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; and three mutants pro22, pro40 and pro41 of S. macrospora, and their corresponding gene-deletions in N. crassa. The results confirm that all three MAP kinase cascades are required for sexual development. However, only the pheromone response and cell-wall integrity MAP kinase pathways, but not the osmoregulatory MAP kinase pathway, are essential for hyphal cell fusion. Evidence of cell fusion-related processes, regulated through MAP kinase signalling, have been identified as novel features important for the construction of fertilisable protoperithecia. These cell-fusion related processes include extracellular matrix deposition, hyphal attachment and envelopment. A novel phenotype of S. macrospora with defective ascogonial septation is presented. This pro22 mutant also has impaired hyphal cell fusion and produces only small, defective protoperithecia. The pro22 gene encodes a protein that is highly conserved throughout eukaryotes. Live-cell imaging revealed that this PRO22 protein is localised in the dynamic tubular and vesicular vacuolar-network of the colony periphery and in ascogonia. PRO22 is absent from the large spherical vacuoles in the vegetative hyphae of the sub-peripheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar-network. Furthermore, the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 A. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 A and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.

Xing, Xiaochen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references. / Abstract also in Chinese. / Abstract (Chinese) --- p.i / Abstract --- p.iii / Contents --- p.v / Acknowledgement --- p.ix / Chapter Chapter1 --- Introduction and background / Chapter 1.1 --- Overview of Studies in Colloid-Polymer mixture --- p.1 / Chapter 1.2 --- Depletion Force in Colloid-Polymer Mixture --- p.1 / Chapter 1.2.1. --- Depletion Interaction in Monodisperse and Neutral Polymer-Colloid Mixtures: Theory --- p.3 / Chapter 1.2.1.1. --- An Exact Result: the Interaction between Parallel Plates due to Ideal Polymer Chains --- p.3 / Chapter 1.2.1.2. --- Penetrable Hard Sphere (PHS) Approach --- p.4 / Chapter 1.2.2. --- Early Experimental Findings of Depletion Interaction --- p.6 / Chapter 1.3 --- References and Notes --- p.8 / Chapter Chapter2 --- Principle of Total Internal Reflection Microscopy (TIRM) and Instrumentation / Chapter 2.1 --- Introduction of Total Internal Reflection Microscopy (TIRM) --- p.10 / Chapter 2.2 --- The Principle of TIRM Technique --- p.11 / Chapter 2.2.1 --- Total Internal Reflection --- p.11 / Chapter 2.2.2 --- Details on Scattering of the Evanescent Wave --- p.13 / Chapter 2.2.3 --- Data Analysis --- p.16 / Chapter 2.3 --- Instrumentation --- p.20 / Chapter 2.3.1 --- Apparatus --- p.20 / Chapter 2.3.2 --- Optical Tweezer --- p.23 / Chapter 2.3.3 --- Cleaning of the Slide Surface --- p.24 / Chapter 2.3.4 --- A Typical Potential Energy Profile --- p.25 / Chapter 2.4 --- Laser Light Scattering (LLS) --- p.26 / Chapter 2.5 --- Zeta-potential Measurements --- p.27 / Chapter 2.6 --- References and Notes --- p.28 / Chapter Chapter3 --- Depletion Attraction between a Polystyrene Sphere and a Hydrophilic Surface in a Pluronic Aqueous Solution / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.34 / Chapter 3.2.1 --- Sample Preparation --- p.34 / Chapter 3.2.2 --- Total Internal Reflection Microscopy --- p.35 / Chapter 3.2.3 --- Laser Light Scattering --- p.36 / Chapter 3.3 --- Results and Discussion --- p.37 / Chapter 3.4 --- Conclusion --- p.48 / Chapter 3.5 --- References and Notes --- p.50 / Chapter Chapter4 --- pH-Controllable Depletion Attraction Induced by Microgel Particles / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Experimental Section --- p.54 / Chapter 4.2.1 --- Sample Preparation --- p.54 / Chapter 4.2.2 --- Total Internal Reflection Microscopy --- p.56 / Chapter 4.3 --- Results and Discussion --- p.58 / Chapter 4.4 --- Conclusion --- p.63 / Chapter 4.5 --- References and Notes --- p.64 / Publication List --- p.65

Single-particle reconstruction technique is one of the major approaches to studying ribosome structure and membrane proteins. In this thesis, I report the use of time-resolved cryo-EM technique to study the structure of short-lived ribosome complexes and conventional cryo-EM technique to study the structure of ribosome complexes and membrane proteins. The thesis consists three parts. The first part is the development of time-resolved cryo-EM technique. I document the protocol for how to capture short-lived states of the molecules with time-resolved cryo-EM technique using microfluidic chip. Working closely with Dr. Lin’s lab at Columbia University Engineering Department, I designed and tested a well-controlled and effective microspraying-plunging method to prepare cryo-grids. I demonstrated the performance of this device by a 3-Å reconstruction from about 4000 particles collected on grids sprayed with apoferritin suspension. The second part is the application of time-resolved cryo-EM technique for studying short-lived ribosome complexes in bacteria translation processes on the time-scale of 10-1000 ms. I document three applications on bacterial translation processes. The initiation project is collaborated with Dr. Gonzalez’s lab at Chemistry Department, Columbia University. The termination and recycling projects are collaborated with Dr. Ehrenberg’s lab at Department of Cell and Molecular Biology, Uppsala University. I captured and solved short-lived ribosome intermediates complexes in these processes. The results demonstrate the power of time-resolved cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. The last part is the application of conventional cryo-EM technique to study ribosome complexes and membrane proteins. This part includes five collaboration projects. Human GABA(B) receptor project is the collaboration with Dr. Fan at Department of Pharmacology, Columbia University. Cyclic nucleotide-gated (CNG) channels project is the collaboration with Dr. Yang at Department of Biological Sciences, Columbia University. The cryo-EM study of Ybit-70S ribosome complex and Cystic fibrosis transmembrane conductance regulator (CFTR) project are the collaboration with Dr. Hunt at Department of Biological Sciences, Columbia University. The cryo-EM study of native lipid bilayer in membrane protein transporter is the collaboration with Dr. Hendrickson at Department of Biochemistry and Molecular Biophysics, Columbia University and Dr. Guo at Department of Medicinal Chemistry, Virginia Commonwealth University.

With the development of ultra-fast laser technology, several new imaging techniques have pushed optical resolution past the diffraction limit for traditional light-based optics. Advancements in lithography have enabled the straightforward creation of micron- and nanometer-sized optical devices. Exposing metal-dielectric structures to light can result in surface plasmon excitation and propagation along the transition interface, creating a surface plasmon polariton (SPP) response. Varying the materials or geometry of the structures, the plasmonic response can be tailored for a wide range of applications. Photoemission electron microscopy (PEEM) has been used to image excitations in micron-sized plasmonic devices. With PEEM, optical responses can be characterized in detail, aiding in the development of new types of plasmonic structures and their applications. We show here that in thin, triangular gold platelets SPPs can be excited and concentrated within specific regions of the material (thickness ~50 nm); resulting in localized photoemission in areas of high electric field intensity. In this regard, the platelets behave as receiver antennas by converting the incident light into localized excitations in specific regions of the gold platelets. The excited areas can be significantly smaller than the wavelength of the incident light (λ≤1µ). By varying the wavelength of the light, the brightness of the excited spots can be changed and by varying the polarization of the light, the brightness and position can be changed, effectively switching the photoemission on or off for a specific region within the triangular gold structure. In this work, the spatial distribution of surface plasmons and the imaging results from photoemission electron microscopy are reproduced in simulation using finite element analysis (FEA). In addition, we show that electromagnetic theory and simulation enable a detailed and quantitative analysis of the excited SPP modes, an explanation of the overall optical responses seen in PEEM images, and prediction of new results.

Typewritten copy Includes bibliographical references (leaves 141-144) Pt. 1. Historical review -- pt. 2. Techniques used in electron microscopy -- pt. 3. Experiments on cilia from Hydrdella australis -- pt. 4. Electron microscope experiments on cilia from the rat trachea -- pt. 5. Electron microscope experiments on cilia from paramecium -- pt. 6. Electron microscope experiments on the trichocysts of paramecium -- pt. 7. Discussion An account of experimental investigations carried out from January 1952 to September 1954.

Three perennial tropical Panicum species were grown under ambient and elevated (900 ppm) carbon dioxide concentrations in especially designed microclimate chambers. The study aimed to investigate the influence of high carbon dioxide concentrations on morphology/anatomy with physiological change among three closely related species possessing distinctly different photosynthetic pathways. The anatomy of the leaf was investigated using light microscopy (LM), transmission electron microscopy (TEM), and graphics image analysis. A suitable schedule for fixation, dehydration and embedding of leaf specimens for both forms of microscopy was developed. The anatomy of the species investigated did not change qualitatively, but there were detectable changes in leaf thickness and tissue proportions of the epidermis, mesophyll and thickened tissues (sclerenchyma, bundle sheath, vascular elements) that differed with species. This study is also relevant to the investigation of the evolution of C4, although species, and the progression involved in plants with characteristics intermediate between those of C3 and C4 species. These intermediate species have been mainly characterized by CO2 exchange and biochemical analysis, but they also display anatomical characteristics in between those of C3 and C4 plants. The evolutionary progression of the C3 to C4 species remains unsolved, although current studies indicate that the evolutionary step was from the C3 plant to the C4. Thus the intermediate C3/C4 plants may not be intermediate in an evolutionary sense and they could be seen as a simple hybridization between a C3 plant and C4 plant. In most of the parameters measured the C3/C4 P. decipiens resembled either the C3 P. tricanthum or the C4 P. antidotale. It may therefore be likened to a physiological chimera rather than to a true intermediate form / Doctor of Philosophy (PhD)

Thesis (Ph.D.)--University of Hong Kong, 1994. / Includes bibliographical references. Also available in print.

Thesis (Ph.D.)--University of Hong Kong, 1994. / Includes bibliographical references. Also available in print.

There is strong evidence that the skeletal muscles of pinnipeds are adapted for an aerobic, lipid-based metabolism under the hypoxic conditions associated with breath-hold diving. However, regional variations in mitochondrial density are unknown, and the few fiber typing studies performed on pinniped skeletal muscles are not consistent with an aerobic physiological profile. Thus, the objectives of this study were to (1) reexamine the fiber type distribution throughout the primary locomotory muscles of the harbor seal, and (2) to better understand the density and distribution of mitochondria in the locomotory muscles. Multiple samples from transverse sections of the epaxial muscles and a single sample of the pectoralis muscle of wild harbor seals were analyzed using immunohistochemical fiber typing and electron microscopy. Fiber typing results indicated that harbor seal epaxial muscles are composed of 47.4% type I (slow twitch, oxidative) fibers and 52.8%, IIa (fast twitch, oxidative) fibers. No fast twitch, glycolytic (type IIb) fibers were detected in the epaxial muscles or the pectoralis muscle. Mean volume density of mitochondria [Vv(mt,f)] was 5.6%, which is elevated over what would be predicted for a terrestrial mammal of similar mass. The elevated Vv(mt,f) had a high proportion of intermyofibrillar mitochondria, a trait not normally found in the muscles of terrestrial mammals with elevated Vv(mt,f). These results provide further evidence that the elevated mitochondrial volume density in pinniped muscle decreases the oxygen diffusion distance between myoglobin and mitochondria to facilitate aerobic respiration in working muscles. In addition, analyses of heterogeneity revealed that the regions of the epaxial muscles that were located deep within the muscle showed a significantly higher Vv(mt,f) relative to those regions that were superficially-located. In contrast, there was no significant heterogeneity of fiber type detected in either plane of the epaxial muscles. Thus, there was a fine-scale pattern of spatial heterogeneity of Vv(mt,f) within the epaxial muscles that does not manifest in fiber type distribution, indicating that the fibers have similar oxidative capacities.