Study on the neuronal circuits implicated in postural tremor and hypokinesia

The effect of various tegmentary lesions at the level of the pontomesenchphalon in monkeys on motor function was observed. The importance of the monoaminergic mechanisms of the brainstem is discussed. The results also show the importance of the descending tegmentary rubral system and the rubroolivocerebellar circuit in controlling peripheral motor activity. The destruction of the sensory motor cortex proves to be a more effective way of eliminating spontaneous or harmaline induced tremor than the complete interruption of the pyramidal system on the level of the cerebral peduncle

Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infectio

In vivo analysis of endocytic and biosynthetic transport to the plant vacuole

The plasma membrane forms the interaction site between a cell and its environment. The proteins in the plasma membrane, namely translocators and receptors, allow for the exchange of nutrients and information. This, however, urges for a stringent regulation of these proteins. One mechanism to control their amount is to transport them into the lytic vacuole via the endocytic pathway. The degradative function of vacuoles depends on proteolytic enzymes, which reach that very organelle through a different route. They are synthesized in the endoplasmic reticulum and transported via the endomembrane system. Vacuolar transport of those soluble proteins depends on sorting receptors, separate them from secretory cargo. To gain a deeper understanding on the trafficking of membrane-bound and soluble cargo to the vacuole, we aimed at characterizing the machinery mediating those processes. For this, we employed nanobody-epitope interactions to create intra-cellular setups, which enabled us to perform transport- and interaction-analyses of proteins via confocal microscopy. We revealed that “Vacuolar Sorting Receptors” (VSRs) interact with their ligands in the endoplasmic reticulum and the Golgi apparatus, but not in the trans-Golgi network and the multivesicular body, by performing “Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer” (FRET-FLIM; Künzl et al., 2016). In order to create the reporters for the compartment-specific FRET-FLIM measurements, we linked the ligand binding domain of the VSRs to marker-proteins via a nanobody-epitope interaction. We demonstrated that VSRs do indeed recycle and identified the cis-Golgi as the destination of their retrograde transport (Früholz et al., 2018). These discoveries were based on the combination of two nanobody-epitope pairs. We used those for post-translational labelling and trapping of vacuolar sorting receptors. Concerning the machinery mediating the transport of to-be-degraded plasma membrane proteins to the vacuole, we analyzed the “Endosomal Sorting Complex Required For Transport II” (ESCRT-II). Here, we employed FRETFLIM to show that “Vacuolar Protein Sorting 22” (VPS22), 25 and 36 interact to form this specific complex. We pushed the limits of nanobody-based approaches by employing membrane-anchored nanobodies in order to import the method of co-immune precipitation into living cells. This enabled us to perform in vivo studies, which showed that ESCRT-II contains two VPS25 moieties (Fäßler et al., prepared manuscript)

Analysis of the bidirectional VSR-mediated transport in the plant endomembrane system

Post-translational regulation of membrane proteins is pivotal for all eukaryotic cells. In particular, the regulation of transporter proteins at the plasma membrane. They are post-translationally regulated by vacuolar degradation after their endocytic removal. For this to occur, the vacuole needs a constant supply of soluble hydrolyzing enzymes. At the heart of this process operate vacuolar sorting receptors (VSRs) mediating the transport of these enzymes towards the lytic vacuole. Based on the research on mammalian cells, it is assumed that VSR transport occurs bidirectional and follows the common principle of receptor-mediated transport: Receptors bind ligands in the donor compartment, thereby forming a receptor-ligand complex that is transported to the acceptor compartment. Upon arrival, ligands are released and receptors recycle back to the donor compartment to reload ligands. It is presumed that this transport occurs between the trans-Golgi network/early endosome (TGN/EE) and the multivesicular bodies/late endosomes (MVBs/LEs) in plants. It now became clear that VSR bind ligand in the early secretory pathway and transport them to the TGN/EE were they are released (Künzl et al., 2016). To analyze the post TGN/EE transport of soluble proteins, we generated a nanobody-based system to follow the fate of soluble proteins lacking vacuolar sorting signals that were placed in the TGN/EE via the endocytic pathway. This enabled us to demonstrated that post TGN/EE transport of ligands to the vacuole occurs independently of VSRs (Künzl et al., 2016). Usage of this system, however, required testing that nanobody-triggered protein-protein interactions between two soluble proteins can occur in the endomembrane system (Früholz and Pimpl, 2017). With the demonstration that VSRs release ligands in the TGN/EE (Künzl et al.,2016) it became clear that if VSRs do recycle, then the TGN/EE would be the starting point for such a recycling. To identify the target compartment of the VSR recycling route, we devised an approach where we employ simultaneously two different nanobody-epitope pairs. One to label VSRs fluorescently in the TGN/EE and a second to trigger the lockdown of recycled VSRs via an endocytosed dual epitope linker to block their further anterograde transport. Using this approach, we demonstrate that VSRs recycle from the TGN/EE to the cis-Golgi and show that recycled VSRs reload ligands there (Früholz et al., in press). Together, we proof that the bidirectional VSR-mediated transport exists and occurs between the TGN/EE and the cis-Golgi

Integrated polarisation rotators

The ability to control and manipulate the state of polarisation of optical signals is becoming an increasingly desirable feature in numerous applications including integrated optical circuits, semiconductor optical amplifiers (SOAs) and optical communication systems. This thesis introduces the design, optimisation, fabrication and operation of two novel integrated reciprocal single-section passive polarisation converter devices based upon mode-beating. The converter designs consist of asymmetric profiled waveguides, which were realised in a single reactive ion dry-etch process step. An in-situ custom built sample holder was utilised to place the samples at a predetermined angle to the incoming ions, which resulted in waveguide profiles with sloped sidewalls. This fabrication technique also allowed the incorporation of adiabatic taper sections within the device design. The converter section waveguide profile of the first design consists of two sloped sidewalls. Devices realised on a GaAs/AlGaAs material structure achieved a converted transverse magnetic (TM) polarisation purity of 81.4% at a device length of 30 μm for a transverse electric (TE) polarised input signal at an operating wavelength of λ = 1064 nm. The convention used is that TE refers to light polarised in the plane of the wafer and TM refers to light polarised perpendicular to the plane of the wafer. The total optical loss imposed by this device was evaluated to be 1.72 dB. This design was also used for the monolithic integration of a passive polarisation converter incorporated within a Fabry-Perot semiconductor laser diode on an unstrained GaAs/AlGaAs double quantum well heterostructure material system. A predominantly TM polarised optical output from the converter facet of greater than 80% is demonstrated for a converter length of 20 μm at an emitting wavelength of 867.1 nm. The about 1.4 mm long fabricated device has a current threshold level of 100 mA and a side mode suppression ratio (SMSR) of 25 dB. The second converter design is based on the modification of an already existing stripe waveguide structure. The converter section is defined by applying the above mentioned angled dry-etch process on a certain length of the stripe waveguide. The fabricated asymmetric waveguide core profile consists of a sloped undercut. A TM polarisation purity of 90% at a device length of 55 μm for a TE polarised input signal at an operating wavelength of λ = 1064 nm was achieved at the output. The total optical loss imposed by this device was evaluated to be 0.47 dB

On-chip high-speed sorting of micron-sized particles for high-throughput analysis

A new design of particle sorting chip is presented. The device employs a dielectrophoretic gate that deflects particles into one of two microfluidic channels at high speed. The device operates by focussing particles into the central streamline of the main flow channel using dielectrophoretic focussing. At the sorting junction (T- or Y-junction) two sets of electrodes produce a small dielectrophoretic force that pushes the particle into one or other of the outlet channels, where they are carried under the pressure-driven fluid flow to the outlet. For a 40mm wide and high channel, it is shown that 6micron diameter particles can be deflected at a rate of 300particles/s. The principle of a fully automated sorting device is demonstrated by separating fluorescent from non-fluorescent latex beads

New insights into the nature of the SMC WR/LBV binary HD 5980

We present the results of optical wavelength observations of the unusual SMC eclipsing binary system HD 5980 obtained in 1999 and 2004--2005. Radial velocity curves for the erupting LBV/WR object (star A) and its close WR-like companion (star B) are obtained by deblending the variable emission-line profiles of N IV and N V lines under the simplistic assumption that these lines originate primarily in the winds of star A and star B. The derived masses M_A=58--79 Mo and M_B=51--67 Mo, are more consistent with the stars' location near the top of the HRD than previous estimates. The presence of a wind-wind interaction region is inferred from the orbital phase-dependent behavior of He I P Cygni absorption components. The emission-line intensities continued with the declining trend previously seen in UV spectra. The behavior of the photospheric absorption lines is consistent with the results of Schweickhardt (2002) who concludes that the third object in the combined spectrum, star C, is also a binary system with P(starC)~96.5 days, e=0.83. The data used in this paper will be made publicly available for further analysis.Comment: 48 pages, 26 figure

Transgenic expression of malaria surface antigens under the control of phaseolin promoter.

Chan Wan Lui Wendy.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 158-162).Abstracts in English and Chinese.Acknowledgements --- p.iiiAbstract --- p.vList of Abbreviations --- p.ixList of Figures --- p.xiiList of Tables --- p.xviTable of Contents --- p.xviiChapter Chapter 1 --- General Introduction --- p.1Chapter Chapter 2 --- Literature review --- p.3Chapter 2.1 --- Malaria --- p.3Chapter 2.2 --- History of malaria --- p.4Chapter 2.3 --- Malaria parasites --- p.4Chapter 2.4 --- Life cycle --- p.5Chapter 2.5 --- Potential use of malaria vaccine --- p.6Chapter 2.6 --- Merozoite surface protein 1 (MSP1) --- p.7Chapter 2.7 --- Potential use of MSPl --- p.8Chapter 2.8 --- Significance of MSPl C-terminal fragments --- p.9Chapter 2.8.1 --- Significance of MSP142 --- p.9Chapter 2.8.2 --- Significance of MSP119 --- p.11Chapter 2.9 --- Production of MSPl C-terminal fragments --- p.12Chapter 2.10 --- Plants as bioreactors --- p.12Chapter 2.11 --- Expression of MSPl C-terminal fragments in transgenic plants --- p.14Chapter 2.12 --- Phaseolin and its sorting signal --- p.19Chapter 2.13 --- Protein targeting signals --- p.20Chapter Chapter 3 --- Material and methods --- p.23Chapter 3.1 --- Introduction --- p.23Chapter 3.2 --- Chemical and enzymes --- p.23Chapter 3.3 --- Cloning --- p.24Chapter 3.3.1 --- MSP142 and MSP119 constructs --- p.24Chapter 3.3.2 --- Protein targeting fusion constructs --- p.24Chapter 3.3.3 --- GUS fusion Constructs --- p.30Chapter (a) --- Particle bombardment --- p.30Chapter (b) --- GUS fusion constructs for plant transformation --- p.32Chapter (c) --- Modified GUS fusion constructs --- p.38Chapter 3.4 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.39Chapter 3.4.1 --- Cloning of pSUNl --- p.40Chapter 3.4.2 --- Primer sequence --- p.45Chapter 3.5 --- Bacterial strains --- p.46Chapter 3.6 --- Particle bombardment --- p.46Chapter 3.6.1 --- Plant materials --- p.46Chapter 3.6.2 --- Microcarrier preparation and coating DNA onto microcarrier --- p.46Chapter 3.6.3 --- GUS assay --- p.48Chapter 3.7 --- Transgenic expression in Arabidopsis thaliana --- p.49Chapter 3.7.1 --- Plant materials --- p.49Chapter 3.7.2 --- Agrobacterium transformation --- p.49Chapter 3.7.3 --- Vacuum infiltration Arabidopsis transformation --- p.49Chapter 3.7.4 --- Selection of successful transformants --- p.50Chapter 3.7.5 --- Selection for homozygous plants --- p.51Chapter 3.8 --- Transgenic expression in tobacco --- p.51Chapter 3.8.1 --- Plant materials --- p.51Chapter 3.8.2 --- Agrobacterium transformation --- p.52Chapter 3.8.2.1 --- Preparation of Agrobacterium tumefaciens LBA4401 competent cells --- p.52Chapter 3.8.3 --- Leaf discs method for tobacco transformation --- p.53Chapter 3.8.4 --- GUS staining --- p.54Chapter 3.9 --- DNA analysis --- p.55Chapter 3.9.1 --- Genomic DNA extraction --- p.55Chapter 3.9.2 --- Genomic PCR --- p.55Chapter 3.9.3 --- Southern blot --- p.55Chapter 3.10 --- RNA analysis --- p.56Chapter 3.10.1 --- RNA extraction --- p.56Chapter 3.10.2 --- Northern blot --- p.56Chapter 3.11 --- Protein analysis --- p.57Chapter 3.11.1 --- Protein extraction --- p.57Chapter 3.11.2 --- Western blot --- p.58Chapter 3.11.3 --- Western blot analysis --- p.58Chapter Chapter 4 --- Results --- p.60Chapter 4.1 --- Transient assay of gene expression of MSP142 and MSPl19 --- p.60Chapter 4.1.1 --- Construction of the GUS fusion constructs --- p.60Chapter 4.1.2 --- Particle Bombardment --- p.63Chapter 4.2 --- Transgenic analysis of MSP142 and MSPl19 expression --- p.70Chapter 4.2.1 --- MSPl42 and MSPl19 constructs and transformation --- p.70Chapter 4.2.2 --- Selection of transgenic plants --- p.71Chapter 4.2.3 --- Southern analysis --- p.75Chapter 4.2.4 --- Northern analysis --- p.77Chapter 4.2.5 --- Western analysis --- p.79Chapter 4.3 --- Expression of the protein-targeting and GUS fused modified MSP1 constructs --- p.81Chapter 4.3.1 --- Construction of the fusion constructs --- p.81Chapter (A) --- Protein-targeting constructs --- p.81Chapter (B) --- GUS fusion constructs --- p.90Chapter B1. --- Constructs for transient assay --- p.90Chapter B2. --- Modification of GUS sequence --- p.96Chapter B3. --- Constructs for tobacco transformation --- p.100Chapter 4.4 --- Transient assay of GUS fused MP42 and MP19 constructs by particle Bombardment --- p.107Chapter 4.4.1 --- The GUS fusion constructs --- p.107Chapter 4.4.2 --- Modification of GUS --- p.112Chapter 4.5 --- Generation of transgenic tobacco --- p.116Chapter 4.6 --- Southern analysis --- p.120Chapter 4.7 --- Northern analysis --- p.126Chapter (A) --- Protein-targeting constructs --- p.126Chapter (B) --- GUS fusion constructs --- p.130Chapter 4.8 --- Western analysis --- p.133Chapter (A) --- Protein-targeting constructs --- p.133Chapter (B) --- GUS fusion constructs --- p.139Chapter Chapter 5 --- Discussion --- p.146Chapter Chapter 6 --- Conclusion --- p.157References --- p.15