Die vorliegende Arbeit liefert wichtige Erkenntnisse über die subzelluläre Verteilung und die Funktion des RS1-Proteins vom Schwein (pRS1), einem Regulator von Plasmamembran-transportern. Das grün fluoreszierende Protein (GFP) wurde mit pRS1 fusioniert und in LLC-PK1 Zellen exprimiert. Das GFP-pRS1 Fusionsprodukt (96 kD) konnte an der Plasmamembran, im Zytosol und im Zellkern entdeckt werden. Bei GFP-Fusion mit trunkierten pRS1-Proteinen zeigte sich, dass der C-Terminus die Kernlokalisierung beeinflusst. Dagegen wurde die Kernlokalisierung durch eine Trunkierung des N-Terminus nicht gestört. Im C-Terminus des pRS1 konnte von AS 579 bis 616 eine Ubiquitin associated domain (UBA) identifiziert werden, die auch in den anderen bisher bekannten RS1-Proteinen aus Mensch, Kaninchen und Maus konserviert vorliegt. Eine Ubiquitin-Affinitätschromatographie zeigte, dass das pRS1-Protein Ubiquitin auf nicht kovalente Weise bindet. Nach der Trunkierung der UBA-Domäne war keine Wechselwirkung des pRS1-Proteins mit Ubiquitin mehr feststellbar. Ein konserviertes Di-Leucin-Endozytose-Motiv (pRS1 AS 366/67) deutet eine Funktion des pRS1-Proteins bei der Internalisierung von Plasmamembranproteinen an. Deshalb wurde das Endozytoseverhalten von pRS1 überexprimierenden LLC-PK1 Zellen untersucht, wobei sich zeigte, dass diese Zellen eine deutlich höhere Aufnahme des Endozytosefarbstoffes RH 414 aufwiesen als Zellen, die pRS1 nicht überexprimierten. Die in dieser Arbeit gesammelten Daten zum RS1-Protein wurden zusammen mit früher erhobenen Ergebnissen zum RS1-Protein im Rahmen eines Modells zusammengefasst. In diesem hypothetischen Modell wird angenommen, dass RS1 ein Adapterprotein ist, welches die ubiquitinabhängige Endozytose von Plasmamembrantransportern vermittelt und als Signalmolekül in den Zellkern gelangen kann, wo es an der Transcriptionsrepression des SGLT1 beteiligt ist. / This work discribes investigations about the subcellular distribution and function of the plasma membrane-associated protein RS1, an regulator of plasma membrane transporters like the Na+-D-glucose cotransporter SGLT1 or the organic cation transporter OCT2 (Vehyl et al., 1993; Reinhardt et al., 1999; Valentin et al., 2000). The green fluorescent protein (GFP) was fused to RS1 from pig (pRS1) and expressed in LLC-PK1 cells. The GFP-pRS1 protein could be detected at the plasma membrane, in the cytoplasma and in the nucleus. Expression of various truncated forms of GFP-pRS1 showed that the N-terminal half of pRS1 (amino acids 1-328) is not necessary for the migration of pRS1 into the nucleus. In contrast, truncations of the C-terminus inhibited translocation into the nucleus. The C-terminus of pRS1 contains a conserved ubiquitin associated domain (UBA) at amino acids 579 to 616. Affinity chromatography with ubiquitin-conjungated Sepharose beads showed a noncovalent binding of pRS1 to immobilized ubiquitin, which was abolished in the presence of an excess of free ubiquitin. Further analysis schowed that the C-terminal 111 amino acids were indispensable for ubiquitin binding. A conserved di-leucine signal (pRS1 336/337) is a well known endocytosis motif and points to an involvement of pRS1 in the internalisation of plasma membrane proteins. This hypothesis was supported by the finding of an increased uptake of the intercalating membrane dye RH 414 in a pRS1-overexpressing LLC-PK1 cell line. Based on this findings together with previous data, a model for the physiological role of RS1 was proposed. In this model, RS1 serves as an adaptor which links ubiquitinated plasma membrane transporters such as SGLT1 to the endocytosis machinery. Moreover RS1 migrates into the nucleus and is involved in the transcriptional suppression of SGLT1.

The integrin α4β7, which mediates the trafficking of T lymphocytes to the gut associated lymphoid tissue (GALT), a site of rapid HIV replication, has been described as an attachment factor for the HIV envelope protein gp120. While differences in binding affinity of early transmitting and chronic gp120s for α4β7 have previously been noted by others, this has not translated to differences in replication. We aimed to investigate what role this binding interaction has in HIV pathogenesis over time and determine the factors that influence α4β7 reactivity. Understanding the subsets on which α4β7 is expressed may indicate the phenotype of the first host cells infected by HIV. The level of expression of α4β7 on Th17 and Treg CD4+ T cells was of interest as both subsets are important in HIV immunity in the GALT and represented in both the GALT and genital mucosa. All-trans retinoic acid-activated CD4+ T cells isolated from whole blood of healthy donors were incubated with or without HP2/1 (anti-α4 monoclonal antibody) or Act-1 (anti-α4β7 monoclonal antibody) prior to adding virus. Sixty infectious envelope clones were prepared using matched envelope genes from 11 individuals in the CAPRISA 002 Acute Infection cohort representing the T/F virus and variants from 1-39 months post infection (p.i.). Replication was monitored by p24 ELISA over 10 days. ATRA-stimulated CD4+ T cells were subjected to intracellular and surface staining for flow cytometric analysis using a FACSAria to distinguish Th17 and Treg CD4+ T cells and to determine the expression of CD45RA, CCR5, p24, α4β7. The dependence on α4β7 for HIV subtype C replication changes over time and varies across individuals. In three individuals, dependence on α4β7 was higher using T/F viruses and decreased sharply during acute infection (1-3 months post-infection). α4β7 dependence showed an increasing trend in chronic infection over time which was slight in the first year. Factors that influence α4β7 reactivity include glycan distance from α4β7-binding motif, glycan density and length of the V1/V2 region of gp120. Several glycans positioned in conserved regions of gp120 including N234, N332 and N334 were present more or less frequently in viruses with high α4β7 reactivity. There was an association between high dependence on α4β7 for replication at transmission and those T/F viruses that have a S/PDI/V α4β7 binding motif as well T/F viruses found in individuals diagnosed with bacterial vaginosis during acute infection. Several cytokines in the cervicovaginal lavage (CVL) of these individuals during early infection (IL-8, IL-7 and IL-1α) positively correlated with α4β7 dependence for replication. Treg CD4+ T cells expressed slightly higher levels of α4β7 as compared to Th17 CD4+ cells. In addition, Th17 cells in this and other studies were shown to rapidly deplete in vitro following HIV infection while Treg CD4+ T cell were shown to expand. Despite this, Treg CD4+ T cells were significantly more permissive to infection as compared to Th17 CD4+ T cells. Collectively, these data suggest that there is a role for α4β7 in HIV pathogenesis and that the interaction is selected for during transmission by a number of bottlenecks, one of which is the presence of bacterial vaginosis and elevated expression of IL-7, IL-8 and IL-1α. Due to high levels of α4β7 expression and the ability to bind gp120, presence in both the genital tract and the GALT, regulation by ATRA similar to α4β7 upregulation, expansion following HIV infection and elevated permissiveness to acute infection, Treg CD4+ T cells may be a robust vector from the genital mucosa to the GALT shortly after transmission. This population of T cells may be more suited for this function than Th17 CD4+ T cells which are more susceptible to depletion either by HIV or bystander effects. As a result of the α4β7 binding motif being in a highly immunogenic region of gp120 and antibodies directed against this region associated with protection in the only effective vaccine trial to date, further understanding of the interaction between the virus and the integrin provides an opportunity for the development of future vaccine and therapeutic strategies.

No description available.

Siu Shu Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 100-106). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Contents --- p.ii / Abstract / 摘要 --- p.iv / Abbreviations --- p.vi / List of figures --- p.vii / List of tables --- p.x / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- "Human EST sequencing project, the role and goal" --- p.1 / Chapter 1.2 --- Human liver cDNA sequencing --- p.2 / Chapter 1.3 --- The role of membrane-associated proteins in hepatocellular functions --- p.3 / Chapter 1.3.1 --- Outline of the liver function --- p.3 / Chapter 1.3.2 --- Basic structure of hepatocyte --- p.4 / Chapter 1.3.3 --- Category of membrane associated proteins --- p.5 / Chapter 1.4 --- Identification of human OAT2 gene --- p.7 / Chapter 1.5 --- The multispecific transporter family --- p.8 / Chapter 1.5.1 --- Classification --- p.8 / Chapter 1.5.2 --- The human OAT family --- p.9 / Chapter 1.6 --- The characteristics of rat multispecific OAT2 --- p.11 / Chapter 1.7 --- Clinical significance of organic anion transport proteins --- p.14 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Human liver EST sequencing project --- p.16 / Chapter 2.1.1 --- Plating out the adult human liver phage library --- p.16 / Chapter 2.1.2 --- PCR detection and amplification of the cDNA clone --- p.17 / Chapter 2.1.3 --- Automatic cDNA sequencing --- p.18 / Chapter 2.2 --- Cloning of hOAT2 gene into TA cloning vector pT-Adv --- p.19 / Chapter 2.2.1 --- Amplification of hOAT2 by PCR --- p.19 / Chapter 2.2.2 --- Ligation reaction --- p.19 / Chapter 2.2.3 --- Transformation of recombinant plasmid into competent cells --- p.20 / Chapter 2.3 --- Sequence analysis and structural prediction --- p.20 / Chapter 2.4 --- Cloning of the hOAT2 gene into the pQE30 expression vector --- p.21 / Chapter 2.4.1 --- PCR amplification and restriction endonuclease cutting --- p.21 / Chapter 2.4.2 --- Gene clean --- p.22 / Chapter 2.4.3 --- Preparation of bacterial competent cells --- p.23 / Chapter 2.5 --- Small scale synthesis of plasmid DNA --- p.24 / Chapter 2.6 --- Large scale synthesis of plasmid DNA --- p.25 / Chapter 2.7 --- Cloning of the hOAT2 gene into the pSecTag2B mammalian expression vector --- p.26 / Chapter 2.8 --- Cloning of the hOAT2 gene into the pEGFP-C2 fluorescent vector --- p.27 / Chapter 2.8.1 --- Tissue culture and transfection --- p.27 / Chapter 2.8.2 --- Fluorescence microscopy examination --- p.28 / Chapter 2.9 --- Chromosomal mapping of the hOAT2 gene --- p.29 / Chapter 2.9.1 --- Somatic cell hybrids mapping --- p.29 / Chapter 2.9.2 --- Radiation hybrids mapping --- p.29 / Chapter 2.10 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.30 / Chapter 2.11 --- Western hybridization --- p.32 / Chapter 2.11.1 --- Preparation of anti-hOAT2 antibodies --- p.32 / Chapter 2.11.1.1 --- Synthetic peptide conjugation --- p.32 / Chapter 2.11.1.2 --- Immunizing rabbit polyclonal antibodies for human OAT2 --- p.32 / Chapter 2.11.1.3 --- Purification of the rabbit polyclonal IgG antibodies --- p.33 / Chapter 2.11.2 --- Western blot analysis --- p.33 / Chapter 2.11.2.1 --- Protein isolation from rat liver --- p.33 / Chapter 2.11.2.2 --- Prote in preparation from cell lysate --- p.34 / Chapter 2.11.2.3 --- Quantitation of total proteins by Bradford protein assay --- p.35 / Chapter 2.11.2.4 --- Blotting and hybridization --- p.35 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Catalogue of the 500 liver ESTs --- p.37 / Chapter 3.2 --- Nomenclature of human NLT gene --- p.47 / Chapter 3.3 --- Cloning and characterization of the hOAT2 sequence --- p.48 / Chapter 3.3.1 --- Isolation of hOAT2 cDNA from human liver cDNA library --- p.48 / Chapter 3.3.2 --- The primary and secondary structural analysis of hOAT2 --- p.53 / Chapter 3.3.3 --- Motif search and prediction --- p.61 / Chapter 3.3.4 --- Homology alignment --- p.64 / Chapter 3.4 --- Chromosomal mapping of hOAT2 gene --- p.67 / Chapter 3.4.1 --- Somatic cell hybrid mapping of hOA T2 gene --- p.67 / Chapter 3.4.2 --- Radiation hybrid mapping of hOA T2 gene --- p.69 / Chapter 3.4.3 --- Identification of partial human genomic sequence --- p.73 / Chapter 3.5 --- Detection of the hOAT2 gene expression in human tissues by RT- PCR assay --- p.76 / Chapter 3.6 --- Detection of subcellular localization of hOAT2 protein by conjugating fluorescence protein --- p.81 / Chapter 3.7 --- Immunodetection of protein extracts from cultured cells --- p.83 / Chapter Chapter 4: --- Discussion / Chapter 4.1 --- Characterization of the hepatocellular ESTs --- p.85 / Chapter 4.1.1 --- Classification and frequency distribution of the 500 ESTs --- p.85 / Chapter 4.1.2 --- The expression pattern of membrane associated proteins --- p.87 / Chapter 4.2 --- Tissue distribution and expression profiles of hOAT2 --- p.88 / Chapter 4.3 --- HOAT2 in fetal development --- p.89 / Chapter 4.4 --- Predicting the topology of membrane proteins --- p.90 / Chapter 4.5 --- Chromosomal mapping of human OAT2 --- p.91 / Chapter 4.6 --- Possible functions of hOAT2 --- p.93 / Chapter 4.6.1 --- Hepato-renal relation --- p.93 / Chapter 4.6.2 --- Substrate diversity --- p.95 / Chapter 4.7 --- Fluorescence detection for subcellular localization --- p.96 / Chapter 4.8 --- Conclusion --- p.97 / Chapter 4.9 --- Further aspects --- p.99 / References --- p.100 / Appendix --- p.107

A novel application of ionic liquid as a charge carrier for the analysis and detection of neutral organometallic complexes using a mass spectrometer has been presented. The mass spectrometer detects only charged compounds which raise a difficulty in analyzing a neutral molecule that lacks a basic site to associate with charge. Therefore, an effective way of providing charge has always been an area of keen interest in the field of mass spectrometry. Ionic liquids have a very fascinating property of forming a cation-? interaction with other molecules to give a charged complex. In order to take advantage of this, it is important to know the geometric structure of the complex. Advanced methodologies like hydrogen-deuterium exchange and computational calculations have been used assisting in better understanding of the structure of the ionic liquid complexes.

"This thesis develops a system for synchronizing two wireless transmitters so that they are able to implement a distributed beamformer in several different channel models. This thesis considers a specific implementation of the system and proposes a metric to quantify its performance. The system's performance is investigated in single-path and multi-path time-invariant channel scenarios, as well as in single-path time-varying channel scenarios. Where prior systems have difficulty in implementing a distributed beamformer in multi-path channels and/or mobile scenarios, the results of this thesis show that the Round-Trip Time-Division distributed beamforming system is able to perform as a beamformer in all three of the channel models considered. "

Cellular membranes were once considered static and passive structures, but are now appreciated as a fluidic and dynamic assembly of macromolecules that play an active role in cellular function. Membrane composition has been proposed to play a critical role in modulating protein function by affecting everything from post-translational modifications to conformation, but the physiologic relevance of the relationship between protein and membrane has been difficult to establish. For example, membrane-associated proteins such as Flotillin-1 (Flot1) have been implicated to scaffold proteins into cholesterol-rich membranes, as well as play a role in a wide array of functions such as endocytosis and axon pathfinding; however, genetic elimination of Flot1 expression had little to no reported consequence, leaving to question the physiologic importance of scaffolding proteins to membrane microdomains. Using genetic and biochemical approaches, I sought to understand how the immediate lipid environment can influence the function of a transmembrane protein, and how this might impact brain function. Specifically, I examined how a cholesterol-rich environment can affect the function of the cell surface neurotransmitter transporter for dopamine, the dopamine transporter (DAT), and how this interaction may influence the ability of an organism to respond to the psychostimulant amphetamine (AMPH). Although neurotransmitter transporters (NTTs) such as DAT and the serotonin transporter (SERT), have been predicted to reside in membrane rafts, it has been difficult to establish the role of microdomain localization in transporter function. DAT localizes to the plasma membrane, where it modulates the strength and duration of neurotransmission by clearing dopamine (DA) from the perisynaptic space. Defects in DAT have been implicated in a range of psychiatric and neurological disorders, from schizophrenia to Parkinson’s disease. Additionally, as a target of psychostimulants, such as AMPH and cocaine (COC), the role of DAT in addiction is of societal interest. Given that Flot1 was required for scaffolding heterologously expressed DAT to cholesterol-rich membranes in cell-based systems, and was selectively necessary for the non-exocytic release of DA through DAT in response to AMPH, I sought to test the hypothesis that the Flot1-mediated membrane localization of DAT was significant for the ability of mice to respond to AMPH. To this end, I created a series of genetic models to determine how the presence of Flot1 impacts DAT function in the brain. I found that Flot1 is not only important for scaffolding DAT into cholesterol-rich membranes, but that the ability of DAT to partition into these membranes was necessary for DAergic neurons, DAT, and ultimately mice, to respond to AMPH. Given that the other parameters of DA neuron function, as well as the ability of the animals to respond to COC was unaffected by DAT partitioning, my findings demonstrate that AMPH and COC exert different mechanisms of action in vivo. Moreover, I found that the cholesterol-rich membrane environment promoted a conformation of DAT that was favorable for reverse transport of DA through DAT, namely increasing the ability of its N-terminus to bind to the phospholipid, PIP2. This dissertation provides the first glimpse into not only how membrane localization can affect protein conformation and function but also the physiologic relevance of these Flot1-dependent membrane microdomains in brain.

Wang, Hao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-93). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.