Neisseria meningitidis is a causative agent of meningitis and septicaemia. Pili are one of the major virulence factors that contribute to the pathogenicity of N. meningitidis. Pili of Neisseria are type IV fimbriae composed primarily of thousands of identical pilin subunits. Pilin of N. meningitidis is post-translationally modified by trisaccharide, phosphorylcholine and -glycerophosphate. The genes involved in pilin expression, pilin glycosylation and phosphorylcholine modification are phase variable (high frequency ON/OFF switching of expression). The function of pilin post-translational modifications and their phase variable expression in host:pathogen interactions is unknown. The phase variable expression of glycosylation in bacteria has been proposed to function in bacterial adherence and immune avoidance. However, the function of pilin glycosylation in N. meningitidis is unclear. Phosphorylcholine is expressed in a number of respiratory organisms including P. aeruginosa (on teichoic acid), S. pneumoniae (on lipoteichoic acid) and H. influenzae (on LPS). Phosphorylcholine in these organisms is important in colonisation of the nasopharynx and invasion of the epithelium. Studies on N. meningitidis pilin post-translational modifications have been restricted by difficulties in purification of pilin protein. In this thesis, we evaluated current pilin purification methods and established an efficient method of purifying pilin from N. meningitidis by Flag-tag purification system. Flag-tag purified pilin is post-translationally modified. The LC-ESI/MS/MS analysis performed in this thesis using Flag-tag purified pilin successfully determined the phosphorylcholine post-translational modification sites. Based on the MS data and the mutagenesis analysis, phosphorylcholine is covalently linked to serine 157 and serine 160 of pilin. The colony immunoblot of a serine 157/160 to alanine mutant revealed that phosphorylcholine modifications of these sites on pilin are the only surface exposed phosphorylcholine and is responsible for binding to TEPC-15 (the monoclonal antibody which binds to phosphorylcholine). In this thesis, molecular modelling demonstrated that surface exposure of pilin phosphorylcholine could be altered by the phase variation of pilin glycosylation on the adjacent pilin monomer. Furthermore, the sites for phosphorylcholine modification are commonly observed in N. meningitidis strains but not in N. gonorrhoeae indicating the importance of phosphorylcholine in pathogenisis of N. meningitidis. In addition, the biosynthesis of phosphorylcholine for pilin post-translational modification still remains a mystery. Bacteria generally obtain choline from the environment. In this thesis, we demonstrated that pilin phosphorylcholine post-translational modification could be endogenously synthesized in N. meningitidis. In summary, this thesis describes the purification method of obtaining pure post-translationally modified pilin from N. meningitidis. The phosphorylcholine post-translation modification sites on pilin have been determined and showed the importance of these sites in antibody binding specificity.

When a biomaterial is inserted into the body, the interaction of the surface with the surrounding biological environment is crucial. Given the importance of the surface, the ability to alter the surface properties to support a compatible environment is therefore desirable. Silicone hydrogel contact lenses (CL) allow for improved oxygen permeability through the incorporation of siloxane functional groups. These groups however are extremely surface active and upon rotation, can impart hydrophobicity to the lens surface, decreasing lens wettability and increasing protein and lipid deposition. Lens biofouling may be problematic and therefore surface modification of these materials to increase compatibility is exceedingly recognized for importance in both industry and research. The current work focuses on the creation of a novel anti-fouling polymer surface by the incorporation of 2-methacryoyloxyethyl phosphorylcholine (MPC), well known for its biomimetic and anti-fouling properties. A controlled polymerization method was used to generate a unique double-grafted architecture to explore the effect of increasing surface density of polyMPC chains on corresponding anti-fouling properties. The novel free polymer was synthesized by a 3-step atom transfer radical polymerization (ATRP). First, poly(2-hydroxyethyl methacrylate) (polyHEMA) was polymerized by ATRP, where the hydroxyl (OH) groups of the polymer then underwent an esterification to create macroinitiating sites. From these sites, a second ATRP of poly(MPC) varying in length occurred, yielding the double-grafted polymer poly(2(2-bromoisobutyryloxy-ethyl methacrylate)-graft-poly(2-methacryloyloxyethyl phosphorylcholine (pBIBEM-g-pMPC). The polymer was designed for resistance to protein adsorption through a possible synergistic effect between the surface induced hydration layer by surrounding PC groups coupled with steric repulsion of the densely grafted chains. To test its potential as a surface modifier, the polymer was grafted from model silicone hydrogel CL through a 4-step surface initiated ATRP (SI-ATRP) in a similar manner to the free polymer. First, the ATRP initiator was immobilized from the HEMA OH groups of the unmodified CL, generating Intermedate-1. A polyHEMA brush was grafted from the initiating sites yielding pHEMA-50, followed by the generation of a second initiator layer (Intermediate- 2). A sequential ATRP of poly(MPC) then generated the target pMPC-50/pMPC-100 surfaces. For the free pBIBEM-g-pMPC polymer analysis, 1H-NMR and GPC determined polymers formed with a predictable MW and low polydispersity (PDI). For surface grafting, using a sacrificial initiator, 1H-NMR and GPC indicated that the pHEMA-50 and pMPC-50/pMPC-100 polymers were well-controlled, with a MW close to the theoretical and a low PDI. For surface chemical composition, ATR-FTIR showed the presence of the ATRP initiator (Intermediate-1 and 2) by the appearance of a C-Br peak and disappearance of the OH peak. XPS confirmed the chemical composition of the 4-step synthesis by a change in the fraction of expected surface elements. Both the surface wettability and EWC of the materials increased upon pMPC modification, further improving upon increasing pMPC chain length. The contact angle was as low as 16.04 ± 2.37º for pMPC-50 surfaces and complete wetting for pMPC-100. Finally, the single protein adsorption using lysozyme and bovine serum albumin (BSA) showed significantly decreased protein levels for pMPC-50/100 lenses, as much as 83% (p 0.00036) for lysozyme and 73% (p 0.0076) for BSA, with no significant difference upon chain length variation. The aforementioned data demonstrates that the novel polymer has potential in providing an anti-fouling and extremely wettable surface, specifically regarding silicone hydrogel CL surfaces. / Thesis / Master of Applied Science (MASc)

Poon Kwok Man. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 118-123). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / DECLARATION --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1. --- Antibody structure and diversity --- p.1 / Chapter 1.2. --- Antibody genes --- p.5 / Chapter 1.3. --- The antibody response to phosphorylcholine --- p.10 / Chapter 1.3.1. --- Group I antibodies --- p.11 / Chapter 1.3.2. --- Group II antibodies --- p.14 / Chapter 1.3.3. --- Fine specificity of group I antibodies --- p.14 / Chapter 1.4. --- Anti-phosphorylcholine antibody structure --- p.15 / Chapter 1.5. --- Recombinant antibody --- p.22 / Chapter 1.5.1. --- Phage biology --- p.24 / Chapter 1.5.2. --- Phage-displayed antibodies --- p.29 / Chapter 1.5.3. --- Helper phage --- p.32 / Chapter 1.6. --- Objectives and scope of study --- p.34 / Chapter CHAPTER 2 --- METHODOLGY / Chapter 2.1. --- Antibody --- p.41 / Chapter 2.1.1. --- Hybridoma culture --- p.41 / Chapter 2.1.2. --- Production of antibody by induction of ascitic fluid --- p.41 / Chapter 2.1.3. --- Antibody purification --- p.41 / Chapter 2.1.3.1. --- Ammonium sulfate precipitation --- p.42 / Chapter 2.1.3.2. --- Affinity purification by Protein A-sepharose --- p.42 / Chapter 2.1.4. --- Production of Fab fragment by papain digestion --- p.43 / Chapter 2.2. --- Antigens --- p.43 / Chapter 2.2.1. --- Preparation of TsAg form infected ICR mouse --- p.44 / Chapter 2.2.2. --- Purification of Trichinella spairalis PC antigen --- p.44 / Chapter 2.2.2.1. --- Preparation of Mab2 affinity column --- p.44 / Chapter 2.2.2.2. --- Purification of TsAg --- p.45 / Chapter 2.2.3. --- Preparation of PC-HSA --- p.45 / Chapter 2.2.3.1. --- Preparation of p-diazonium phenylphosphorylcholine (DPPC) --- p.45 / Chapter 2.2.3.2. --- Conjugation of PC to HSA --- p.45 / Chapter 2.2.4. --- Commercial available antigens --- p.46 / Chapter 2.2.4.1. --- Pneumovax® 23 --- p.46 / Chapter 2.2.4.2. --- Lipopolysaccharide --- p.46 / Chapter 2.2.5. --- Standardization of PC-antigens --- p.46 / Chapter 2.3. --- Cloning of Mab2-scFv into phage display form --- p.47 / Chapter 2.3.1. --- Total RNA extraction --- p.50 / Chapter 2.3.2. --- cDNA synthesis --- p.50 / Chapter 2.3.3. --- Heavy chain variable region gene amplification --- p.51 / Chapter 2.3.4. --- Light chain variable region gene amplification --- p.51 / Chapter 2.3.5. --- Joining of heavy and light chain gene with linker --- p.52 / Chapter 2.3.6. --- Ligation of scFv gene with pCANTAB-5E vector --- p.52 / Chapter 2.3.7. --- Transformation --- p.53 / Chapter 2.3.7.1. --- E.coli strains --- p.53 / Chapter 2.3.7.2. --- E.coli cell preparation for electroporation --- p.54 / Chapter 2.3.7.3. --- Electroporation --- p.54 / Chapter 2.3.7.4. --- Competent E.coli preparation by CaCl2 --- p.55 / Chapter 2.3.7.5. --- Heat shock --- p.55 / Chapter 2.4. --- Expression of phage display scFv --- p.55 / Chapter 2.5. --- Enrichment and screening of Mab2-scFv phage --- p.56 / Chapter 2.5.1. --- Biopanning --- p.56 / Chapter 2.5.2. --- Restricition fragment analysis --- p.58 / Chapter 2.5.3. --- PCR screening --- p.58 / Chapter 2.5.4. --- DNA sequencing --- p.58 / Chapter 2.5.4.1. --- Manual sequencing --- p.58 / Chapter 2.5.4.2. --- Auto sequencing --- p.59 / Chapter 2.6. --- Mutagenesis --- p.59 / Chapter 2.6.1. --- Preparation of Uracil containing ssDNA --- p.60 / Chapter 2.6.2. --- Phosphorylation of mutagenic oligonucleotide --- p.60 / Chapter 2.6.3. --- Hybridization and secondary strand synthesis...…… --- p.60 / Chapter 2.6.4. --- Transfection and screening of mutants --- p.61 / Chapter 2.7. --- Expression of soluble scFv-E-tag --- p.61 / Chapter 2.7.1. --- SDS-PAGE analysis --- p.62 / Chapter 2.7.2. --- Anti-E-tag ELISA --- p.62 / Chapter 2.8. --- ELISA binding assay --- p.63 / Chapter 2.8.1. --- Specificity of Mab2 antibody Fab --- p.63 / Chapter 2.8.1.1. --- Carrier specifcity assay --- p.63 / Chapter 2.8.1.2. --- Free hapten inhibition assay --- p.64 / Chapter 2.8.2. --- Specificity of the scFv --- p.64 / Chapter 2.8.2.1. --- Antigen binding assay --- p.65 / Chapter 2.8.2.2. --- Free hapten inhibition assay --- p.65 / Chapter 2.8.2.3. --- Inhibition on Ts2 and Mab2 antibody assay --- p.65 / Chapter 2.9. --- Affinity assay --- p.66 / Chapter 2.10. --- Mutants analysis --- p.66 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1. --- Cloning VH and VL gene of Mab2 into scFv --- p.67 / Chapter 3.1.1. --- Amplification of variable region of H and L chain --- p.67 / Chapter 3.1.2. --- Biopanning --- p.70 / Chapter 3.1.3. --- Genetic composition of isolated clones --- p.70 / Chapter 3.2. --- Mutagenesis --- p.84 / Chapter 3.3. --- Expression and characterisation of wild-type scFv --- p.88 / Chapter 3.3.1. --- ScFv soluble protein --- p.88 / Chapter 3.3.2. --- Phage displayed scFv --- p.91 / Chapter 3.3.3. --- Standardization of PC antigens --- p.91 / Chapter 3.3.4. --- Binding acticity of scFv --- p.94 / Chapter 3.3.4.1. --- Influence of the avidity on carrier specificity binding --- p.96 / Chapter 3.4. --- Antigen specificity --- p.99 / Chapter 3.4.1. --- Free hapten inhibiton --- p.99 / Chapter 3.4.2. --- Inhibition on the binding of Ts2 --- p.102 / Chapter 3.4.3. --- Binding affinity --- p.104 / Chapter 3.5. --- Binding activities of mutants --- p.106 / Chapter CHAPTER 4 --- GENERAL DISCUSSION --- p.109 / REFERENCE --- p.118

<p>In order to develop blood biocompatible materials a heparin surface and a phosphorylcholine (PC) functionalized polymer surface were characterized using photoelectron spectroscopy (PES). The formation of the heparin surface was studied by quartz crystal microbalance with dissipation monitoring (QCM-D). This heparin surface consists of heparin conjugates deposited on a conditioning layer, applied once or twice. The PC functionalized polymer, poly(trimethylene carbonate), was linked to a silicon substrate through 3-amino- propyltrimethoxysilane (APTMS), also studied using PES. </p><p>Synchrotron radiation based PES showed that the thicker heparin film resulted in complete coverage of the substrate, while the thinner did not. This could explain the difference in blood biocompatibility between the two films, as observed by others. It was also found that the heparin chains bend down towards the substrate (under vacuum). </p><p>For the thinner heparin film the modifications, resulting from extensive irradiation of the sample, were studied with synchrotron radiation based PES. This was done at a pressure of about 10^-7 mbar and in 0.5 mbar water vapor. It was found that the modification is slower under water vapor than at low pressures and that the damaged film incorporates water upon exposure.</p><p>The heparin coating was found to be stable and wear resistant enough to still be present on artificial heart valves after three weeks testing in circulating plasma. It then had about the same antithrombin uptake as a non-tested surface. The film was, however, partly destroyed by the durability test and plasma proteins were deposited. </p><p>The PC functionalized, APTMS linked polymer was found to be much shorter than could be expected from random reactions. One plausible explanation is an interaction between the PC group and the silane surface, favoring aminolysis close to the PC group. This is consistent with our finding that the PC group bends down towards the surface.</p>

In order to develop blood biocompatible materials a heparin surface and a phosphorylcholine (PC) functionalized polymer surface were characterized using photoelectron spectroscopy (PES). The formation of the heparin surface was studied by quartz crystal microbalance with dissipation monitoring (QCM-D). This heparin surface consists of heparin conjugates deposited on a conditioning layer, applied once or twice. The PC functionalized polymer, poly(trimethylene carbonate), was linked to a silicon substrate through 3-amino- propyltrimethoxysilane (APTMS), also studied using PES. Synchrotron radiation based PES showed that the thicker heparin film resulted in complete coverage of the substrate, while the thinner did not. This could explain the difference in blood biocompatibility between the two films, as observed by others. It was also found that the heparin chains bend down towards the substrate (under vacuum). For the thinner heparin film the modifications, resulting from extensive irradiation of the sample, were studied with synchrotron radiation based PES. This was done at a pressure of about 10-7 mbar and in 0.5 mbar water vapor. It was found that the modification is slower under water vapor than at low pressures and that the damaged film incorporates water upon exposure. The heparin coating was found to be stable and wear resistant enough to still be present on artificial heart valves after three weeks testing in circulating plasma. It then had about the same antithrombin uptake as a non-tested surface. The film was, however, partly destroyed by the durability test and plasma proteins were deposited. The PC functionalized, APTMS linked polymer was found to be much shorter than could be expected from random reactions. One plausible explanation is an interaction between the PC group and the silane surface, favoring aminolysis close to the PC group. This is consistent with our finding that the PC group bends down towards the surface.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination. / Ph. D.

Les cellules dérivées de la moelle osseuse, principalement les cellules endothéliales progénitrices, sont réduites chez les patients souffrant de maladies cardiovasculaires. Leur mobilisation et leur incorporation aux sites de lésion vasculaire sont des évènements prépondérants dans l’accélération des processus de réendothélialisation. Dans un modèle murin, le 17β-estradiol favorise les processus de guérison vasculaire par la mobilisation et le recrutement des cellules endothéliales progénitrices dérivées de la moelle osseuse. Il existe présentement plusieurs stratégies afin d’augmenter la mobilisation des cellules progénitrices ainsi que leur incorporation à la paroi vasculaire. Cependant, peu d’études privilégient la livraison locale d’un nombre élevé de cellules progénitrices fonctionnelles par un véhicule biodégradable et leur maintien au site de lésion afin de favoriser la réendothélialisation ciblée. Un polymère d’intérêt pour cette application s’avère être le chitosan. Ce biopolymère non toxique et biodégradable est couramment utilisé dans l’ingénierie tissulaire et, depuis peu, est utilisé dans la guérison vasculaire. Le chitosan complexé à la phosphorylcholine voit sa solubilité s’accroître dans les solutions aqueuses ainsi que sa biocompatibilité cellulaire en condition physiologique. Le projet de ce mémoire visait donc : 1) à étudier in vitro, la capacité d’un polymère de chitosan complexé à la phosphorylcholine à influencer l’adhésion, la survie, la différenciation et la fonctionnalité cellulaire dans un modèle murin de culture mixte de cellules dérivées de la moelle osseuse et 2) de déterminer l’impact de la présence du 17β-estradiol sur ces mêmes comportements cellulaires. Nos travaux démontrent que la matrice de chitosan-phosphorylcholine s’avère compatible avec notre modèle de culture cellulaire. En effet, ce polymère est capable de promouvoir l’organisation et le développement des cellules dérivées de la moelle osseuse de façon comparable à la matrice normalement utilisée dans la croissance in vitro des cellules endothéliales progénitrices, la fibronectine. De plus, ce polymère n’a nullement compromis l’activité migratoire des cellules, laissant supposer qu’il pourrait éventuellement être un véhicule approprié pour effectuer une livraison cellulaire à un site de lésion. Il s’avère que le 17β-estradiol, lorsqu’ajouté au milieu de culture ou complexé au polymère de chitosan phosphorylcholine, est capable de moduler le comportement cellulaire, et ce, de façon différente. Le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine démontre, par rapport à sa forme soluble, une plus grande aptitude à accroître le nombre de cellules hématopoïétiques ainsi que des cellules endothéliales progénitrices dérivées de la moelle osseuse in vitro. De plus, le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine permet une amplification marquée des cellules endothéliales progénitrices et leur offre un support adéquat afin de favoriser la guérison vasculaire. L’ensemble de nos travaux suggère que le polymère de chitosan complexé à la phosphorylcholine en présence ou non de 17β-estradiol est une matrice compatible avec les cellules progénitrices dérivées de la moelle osseuse in vitro. Le 17β-estradiol complexé au polymère est toutefois plus efficace que sa forme soluble à promouvoir l’amplification du nombre de cellules progénitrices. Ce polymère représente un outil thérapeutique attrayant et une matrice de livraison d’agent bioactif prometteuse pour le recrutement cellulaire dans l’accélération de la guérison vasculaire. / Bone marrow derived cells, including endothelial progenitor cells, are reduced in numbers in patient with cardiovascular disease or risk factors. Mobilization and incorporation of these cells at the vascular lesion site are important events in the reendothelialization process. 17β-estradiol was shown in a mouse model of injury, to favour this healing process through mechanisms which involve the mobilization and incorporation of endothelial progenitor cells derived from the bone marrow. At the moment, there are many strategies to increase endothelial progenitor cells mobilization as well as recruitment into the vascular wall. However, few studies favour local delivery of a large number of functional progenitor cells on a biodegradable scaffold and to maintain them at the lesion site in order to promote reendothelialization. An interesting biopolymer for this application is chitosan. This non toxic and biodegradable biopolymer is commonly used in tissue engineering and was recently used in vascular healing. Phosphorylcholine modified chitosan can increase the water solubility and cell biocompatibility of the biopolymer in physiological condition. This master project was thus designed to :1) evaluate, in vitro, the capacity of phosphorylcholine modified chitosan to influence cell adhesion, survival, differentiation and functionality in a mouse model of bone marrow mixed culture and 2) determine the impact of 17β-estradiol on these cell behaviours. Our results suggest an adequate biocompatibility of phosphorylcholine modified chitosan with our cell culture system. Indeed, this polymer was able to promote cell organization and development of bone marrow derived cells in the same way that fibronectin, the most commonly matrix used in the progenitor cells in vitro culture. Moreover, cell migratory activity was not compromised by the chitosan polymer. It appears that 17β-estradiol, when added to cell culture media or attached on phosphorylcholine modified chitosan is able to modulate differently cell behaviour. Our data suggest that 17β-estradiol coupled to the chitosan polymer was superior to increase the number of haematopoietic and endothelial progenitor cells derived from bone marrow in vitro compared to the soluble form. 17β-estradiol coupled to the polymer of phosphorylcholine modified chitosan allowed an increased amplification of progenitor cell number and provided adequate scaffold to favour vascular healing. We propose that phosphorylcholine modified chitosan in presence or not of 17β-estradiol is a compatible matrix with bone marrow derived progenitor cells in vitro. 17β-estradiol enhances the amplification of progenitor cell in vitro when associated to the polymer compared to its soluble form. This biopolymer may be an attractive matrix and a promising vehicle in a drug delivery therapeutic system for progenitor cells recruitment and to promote vascular healing.

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