by Liu Chun Ki, Kevin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 126-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of figures --- p.viii / List of tables --- p.x / List of abbreviations --- p.xi / Chapter Chapter 1 --- Introduction Epstein-Barr Virus / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- Classification --- p.2 / Chapter 1.3 --- Virus and genome structure --- p.3 / Chapter 1.4 --- Epidemiology --- p.6 / Chapter 1.4.1 --- Prevalence of infection --- p.6 / Chapter 1.4.2 --- Modes of transmission --- p.7 / Chapter 1.5 --- Pathogenesis of EBV --- p.7 / Chapter 1.5.1 --- "Adsorption, penetration and dissemination" --- p.7 / Chapter 1.5.2 --- Lytic infection cycle --- p.8 / Chapter 1.5.3 --- Latent infection cycle --- p.9 / Chapter 1.5.4 --- Functions of the EBV-specific proteins associated with latent infection cycle proteins --- p.10 / Chapter 1.5.4.1 --- EBNA1 --- p.10 / Chapter 1.5.4.2 --- EBNA2 --- p.11 / Chapter 1.5.4.3 --- "EBNA 3A, 3B and 3C" --- p.11 / Chapter 1.5.4.4 --- EBNA LP --- p.12 / Chapter 1.5.4.5 --- LMP1 --- p.13 / Chapter 1.5.4.6 --- Characteristics of EBV LMP 2 gene --- p.14 / Chapter 1.5.4.7 --- Functions of LMP 2A --- p.15 / Chapter 1.5.4.8 --- Functions of LMP 2B --- p.18 / Chapter 1.6 --- Clinical significance of EBV --- p.20 / Chapter 1.6.1 --- Infectious mononucleosis (IM) --- p.20 / Chapter 1.6.2 --- Burkitt's lymphoma (BL) --- p.20 / Chapter 1.6.3 --- Nasopharyngeal carcinoma (NPC) --- p.21 / Chapter 1.6.4 --- Hodgkin's lymphoma (HL) --- p.21 / Chapter 1.7 --- Immune response to EBV infection --- p.22 / Chapter 1.7.1 --- Humoral immune response --- p.22 / Chapter 1.7.2 --- Cellular immune response --- p.22 / Chapter 1.8 --- Diagnosis of EBV infection --- p.26 / Chapter 1.9 --- Treatment and prevention --- p.27 / Chapter 1.10 --- Nasopharygneal Carcinoma (NPC) --- p.28 / Chapter 1.10.1 --- Epidemiology --- p.28 / Chapter 1.10.2 --- Etiology --- p.28 / Chapter 1.10.2.1 --- Environmental factor associated with NPC --- p.30 / Chapter 1.10.2.2 --- Genetic factors associated with NPC --- p.31 / Chapter 1.10.2.3 --- Association of NPC and EBV --- p.31 / Chapter 1.10.3 --- Diagnosis ofNPC --- p.32 / Chapter 1.10.4 --- Treatment --- p.33 / Chapter 1.11 --- Objective of the project --- p.34 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- EBV-containing cell cultures --- p.35 / Chapter 2.2 --- Extraction of total RNA --- p.36 / Chapter 2.2.1 --- Cell lysis --- p.36 / Chapter 2.2.2 --- Protein digestion --- p.36 / Chapter 2.2.3 --- DNA digestion --- p.37 / Chapter 2.2.4 --- Elution of total RNA --- p.37 / Chapter 2.2.5 --- Purity and electrophoresis analysis of total RNA --- p.38 / Chapter 2.3 --- First strand cDNA synthesis --- p.38 / Chapter 2.4 --- PCR amplification of LMP 2 cDNA --- p.39 / Chapter 2.5 --- Isolation of the PCR amplified LMP 2 cDNA --- p.40 / Chapter 2.6 --- Purification of the PCR amplified LMP 2 cDNA --- p.41 / Chapter 2.7 --- Confirmation of the PCR amplified cDNA --- p.42 / Chapter 2.7.1 --- Nested PCR --- p.42 / Chapter 2.7.2 --- Restriction enzyme digestion --- p.44 / Chapter 2.8 --- Ligation of insert LMP 2 cDNA with vector --- p.45 / Chapter 2.9 --- Transformation of competent cells JM109 --- p.45 / Chapter 2.10 --- Screening of the recombinant clones --- p.47 / Chapter 2.11 --- Small scale purification of plasmid DNA --- p.47 / Chapter 2.12 --- Determination of the size of the insert DNA --- p.48 / Chapter 2.13 --- DNA sequencing --- p.48 / Chapter 2.13.1 --- The cycle sequencing reaction --- p.48 / Chapter 2.13.2 --- Preparation of the acrylamide gel and TBE buffer --- p.51 / Chapter 2.13.3 --- Running conditions of the electrophoresis --- p.52 / Chapter 2.13.4 --- "Processing, editing and exporting the sequences" --- p.52 / Chapter 2.14 --- Data analysis --- p.53 / Chapter 2.14.1 --- Sequence analysis --- p.53 / Chapter 2.14.2 --- Amino acid analysis --- p.53 / Chapter 2.14.3 --- Protein secondary structure analysis --- p.53 / Chapter 2.14.4 --- Hydrophobicity analysis --- p.54 / Chapter 2.14.5 --- Isoelectric point analysis --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Cell Cultures --- p.55 / Chapter 3.2 --- Extraction of total RNA --- p.56 / Chapter 3.3 --- PCR amplification --- p.61 / Chapter 3.4 --- Isolation of PCR amplified LMP 2 cDNA --- p.62 / Chapter 3.5 --- Confirmation of the PCR amplified cDNA --- p.66 / Chapter 3.5.1 --- Nested PCR --- p.66 / Chapter 3.5.2 --- Restriction enzyme digestion --- p.71 / Chapter 3.6 --- Transformation and screening --- p.77 / Chapter 3.7 --- Extraction of plasmid DNA and its digestion with restriction enzyme --- p.78 / Chapter 3.8 --- DNA sequencing --- p.83 / Chapter 3.8.1 --- DNA sequence comparison --- p.84 / Chapter 3.9 --- Amino acid sequence homology --- p.89 / Chapter 3.9.1 --- Amino acid sequence comparison --- p.90 / Chapter 3.10 --- Hydrophobicity analysis --- p.92 / Chapter 3.10.1 --- Comparison of hydrophobicity of B95-8 derived LMP2 with GeneBank --- p.93 / Chapter 3.10.2 --- Comparison of hydrophobicity of CB 14022-derived LMP2 with GeneBank --- p.95 / Chapter 3.10.3 --- Comparison of hydrophobicity of Raji-derived LMP2 with GeneBank --- p.97 / Chapter 3.11 --- Protein secondary structure analysis --- p.100 / Chapter 3.11.1 --- Comparison of secondary structure of B95-8-derived LMP2 with GeneBank --- p.100 / Chapter 3.11.2 --- Comparison of secondary structure of CB 14022-derived LMP2 with GeneBank --- p.100 / Chapter 3.11.3 --- Comparison of secondary structure of Raji-derived LMP2 with GeneBank --- p.101 / Chapter 3.12 --- Isoelectric point analysis --- p.103 / Chapter Chapter 4 --- Discussions / Chapter 4.1 --- Overall strategy for the cloning and sequencing of EBV LMP 2 gene --- p.106 / Chapter 4.2 --- Implications of the results obtained in sequencing --- p.107 / Chapter 4.3 --- Results interpretation --- p.108 / Chapter 4.3.1 --- Cell culture --- p.108 / Chapter 4.3.2 --- Extraction of total RNA --- p.108 / Chapter 4.3.3 --- PCR amplification --- p.109 / Chapter 4.3.4 --- Confirmation of the PCR amplified cDNAs using nested PCR --- p.109 / Chapter 4.3.5 --- Confirmation of the PCR amplified cDNAs using restriction enzyme digestion --- p.110 / Chapter 4.3.6 --- Ligation of EBV LMP 2 cDNA to pGEM-T Easy Vector --- p.111 / Chapter 4.3.7 --- Transformation and screening --- p.114 / Chapter 4.3.8 --- Extraction of plasmid DNA and digestion with restriction enzyme --- p.115 / Chapter 4.4 --- DNA sequencing and sequence homology --- p.116 / Chapter 4.5 --- Amino acid sequence homology --- p.117 / Chapter 4.6 --- Hydrophobicity analysis --- p.119 / Chapter 4.7 --- Protein secondary structure analysis --- p.120 / Chapter 4.8 --- Isoelectric point analysis --- p.122 / Chapter 4.9 --- Summary of results --- p.122 / Chapter 4.10 --- Conclusions --- p.124 / References --- p.126

During the past several decades polymer materials become widely used as components of medical devices and implants such as hemodialysers, bioartificial organs as well as vascular and recombinant surgery. Most of the devices cannot avoid the blood contact in their use. When the polymer materials come in contact with blood they can cause different undesired host responses like thrombosis, inflammatory reactions and infections. Thus the materials must be hemocompatible in order to minimize these undesired body responses. The earliest and one of the main problems in the use of blood-contacting biomaterials is the surface induced thrombosis. The sequence of the thrombus formation on the artificial surfaces has been well established. The first event, which occurs, after exposure of biomaterials to blood, is the adsorption of blood proteins. Surface physicochemical properties of the materials as wettability greatly influence the amount and conformational changes of adsorbed proteins. In turn the type, amount and conformational state of the adsorbed protein layer determines whether platelets will adhere and become activated or not on the artificial surface and thus to complete the thrombus formation. The adsorption of fibrinogen (FNG), which is present in plasma, has been shown to be closely related to surface induced thrombosis by participating in all processes of the thrombus formation such as fibrin formation, platelet adhesion and aggregation. Therefore study the FNG adsorption to artificial surfaces could contribute to better understanding of the mechanisms of platelet adhesion and activation and thus to controlling the surface induced thrombosis. <br /> <br /> Endothelization of the polymer surfaces is one of the strategies for improving the materials hemocompatibility, which is believed to be the most ideal solution for making truly blood-compatible materials. Since at physiological conditions proteins such as FNG and fibronectin (FN) are the usual extracellular matrix (ECM) for endothelial cells (EC) adhesion, precoating of the materials with these proteins has been shown to improve EC adhesion and growth in vitro. ECM proteins play an essential role not only like a structural support for cell adhesion and spreading, but also they are important factor in transmitting signals for different cell functions. The ability of cells to remodel plasma proteins such as FNG and FN in matrix-like structures together with the classical cell parameters such as actin cytoskeleton and focal adhesion formation could be used as an criteria for proper cell functioning. The establishment and the maintaining of delicate balance between cell-cell and cell-substrate contacts is another important factor for better EC colonization of the implants. The functionality of newly established endothelium in order to produce antithromotic substances should be always considered when EC seeding is used for improving the hemocompatibility of the polymer materials. <br /> <br /> Controlling the polymer surface properties such as surface wettability represents a versatile approach to manipulate the above cellular responses and therefore can be used in biomaterial and tissue engineering applications for producing better hemocompatible materials.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type II collagen is the major extracellular matrix (ECM) protein present in cartilage and is detected in non-chondrogenic tissues such as the heart and the neural tube during developmental stages involving rapid tissue morphogenesis indicating an active role played by the collagen in embryogenesis. Type II collagen is synthesized as a procollagen precursor which has amino- and carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central triple helical domain. Two isoforms of type II procollagen are generated by alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous to those present in regulators of the bone morphogenetic protein (BMP) signaling such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv). Previous in vitro binding assays and overexpression studies in frog embryo suggest that the CR domain of IIA antagonized BMP signaling. In order to give a better understanding of the function of IIA in embryonic development and cellular signaling, several approaches including expression pattern analyses, phenotypic analyses of null mutant and gain of function studies are employed in this study. Expression studies of IIA mRNA in early postimplantation mouse embryos find that it is present in the axial mesendoderm (including the anterior definitive endoderm [ADE] and the prechordal plate) which is a critical head organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II collagen (Col2a1) gene by homologous recombination, indeed reveals that the anterior-most neural tissue is deficient at early somitogenesis denoted by reduction/loss of the forebrain/optic cup markers. Marker studies indicate that the ADE may already be affected at the neural plate stage in IIA-/-. The neural phenotype of IIA-/- displays significant similarities with mutants deficient in BMP pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting that IIA plays a role in maintaining the specification and/or regulating the signaling properties of the anterior midline tissue which involves regulation of BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos suggest that IIA regulate BMP and the related Nodal signaling pathways in a context dependent manner which has significant implications in normal anterior neural plate development. Based on the work described in this thesis and the body of existing evidence, a model is presented which suggests that IIA promote/maintain anterior neural plate development by regulating the range and extent of BMP signaling in the anterior neural plate. This study sheds light on the role of an ECM component in regulating tissue patterning and cellular signaling during early mouse development and also provides putative function for the CR domain of other fibrillar procollagens including type I, III and V which is poorly understood currently. This work will provide the framework for the design of subsequent studies in re-examining the role of these fibrillar procollagens in embryogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

In vitro cell culture is an essential part of many cell and tissue engineering approaches. In particular, monolayer culture of mammalian cells is a key tool for applications such as cell therapy. Novel bioreactors like the Cellerator(TM) allow for expansion of cell populations on mechanically stimulated surfaces coated with proteins. This thesis constitutes a preliminary study which focused on cell-matrix interactions in the absence of stretch. The aim was to establish standard protocols for protein coating on poly (dimethyl siloxane) (PDMS) and for measuring cell proliferation. Specifically, the proliferation of rat pulmonary artery vascular smooth muscle (PAC1) cells on type I collagen and soluble fibronectin was studied. Growth curves were obtained and the doubling time for subconfluent cultures was computed. Although cell-matrix interactions do not enhance proliferation of PAC1 cells, it was found that a preliminary sulphuric acid treatment is necessary to yield a well-behaved culture.

The extracellular matrix is a vital component of the cardiovascular system, in that, it not only provides structural support but also plays a critical role in the maintenance of cellular stability. One of the major components of the vascular matrix is elastin, which confers vessels with the specialized property of stretch and recoil. Elastin deficiency has been implicated in many vascular diseases and determined experimentally to be a negative regulator of smooth muscle cell proliferation. In zebrafish, two elastin genes have been identified, which are actively expressed during development. Based on this finding, protein production and spatial localization for the two elastin proteins was studied by immunohistochemistry with specific antibodies. Results revealed a global distribution for elastin 1 in the ventral aorta and swim bladder, whereas elastin 2 was preferentially localized to the bulbus arteriosus indicating a possible specialized function of elastin 2 in this structure. This observation, and the unique physiological property of this structure, suggests a possible reason for the preservation of both elastin genes during evolution. / In the second part of this study, elastin-null mice were studied to uncover the impact of the loss of elastin on the expression of other elastic fiber-associated proteins. The expression of fibrillin-1, the major component of microfibrils, was not altered in the absence of elastin, implying that elastin is not necessary for the formation of microfibrils. On the other hand, both fibulin-2 and -5 were upregulated in the absence of elastin, suggesting that expression of these genes are controlled by elastin. Overall, this study highlights the importance of elastin in evolution, as well as its potential role in the regulation of expression of other matrix molecules.

No description available.

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 2002. / Includes bibliographical references (leaves 292-352).