by Chan Po Kwok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 132-140). / Abstract --- p.i / Acknowledgement --- p.iii / Publication --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter Section a --- Blood Coagulation --- p.1 / Chapter Section b --- The role of factor XII --- p.4 / Chapter Section c --- Genomic organisation of the human factor XII gene --- p.8 / Chapter Section d --- Protein structure of the human factor XII --- p.12 / Chapter Section e --- Mutations in factor XII --- p.16 / Chapter Section f --- Methods to detect mutations --- p.25 / Chapter Section g --- About the patient with factor XII deficiency --- p.29 / Chapter Section h --- Strategies used in this study --- p.30 / Chapter Chapter 2 --- Methods and Materials / Chapter Section a --- Genomic DNA extraction --- p.33 / Chapter Section b --- Polymerase chain reaction amplification --- p.34 / Chapter Section c --- Cycle sequencing --- p.35 / Chapter Section d --- Restriction enzyme digestion and cloning of PCR products --- p.36 / Chapter Section e --- Reverse transcription and polymerase chain reaction of factor XII ectopic transcript --- p.36 / Chapter Section f --- Subcloning of 95-bp novel fragment --- p.38 / Chapter Section g --- Gel mobility shift assay --- p.39 / Chapter Chapter 3 --- Results / Chapter Section a --- Analysis of the catalytic region of the human factor XII --- p.41 / Chapter Section b --- Ectopic transcript of factor XII in peripheral blood lymphocytes --- p.65 / Chapter Section c --- Analysis of 3'end untranslated region of factor XII gene --- p.70 / Chapter Section d --- Analysis of 5' flanking region of factor XII gene --- p.75 / Chapter Section e --- Analysis of intron B --- p.94 / Chapter Section f --- Analysis of 5'-b PCR product --- p.98 / Chapter Chapter 4 --- Discussions / Chapter Section a --- Mutations in the coding sequence of factor XII gene --- p.107 / Chapter Section b --- Ectopic transcript of factor XII in lymphocytes --- p.113 / Chapter Section c --- The 3'untranslated region of factor XII gene --- p.116 / Chapter Section d --- The 5'flanking region of factor XII gene --- p.117 / Chapter Section e --- Other possible explanations of undetectable factor XII mRNA --- p.120 / Chapter Section f --- The 95-bp novel sequence in ´5ة flanking region --- p.123 / Chapter Section g --- Technical difficulties --- p.126 / Chapter Section h --- Future study --- p.130 / Chapter Chapter 5 --- References --- p.132 / List of Tables --- p.137 / List of Figures --- p.138

Thesis (M.Sc. (Physiology)) -- University of Limpopo, 1993. / Refer to the document / University of Limpopo Research Office

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

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

Histidine-rich glycoprotein (HRG) is a plasma protein that regulates key cardiovascular processes such as coagulation, angiogenesis and immune response. The protein consists of six distinct functional domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a central histidine-rich region (HRR) and a C-terminal domain. The HRR binds Zn²⁺, which alters the affinity of HRG towards various ligands including the anticoagulant, heparin. A key aim of this study was to structurally characterise HRG. The 1.93 Å crystal structure of the HRG N2 domain presented here represents the first crystallographic snapshot of the molecule. The N2 domain is cystatin-like and N-glycosylated at Asn184. An S-glutathionyl adduct was observed at Cys185, providing in vivo evidence that release of an anti-angiogenic HRR/PRR fragment is controlled in part by a redox mechanism, representing a novel further role for GSH in regulation of angiogenesis. Since Zn²⁺ regulates some of the functions of HRG, the dynamics of Zn²⁺ in plasma were investigated using a combination of ITC, ELISA and thrombin assay systems. Zn²⁺ is normally associated with albumin in circulation, but its ability to bind Zn²⁺ is allosterically inhibited upon fatty acids binding to albumin. Elevated plasma fatty acid levels are associated with some disease states. It is proposed that this may alter the proportion of Zn²⁺ bound to HRG, which could in turn activate thrombin to promote coagulation. These studies provide evidence to suggest that Zn²⁺-dependent activation of HRG (following fatty acid binding to albumin) may play a role in the development of haemostatic complications in susceptible individuals. Finally, the Zn²⁺ binding ability of albumin was probed in order to locate unidentified sites using recombinant albumin mutants. H9A, H67A, E252A, D256A and H288A mutants all exhibited diminished Zn²⁺ binding ability, indicating that these residues are involved directly or indirectly in Zn²⁺ binding.

Ho, Sui Fan Tong. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-71). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- OBJECTIVES --- p.12 / Chapter 3. --- MATERIALS AND METHODS --- p.13 / Chapter 3.1 --- Materials --- p.13 / Chapter 3.1.1 --- Enzymes --- p.13 / Chapter 3.1.2 --- DNA Markers --- p.13 / Chapter 3.1.3 --- General Reagents --- p.13 / Chapter 3.2 --- General Methods --- p.15 / Chapter 3.2.1 --- Phenol and Phenol/Chloroform (1:1) Preparation --- p.15 / Chapter 3.2.2 --- Buffer Preparation --- p.15 / Chapter 3.2.3 --- Agarose Gel Electrophoresis --- p.18 / Chapter 3.2.4 --- Polyacrylamide Gel Electrophoresis --- p.18 / Chapter 3.3 --- DNA Study --- p.19 / Chapter 3.3.1 --- Haemophilia B Patient --- p.19 / Chapter 3.3.2 --- Blood Collection --- p.20 / Chapter 3.3.3 --- DNA Extraction --- p.20 / Chapter 3.3.4 --- DNA Quantitation --- p.21 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.22 / Chapter 3.3.6 --- Purification of PCR Products --- p.28 / Chapter 3.3.7 --- Sequencing --- p.32 / Chapter 3.3.8 --- Cloning --- p.37 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- DNA Extraction --- p.40 / Chapter 4.2 --- Calibration of the Coy TempCycler --- p.42 / Chapter 4.3 --- Optimization of PCR --- p.44 / Chapter 4.3.1 --- PCR-1 --- p.44 / Chapter 4.3.2 --- PCR-2 --- p.46 / Chapter 4.3.3 --- PCR-3 --- p.46 / Chapter 4.3.4 --- PCR-4 --- p.48 / Chapter 4.3.5 --- PCR-5 --- p.49 / Chapter 4.3.6 --- PCR-6 --- p.50 / Chapter 4.3.7 --- PCR-7 --- p.51 / Chapter 4.4 --- Purification of PCR Product --- p.52 / Chapter 4.4.1 --- GC-1 --- p.52 / Chapter 4.4.2 --- GC-2 --- p.52 / Chapter 4.4.3 --- GC-3 --- p.53 / Chapter 4.4.4 --- PAGE-1 --- p.54 / Chapter 4.4.5 --- PAGE-2 --- p.54 / Chapter 4.4.6 --- Agarose Gel Extraction with Glasswool Exclusion --- p.55 / Chapter 4.5 --- Direct Sequencing of PCR Products --- p.55 / Chapter 4.6 --- Cloning --- p.55 / Chapter 5. --- DISCUSSION --- p.57 / Chapter 5.1 --- DNA Extraction --- p.57 / Chapter 5.2 --- Polymerase Chain Reaction --- p.57 / Chapter 5.3 --- Purification of PCR Products --- p.58 / Chapter 5.4 --- Sequencing --- p.61 / Chapter 5.5 --- Cloning --- p.61 / Chapter 6. --- CONCLUSION --- p.67 / Chapter 7. --- PHOTOGRAPHS --- p.64 / Chapter 8. --- REFERENCES --- p.68

[Truncated abstract] Every year thousands of individuals suffer from thrombotic related complications that in some cases can be fatal and every year millions of women take some form of hormonal contraceptive. In some cases, there is a cause and effect relationship between the two as users of the combined oral contraceptive pill have an increased risk of developing a thrombotic event. Increased circulating levels of oestrogen cause a prothrombotic shift in the coagulation cascade resulting from upregulation of several procoagulant proteins and a decrease of key anticoagulant proteins. One of the most oestrogen sensitive anticoagulants is Protein S (PS), a product of the PROS1 gene. PS acts as a cofactor to activated protein C (aPC) and the PS-aPC complex serves to downregulate clot formation by deactivating the tenase and prothrombinase complexes via proteolytic cleavage of activated factors VIII and V, respectively. As such, low PS levels are associated with an increased risk of developing thrombotic disorders such as pulmonary embolism, stroke or coronary thrombosis and deep vein thrombosis. During pregnancy when oestrogen levels increase, a steady decline in PS is evident in the early weeks of gestation and continues to decrease to below the normal range in the 2nd trimester, remaining there until post-partum. In addition, reduced free and total PS levels are observed in users of the combined oral contraceptive (COC) pill that contains an oestrogen and a progestin. Interestingly, users of 3rd generation COCs have significantly greater reductions of PS than do 2nd generation COC users. The difference between the two forms is the type of progestin, not the oestrogen, which is predominantly ethinyl oestradiol in the majority of commercially available preparations. At present, a mechanism to describe the relationship between oestrogen and/or progesterone associated with the observed in vivo changes in the levels of PS has not been identified. The aim of this thesis was to define the molecular mechanisms involved in the regulation of PS expression by oestrogen and progesterone. In this study, a Combined Single-stranded conformational analysis and Heteroduplex Analysis (CSHA) iv methodology was optimised for screening both PROS1 DNA and mRNA for the detection of mutations. '...' This may explain why users of 3rd generation COCs display a greater reduction in circulating PS levels compared to 2nd generation users. To investigate potential PS interactions with other proteins that could be hormonally regulated, a yeast-2-hybrid (Y-2-H) screen was performed using the PS molecule as a 'bait' against molecules derived from liver and bone marrow cDNA libraries. A clone that contained a portion of another haemostatic protein, Protein Z (PZ) was isolated and confirmed via sequencing. As no full length PZ clones were identified, a second Y-2-H screen was performed once again using the PS molecule as bait and the PZ molecule as the fish. Interaction between the two proteins was shown to be possible via the successful growth of colonies on triple knock out selective media and by positive ß-galactosidase activity.

The role of the nonionic surfactant Tween 80 on the behavior of the therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and air/water interfaces. In order to provide a model system to compare results obtained for the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme was used. The experimental scheme involved the introduction of the protein and Tween to the adsorption substrate in different manners, either lysozyme Tween together or in sequence as lysozyme followed by Tween or vice versa. It was observed that the addition of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces, while no such reduction was observed on hydrophilic surfaces. A high Tween concentration was required to effect the removal of the lysozyme molecules from the hydrophobic surface and Tween was not effective in removing lysozyme from the hydrophilic surface at any concentration. These results suggest that the Tween-surface interaction is important in determining lysozyme adsorption. Similar observations were made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In this case, the presence of interfacial and solution Tween together resulted in complete prevention of rFVIII adsorption. Electrostatic forces were observed to be play an important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces were also evaluated using intrinsic fluorescence and biological activity measurements. Results obtained with respect rFVIII adsorbed mass, and structure or biological activity change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly ordered and oriented in a manner that retains the solvent accessibility of the active sites in rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at the air/water interface was investigated separately. Surface tension data recorded for rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the Tween concentration was increased. Reduced interface-induced unfolding was observed at high Tween concentrations. These results were also thought to contribute to the reduction in rFVIII aggregation typically observed as a result of exposure to the air/water interface. / Graduation date: 2006

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.

Haemophilia B is a bleeding disorder caused by various mutations of the coagulation Factor IX gene (F9) resulting in qualitative or quantitative Factor IX protein (FIX) deficiency. Factor replacement therapy is the current standard of care. Cure may be possible in the near future by gene therapy — the transfer of normal copies of F9 to patients with haemophilia, causing establishment of FIX production and correction of the bleeding phenotype. Mesenchymal stem cells (MSC) are potential vehicles for gene delivery through ex vivo gene transfer and subsequent transplantation to the patient. Lentiviral vectors can transduce MSC effectively and mediate long term gene expression. However, gene expression may decline with time due to transgene silencing. Ubiquitous Chromatin Opening Element (UCOE) is a set of genetic sequences cloned from housekeeping genes that can maintain a transcriptionally competent, open chromatin structure and was shown to prevent gene silencing by resisting DNA methylation. We tested human F9 expression and FIX protein secretion by transducing MSC with lentiviral vectors that carry the FIX gene under the control of A2UCOE (A2UCOE-hF9). A2UCOE is a 2.2 kb sequence cloned from the HNRPA2B1–CBX3 gene loci that harbour UCOE function. A2UCOE-eGFP, an enhanced Green Fluorescent Protein (eGFP) gene expression construct, was used to assist in vector titration of A2UCOE-hF9 by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MSC were transduced at various Multiplicities of Infection (MOIs) by A2UCOE-hF9 lentiviral vector. Upon transduction, F9 mRNA expression and FIX secretion were measured by qRT-PCR and ELISA respectively. Osteogenic and adipogenic differentiation assay were performed to compare differentiation potential before and after transduction at an MOI of 1. F9 mRNA expression and FIX secretion were both undetectable in untransduced MSC. Upon transduction, vector dose-dependent increase in F9 mRNA expression and FIX secretion were detected at MOIs of 1, 2, 4 and 8. The level of secreted FIX ranges from 20 to 150 μIU in 72 hours. Osteogenic and adipogenic differentiation were not affected post-transduction at an MOI of 1. In conclusion, FIX secretion by MSC was detected upon A2UCOE-hF9 lentiviral transduction. However, the level of FIX appeared to be low compared to published studies. Further studies are required to determine the cause of low FIX expression, develop methods to maximize FIX expression and confirm whether A2UCOE can prevent gene silencing and maintain sustainable gene expression. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine