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Identification of plasma proteins associated with pre-eclampsia by a proteomic approach.January 2005 (has links)
Yim Ka Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 93-102). / Abstracts in English and Chinese. / Statement --- p.I / Abstract --- p.Ii / Acknowledgements --- p.Iv / Publications and awards --- p.Vii / General abbreviations --- p.Viii / Technical abbreviations --- p.Ix / Abbreviations of chemicals --- p.X / List of figures --- p.Xi / List of tables --- p.Xiii / Table of contents --- p.Xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Pre-eclampsia --- p.3 / Chapter 2.1 --- Inadequate Uteroplacental Circulation --- p.5 / Chapter 2.2 --- Placenta Ischaemia --- p.7 / Chapter 2.3 --- Oxidative Stress --- p.7 / Chapter 2.4 --- Systemic Inflammatory Response --- p.9 / Chapter 2.5 --- The Continuum Theory of Clinical Syndromes --- p.12 / Chapter 2.6 --- Origin of Stimuli to Inflammatory Response --- p.13 / Chapter Chapter 3 --- Proteomic Analysis --- p.16 / Chapter 3.1 --- Methodology in Proteomic Research --- p.17 / Chapter 3.1.1 --- 2D-PAGE --- p.17 / Chapter 3.1.2 --- Mass Spectrometry --- p.18 / Chapter 3.1.2.1 --- Peptide Mass Fingerprinting --- p.18 / Chapter 3.1.2.2 --- Product-ion Data --- p.19 / Chapter 3.1.2.3 --- Matrix-assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS) --- p.19 / Chapter 3.1.3 --- Bioinformatics Tools --- p.20 / Chapter 3.2 --- Applications of Proteomic Analysis --- p.20 / Chapter Chapter 4 --- Materials and Methods --- p.21 / Chapter 4.1 --- Overview --- p.21 / Chapter 4.2 --- Patients --- p.23 / Chapter 4.2.1 --- Pre-eclampsia --- p.23 / Chapter 4.2.2 --- Recruitment --- p.23 / Chapter 4.3 --- Stage I: Comparative Proteomic Analysis --- p.24 / Chapter 4.3.1 --- Sample Processing --- p.24 / Chapter 4.3.2 --- Modified Bradford's Method --- p.24 / Chapter 4.3.3 --- Albumin Depletion --- p.25 / Chapter 4.3.4. --- Comparative Proteomic Analysis --- p.26 / Chapter 4.3.4.1 --- First Dimension: Isoelectric Focusing (IEF) --- p.28 / Chapter 4.3.4.2 --- Reduction and Alkylation --- p.29 / Chapter 4.3.4.3 --- Second Dimension: SDS-PAGE --- p.30 / Chapter 4.3.4.4 --- Gel Imaging --- p.31 / Chapter 4.3.5 --- 2D-PAGE Data Analysis --- p.32 / Chapter 4.3.5.1 --- Gaussian Spot --- p.32 / Chapter 4.3.5.2 --- Matchset --- p.33 / Chapter 4.3.5.3 --- Normalization --- p.33 / Chapter 4.3.5.4 --- Significance Analysis of Microarrays --- p.34 / Chapter 4.4 --- Stage II: Protein Identification --- p.35 / Chapter 4.4.1 --- Tryptic Peptide Fingerprinting --- p.37 / Chapter 4.4.1.1 --- Removal of Silver Ions --- p.37 / Chapter 4.4.1.2 --- Reduction and Alkylation --- p.38 / Chapter 4.4.1.3 --- In-gel Digestion --- p.38 / Chapter 4.4.1.4 --- Clean up of Peptides --- p.39 / Chapter 4.4.1.5 --- Mass Spectrometric Analysis --- p.39 / Chapter 4.4.2 --- Database search: Profound-Peptide Mapping --- p.40 / Chapter 4.4.3 --- Database search: Mascot-MS/MS Ion Search --- p.40 / Chapter 4.5 --- Stage III: Immunoassays --- p.41 / Chapter 4.6 --- Sample Size and Power Calculations --- p.42 / Chapter 4.7 --- Statistical Analysis --- p.42 / Chapter Chapter 5 --- Results --- p.43 / Chapter 5.1 --- Patients' Demographic Characteristics and Obstetric Outcomes for 2D-PAGE Analysis --- p.43 / Chapter 5.2 --- Plasma Protein Quantity --- p.45 / Chapter 5.3 --- 2D-PAGE --- p.47 / Chapter 5.4 --- SAM Analysis --- p.47 / Chapter 5.5 --- Spot # 5204 and Spot # 6210 --- p.50 / Chapter 5.6 --- Protein Identification --- p.54 / Chapter 5.6.1 --- Identification of Protein Spot # 5204 --- p.54 / Chapter 5.6.2 --- Identification of Protein Spot # 6210 --- p.61 / Chapter 5.7 --- Patients' Demographic Characteristics and Obstetric Outcomes for Immunoassays --- p.68 / Chapter 5.8 --- Immunoassays --- p.70 / Chapter Chapter 6 --- Discussion --- p.81 / Chapter 6.1 --- Role of ficolins and MBL in complement activation pathway --- p.82 / Chapter 6.2 --- Structure of ficolins and MBL --- p.83 / Chapter 6.2.1 --- H-ficolin --- p.85 / Chapter 6.2.2 --- L-ficolin --- p.86 / Chapter 6.3 --- Immune Response and Pre-eclampsia --- p.86 / Chapter 7 --- Conclusion --- p.89 / Appendix --- p.90 / References --- p.93
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Determination of Fe, Cu and Zn in human plasma by energy dispersive X-ray fluorescence spectrometry.January 1993 (has links)
by Chan Wing-yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 93-95). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- "Clinical Significance of Fe, Cu and Zn" --- p.2 / Chapter 1.3 --- Alternative Methods of Analysis --- p.5 / Chapter 1.4 --- Principles of Energy Dispersive X-ray Fluorescence Spectrometry --- p.11 / Chapter 1.5 --- Research Plan --- p.20 / Chapter CHAPTER 2 --- EXPERIMENTAL --- p.22 / Chapter 2.1 --- Energy Dispersive X-ray Fluorescence Analysis --- p.22 / Chapter 2.1.1 --- Apparatus --- p.22 / Chapter 2.1.2 --- Reagents --- p.25 / Chapter 2.1.3 --- Procedure --- p.28 / Chapter 2.2 --- Atomic Absorption Spectrometric Analysis --- p.32 / Chapter 2.2.1 --- Apparatus --- p.32 / Chapter 2.2.2 --- Reagents --- p.32 / Chapter 2.2.3 --- Procedure --- p.34 / Chapter CHAPTER 3 --- RESULTS AND DISCUSSION --- p.38 / Chapter 3.1 --- Optimisation of Excitation Conditions --- p.38 / Chapter 3.1.1 --- Effect of Filter --- p.38 / Chapter 3.1.2 --- Effect of Tube Voltage --- p.43 / Chapter 3.1.3 --- Effect of Tube Current --- p.44 / Chapter 3.2 --- Optimisation of Preconcentration Procedure --- p.46 / Chapter 3.2.1 --- Effect of Sample Area and Collimator Size --- p.46 / Chapter 3.2.2 --- Effect of pH --- p.51 / Chapter 3.2.3 --- Effect of Ligand Concentration --- p.54 / Chapter 3.2.4 --- Effect of Mixing Time --- p.57 / Chapter 3.2.5 --- Effect of Standing Time --- p.59 / Chapter 3.2.6 --- Study of Sample Homogeneity --- p.61 / Chapter 3.3 --- Optimisation for Deproteination --- p.63 / Chapter 3.3.1 --- Effect of Different Protein Precipitants --- p.63 / Chapter 3.3.2 --- Effect of Trichloroacetic Acid Concentration --- p.65 / Chapter 3.3.3 --- Effect of Hydrochloric Acid Concentration --- p.67 / Chapter 3.3.4 --- Effect of Temperature --- p.69 / Chapter 3.3.5 --- Effect of Incubation Time --- p.71 / Chapter 3.4 --- Study of Blanks --- p.74 / Chapter 3.5 --- Construction of Calibration Curves --- p.77 / Chapter 3.6 --- Determination of Detection Limit and Sensitivity --- p.84 / Chapter 3.7 --- Accuracy and Reproducibility Tests --- p.86 / Chapter 3.8 --- Parallel Check --- p.89 / Chapter CHAPTER 4 --- CONCLUSION --- p.92 / REFERENCES --- p.93
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Structural and functional studies of histidine-rich glycoprotein in relation to its roles in angiogenesis and coagulationKassaar, Omar January 2014 (has links)
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.
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Distribution and function of the hemolymph proteins, hemoecdysin and hemocyanin, in relation to the molt cycle of the juvenile Dungeness crab, Cancer magester [i.e. magister], and size-specific molting and reproductive capability of the adult female Cancer magisterOtoshi, Clete Asa January 1994 (has links)
Typescript.
Includes vita and abstract.
Bibliography: Includes bibliographical references (leaves 93-101).
Description: xi, 101 leaves : ill. ; 29 cm.
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Plasma DNA sequencing: a tool for noninvasive prenatal diagnosis and research into circulating nucleic acids. / CUHK electronic theses & dissertations collectionJanuary 2010 (has links)
In the first part of this thesis, two chromosome Y specific genes ( SRYand TSPY) were chosen as the molecular targets to investigate the characteristics of fetal-specific DNA fragments in maternal plasma. By employing the touch down ligation-mediated PCR coupled with cloning and sequencing, the end property and the fragment species of fetal DNA were studied. / Noninvasive prenatal detection of fetal chromosomal aneuploidies is a much sought-after goal in fetomaternal medicine. The discovery of fetal DNA in the plasma of pregnant women has offered new opportunities for this purpose. However, the fact that fetal DNA amounts to just a minor fraction of all DNA in maternal plasma makes it challenging for locus-specific DNA assays to detect the small increase in sequences derived from a trisomic chromosome. On the other hand, although the clinical applications of plasma DNA for prenatal diagnosis are expanding rapidly, the biological properties of circulating DNA in plasma remain unclear. Recently, next-generation sequencing technologies have transformed the landscape of biomedical research through the ultra-high-throughput sequence information generated in a single run. Massively parallel sequencing allows us to study plasma DNA at an unprecedented resolution and also precisely detect fetal chromosomal aneuploidies in a locus-independent way. / Our group has demonstrated the use of massively parallel sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. In the second part of this thesis, the clinical utility of this new sequencing approach was extended to the prenatal detection of fetal trisomy 18 and 13. A region-selection method was developed to minimize the effects of GC content on the diagnostic sensitivity and precision for the prenatal diagnosis of trisomy 13. To facilitate the next-generation sequencing-based maternal plasma DNA analysis for clinical implementation, two measures, i.e., lowering the starting volume of maternal plasma and barcoding multiple maternal plasma samples, were investigated. / Taken together, the results presented in this thesis have demonstrated the clinical utility of massively parallel sequencing of maternal plasma DNA and have also provided us a better understanding of the biology of circulating DNA molecules. / The third part of this thesis focuses on the massively parallel paired-end sequencing of plasma DNA. By analyzing millions of sequenced DNA fragments, the biological properties of maternal plasma DNA were elucidated, such as the size distribution of fetal-derived and maternally-contributed DNA molecules and the potential effect of epigenetic modification on DNA fragmentation. Moreover, the plasma DNA from hematopoietic stem cell transplant patients was characterized by paired-end sequencing approach. These sequencing data not only confirmed the predominant hematopoietic origin of cell-free DNA but also revealed the size difference between hematologically-derived and other tissue-derived DNA molecules in plasma. / Zheng, Wenli. / Adviser: Lo Yu Ming Dennis. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 261-275). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Development of bioinformatics algorithms for trisomy 13 and 18 detection by next generation sequencing of maternal plasma DNA.January 2011 (has links)
Chen, Zhang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 109-114). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATIONS --- p.VI / CONTRIBUTORS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XIV / LIST OF ABBREVIATIONS --- p.XVI / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY BY NEXT GENERATION SEQUENCING TECHNOLOGY --- p.2 / Chapter 1.1 --- FETAL TRISOMY --- p.2 / Chapter 1.2 --- CONVENTIONAL PRENATAL DIAGNOSIS OF FETAL TRISOMIES --- p.3 / Chapter 1.3 --- CELL FREE FETAL D N A AND ITS APPLICATION IN PRENATAL DIAGNOSIS --- p.5 / Chapter 1.4 --- NEXT GENERATION SEQUENCING TECHNOLOGY --- p.5 / Chapter 1.5 --- SUBSTANTIAL BIAS IN THE NEXT GENERATION SEQUENCING PLATFORM --- p.9 / Chapter 1.6 --- PRENATAL DIAGNOSIS OF TRISOMY BY NEXT GENERATION SEQUENCING --- p.10 / Chapter 1.7 --- AIMS OF THIS THESIS --- p.11 / Chapter SECTION I I : --- MATERIALS AND METHODS --- p.13 / Chapter CHAPTER 2: --- METHODS FOR NONINVASIVE PRENATAL DIAGNOSIS OF FETAL TRISOMY MATERNAL PLASMA DNA SEQUENCING --- p.14 / Chapter 2.1 --- STUDY DESIGN AND PARTICIPANTS --- p.14 / Chapter 2.1.1 --- Ethics Statement --- p.14 / Chapter 2.1.2 --- "Study design, setting and participants" --- p.14 / Chapter 2.2 --- MATERNAL PLASMA D N A SEQUENCING --- p.17 / Chapter 2.3 --- SEQUENCING DATA ANALYSIS --- p.18 / Chapter SECTION I I I : --- TRISOMY 13 AND 18 DETECTION BY THE T21 BIOINFORMATICS ANALYSIS PIPELINE --- p.21 / Chapter CHAPTER 3: --- THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.22 / Chapter 3.1 --- INTRODUCTION --- p.22 / Chapter 3.2 --- METHODS --- p.23 / Chapter 3.2.1 --- Bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Performance of the T21 bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3.2 --- The precision of quantifying chrl 3 and chrl 8 --- p.27 / Chapter 3.4 --- DISCUSSION --- p.29 / Chapter SECTION IV : --- IMPROVING THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.30 / Chapter CHAPTER 4: --- IMPROVING THE ALIGNMENT --- p.31 / Chapter 4.1 --- INTRODUCTION --- p.31 / Chapter 4.2 --- METHODS --- p.32 / Chapter 4.2.1 --- Allowing mismatches in the index sequences --- p.32 / Chapter 4.2.2 --- Calculating the mappability of the human reference genome --- p.33 / Chapter 4.2.3 --- Aligning reads to the non-repeat masked human reference genome --- p.34 / Chapter 4.2.4 --- Trisomy 13 and 18 detection --- p.34 / Chapter 4.3 --- RESULTS --- p.34 / Chapter 4.3.1 --- Increasing read numbers by allowing mismatches in the index sequences --- p.34 / Chapter 4.3.2 --- Increasing read numbers by using the non-masked reference genome for alignment . --- p.38 / Chapter 4.3.3 --- Allowing mismatches in the read alignment --- p.42 / Chapter 4.3.4 --- The performance of trisomy 13 and 18 detection after improving the alignment --- p.47 / Chapter 4.4 --- DISCUSSION --- p.50 / Chapter CHAPTER 5: --- REDUCING THE GC BIAS BY CORRECTION OF READ COUNTS --- p.53 / Chapter 5.1 --- INTRODUCTION --- p.53 / Chapter 5.2 --- METHODS --- p.54 / Chapter 5.2.1 --- Read alignment --- p.54 / Chapter 5.2.2 --- Calculating the correlation between GC content and read counts --- p.55 / Chapter 5.2.3 --- GC correction in read counts --- p.55 / Chapter 5.2.4 --- Trisomy 13 and 18 detection --- p.56 / Chapter 5.3 --- RESULTS --- p.56 / Chapter 5.3.1 --- GC bias in plasma DNA sequencing --- p.56 / Chapter 5.3.2 --- Correcting the GC bias in read counts by linear regression --- p.59 / Chapter 5.3.3 --- Correcting the GC bias in read counts by LOESS regression --- p.65 / Chapter 5.3.4 --- Bin size --- p.72 / Chapter 5.4 --- DISCUSSION --- p.75 / Chapter CHAPTER 6: --- REDUCING THE GC BIAS BY MODIFYING THE GENOMIC REPRESENTATION CALCULATION --- p.77 / Chapter 6.1 --- INTRODUCTION --- p.77 / Chapter 6.2 --- METHODS --- p.78 / Chapter 6.2.1 --- Modifying the genomic representation calculation --- p.78 / Chapter 6.2.2 --- Trisomy 13 and 18 detection --- p.78 / Chapter 6.2.3 --- Combining GC correction and modified genomic representation --- p.78 / Chapter 6.3 --- RESULTS --- p.79 / Chapter 6.3.1 --- Reducing the GC bias by modifying genomic representation calculation --- p.79 / Chapter 6.3.2 --- Combining GC correction and modified genomic representation --- p.86 / Chapter 6.4 --- DISCUSSION --- p.89 / Chapter CHAPTER 7: --- IMPROVING THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION --- p.91 / Chapter 7.1 --- INTRODUCTION --- p.91 / Chapter 7.2 --- METHODS --- p.92 / Chapter 7.2.1 --- Comparing chrl 3 or chrl8 with other chromosomes within the sample --- p.92 / Chapter 7.2.2 --- Comparing chrl 3 or chrl 8 with the artificial chromosomes --- p.92 / Chapter 7.3 --- RESULTS --- p.93 / Chapter 7.3.1 --- Determining the trisomy 13 and 18 status by comparing chromosomes within the samples --- p.93 / Chapter 7.3.2 --- Determining the trisomy 13 and 18 status by comparing chrl3 or chrl 8 with artificial chromosomes --- p.97 / Chapter 7.4 --- DISCUSSION --- p.100 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.102 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.103 / Chapter 8.1 --- THE PERFORMANCE OF THE T21 BIOINFORMATICS ANALYSIS PIPELINE DEVELOPED FOR TRISOMY 21 DETECTION IS SUBOPTIMAL FOR TRISOMY 13 AND 18 DETECTION --- p.103 / Chapter 8.2 --- THE ALIGNMENT COULD BE IMPROVED BY ALLOWING ONE MISMATCH IN THE INDEX AND USING THE NON-REPEAT MASKED HUMAN REFERENCE GENOME AS THE ALIGNMENT REFERENCE --- p.104 / Chapter 8.3 --- THE PRECISION OF QUANTIFYING CHR13 AND CHR18 COULD BE IMPROVED BY THE G C CORRECTION OR THE MODIFIED GENOMIC REPRESENTATION --- p.104 / Chapter 8.4 --- THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION COULD BE IMPROVED BY COMPARING CHR13 OR CHR18 WITH ARTIFICIAL CHROMOSOMES WITHIN THE SAMPLE --- p.105 / Chapter 8.5 --- PROSPECTS FOR FUTURE WORK --- p.106 / REFERENCE --- p.109
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