In 2010, Bright et al. created two person mixtures to determine how effective traditional differential extraction was in determining mixtures by examining mixture proportion variation by using the peak heights from each sample. This project aims to follow that method, however, in this case using a Temperature Controlled Differential Extraction (TCDE) to analyze post coital swabs in place of a traditional differential extraction. The project also aims to determine how efficient the separation of sperm cells from epithelial cells was by comparing the mixture proportion mean of male deoxyribonucleic acid (DNA) from an Acrosolv digest that did not undergo the TCDE to the proportion of male DNA from the TCDE. The amount of DNA remaining on a swab after undergoing the TCDE was also assessed as a material fraction. Many of the material fractions generated a mixture in their profiles and thus enough DNA to generate a male profile was remaining on the swab after the TCDE in almost all cases. The sperm fractions were mostly single source male profiles or profiles with the male DNA as a major contributor and the female DNA as a minor contributor.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

In conclusion, two new methods of cccDNA quantitation were developed and validated. The two assays are complementary to each other and may be used in patients with extreme HBV DNA levels. These cccDNA assays should be further validated in larger studies and may become important tests for diagnostic, prognostic and treatment monitoring purposes. / Over 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, which leads to many cases of cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a critical intracellular replicative intermediate and cannot be eliminated during antiviral therapy. Current methods for cccDNA detection are limited by false positive detection due to the interference by HBV relaxed circular DNA (rcDNA). The tests also have limited sensitivity to detect cccDNA at low concentrations. Hence, we aimed to develop a highly sensitive and highly specific assay for cccDNA detection with wide linear range. / The modified Bowden's assay had the highest intrahepatic cccDNA detection rate (60 positive results out of 61 cases). The detection rate of the modified Bowden's assay is significantly higher than that of the Bowden's assay. On the other hand, the cccDNA detection rate in serum samples was low at 20--27% by all 3 assays. In 5 samples in which cccDNA was undetectable by the Bowden's assay but detectable by the other two assays, a point mutation in the HBV genome was found in the forward primer binding site of the Bowden's assay. This partly explained the false negative results. / The quantification result of cccDNA by the bisulfite conversion assay was significantly lower than that by the Bowden's assay assay (P=0.001) and the modified Bowden's assay (P=0.003). When the total HBV DNA was higher than 107 copies/ml, the serum cccDNA level detected by the bisulfite conversion assay was significantly lower than that detected by the Bowden's assay (P=0.008) and the modified Bowden's assay (P=0.046). When the total HBV DNA is less than 107 copies/ml, there were no significant differences. This suggests that the bisulfite conversion assay was less affected by rcDNA even in samples containing a high viral load. / With this background, two new cccDNA assays were developed and optimized. Bowden's assay was used as a standard to evaluate the performance of new assays. The first new assay (modified Bowden's assay) involved the use of new primers and probes that targeted more conserved regions in the HBV genome. The second assay adopted the bisulfite conversion method, which introduced gene sequence changes into the HBV genome and thereby enhance the specificity of the assay. Capillary sequencing was performed to find mutations in primers and probe range of different assays. / Yu, Ling. / Advisers: Vincent Wai-Sun Wang; Joseph Jao-Yiu Sung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 105-111). / 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.

腎細胞癌是一種成人惡性腫瘤，治療效果不理想且常發生腫瘤轉移。目前對腎細胞癌的研究主要集中於鑒定並驗證可用於癌癥早期診斷和預後判斷的新型潛在生物標誌物。擬遺傳學變化尤其是啟動子CpG二核苷酸甲基化所導致的抑癌基因功能喪失已被廣泛認為是腫瘤發生的一個主要機理。迄今為止，已有許多抑癌基因在腎細胞癌中被報道出現啟動子甲基化。這些發現為腎癌發生的分子機制及潛在生物標誌物提供了新的思路。本課題旨在探索ZNF382和BCL6B這兩個鋅指蛋白抑癌基因在腎細胞癌中的啟動子甲基化情況，及其與腫瘤抑制有關的生物學功能和可能的分子機制。 / 鋅指蛋白轉錄抑制子ZNF382已在多種癌癥中被報道為功能性抑癌基因， 且常伴隨有啟動子甲基化導致的基因沈默，但其在腎細胞癌中尚未被報道。我們發現ZNF382在腎癌細胞系中由於啟動子CpG甲基化而致表達下調或基因沈默，並且其表達下調或沈默可被去甲基化藥物逆轉，在正常細胞系中則觀察不到這一現象。腎癌原發腫瘤組織中也廣泛檢測到ZNF382異常甲基化。在ZNF382沈默的腎細胞癌細胞系中，外源表達的ZNF382顯著地抑制了腫瘤細胞集落形成和細胞遷移，並且誘導細胞發生雕亡。而且，我們發現ZNF382在腎癌細胞系中可抑制多種致瘤基因和幹細胞標誌基因的表達。因此，本研究證明ZNF382通過抑制下遊致癌基因和幹細胞標誌基因的表達從而發揮抑制腫瘤的功能，並且其在腎細胞癌中常因啟動子高度甲基化而導致基因失活。 / 另一個鋅指蛋白基因BCL6B（ZNF62）已被證實可通過招募組蛋白去乙酰化酶抑制靶基因的轉錄，但其在腎細胞癌中的表達情況和生物學功能尚不清楚。我們發現，BCL6B基因在正常腎組織和正常細胞系中穩定表達， 但在腎癌細胞系中由於啟動子甲基化其表達下調或沈默。去甲基化藥物可以重新激活BCL6B的表達，同時伴隨其啟動子的去甲基化。BCL6B甲基化在腎癌原發腫瘤組織中也被頻繁檢測到。在腎癌細胞系中，外源表達BCL6B顯著抑制了腫瘤細胞集落形成和細胞遷移，並且誘導腫瘤細胞雕亡。我們進一步發現，BCL6B作為功能性轉錄抑制子在腎癌細胞系中抑制多種致癌基因和幹細胞標誌基因的表達。這些結果表明BCL6B是腎細胞癌的一個抑癌基因且其在腎癌中常被甲基化。 / 綜上所述，本課題從擬遺傳學和生物學功能兩個方面分別鑒定了腎癌中的兩個鋅指蛋白抑癌基因，ZNF382 和BCL6B。此研究可以幫助更好地了解腎癌發生的分子機理，並且為發展新的腎癌標誌物提供了更多思路。 / Renal cell carcinoma (RCC) is a malignant cancer in adults, often with poor outcome and frequent metastasis. Recent studies on this disease focus on the identification and verification of novel potential biomarkers for early detection and prognostic prediction of cancer. Epigenetic alterations, especially promoter CpG methylation, leading to the loss of tumor suppressor gene (TSG) function have been widely recognized as a major cause for tumor pathogenesis. To date, a number of TSGs with aberrant promoter methylation have been reported in RCC, which provides new insights into the molecular mechanism of renal cancer and the potential as biomarkers. The aim of this study is to characterize promoter methylation of two zinc finger tumor suppressors, ZNF382 and BCL6B, their biological functions and underlying molecular mechanisms in RCC. / Transcription repressor ZNF382 (zinc finger protein 382) was reported as a functional TSG with frequent inactivation by promoter methylation in multiple carcinomas, but not studied in RCC yet. I found that ZNF382 was silenced or downregulated in RCC cell lines due to promoter CpG methylation which could be reversed by pharmacologic demethylation treatment, but not in normal renal cell lines. Aberrant methylation of ZNF382 was also frequently detected in the RCC primary tumors. Ectopic expression of ZNF382 in the silenced RCC cells strongly inhibited their clonogenicity and migration, as well as promoted cell apoptosis. Moreover, I found that ZNF382 repressed the expression of multiple oncogenes and stem cell markers in RCC cells. Therefore, my results demonstrate ZNF382 exerts the tumor suppressive function through repressing the downstream target oncogenes and stem cell markers, and is often epigenetically inactivated by promoter methylation in RCC. / Another zinc finger protein, B cell CLL/lymphoma 6 member B (BCL6B, ZNF62) has been identified to repress transcription of its target genes by recruiting histone deacetylases, but its expression and biological function in RCC remain largely unknown. BCL6B was readily expressed in normal kidney tissue and renal cell line. BCL6B was silenced or downregulated by promoter CpG methylation in RCC cell lines. Pharmacologic demethylation reactivated BCL6B expression along with concomitant promoter demethylation. BCL6B methylation was also frequently detected in RCC primary tumors. Ectopic expression of BCL6B in RCC cells significantly inhibited tumor clonogenicity and migration of RCC cells, and induced tumor cell apoptosis. We further found that BCL6B as functional repressor suppressed the expression of multiple oncogenes and stem cell markers. These data indicated BCL6B was a functional tumor suppressor frequently methylated in RCC. / In summary, my study identified two zinc finger tumor suppressors, ZNF382 and BCL6B, in RCC from both epigenetical and functional aspects. This work may contribute to a better understanding of the molecular mechanisms of renal cancer pathogenesis and also give more clues to the discovery of novel biomarkers for RCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Rong, Rong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 110-128). / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of abbreviations --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / List of Publications --- p.xviii / Chapter Chapter 1 --- Literature Reviews --- p.1 / Chapter 1.1 --- Molecular basis of cancer --- p.1 / Chapter 1.1.1 --- Oncogenes and TSGs --- p.2 / Chapter 1.1.2 --- Cancer genetics --- p.3 / Chapter 1.1.3 --- Cancer epigenetics --- p.4 / Chapter 1.1.4 --- DNA methylation --- p.4 / Chapter 1.1.4.1 --- Mechanism of DNA methylation --- p.5 / Chapter 1.1.4.2 --- DNA methylation and gene transcription --- p.6 / Chapter 1.1.4.3 --- Types of DNA methylation in human cancers --- p.7 / Chapter 1.1.4.3.1 --- Hypomethylation in cancer genome --- p.8 / Chapter 1.1.4.3.2 --- Hypermethylation of TSGs in cancer --- p.8 / Chapter 1.1.5 --- The link between cancer genetics and cancer epigenetics --- p.9 / Chapter 1.2 --- Renal cell carcinoma (RCC) --- p.10 / Chapter 1.2.1 --- Epidemiology of RCC --- p.10 / Chapter 1.2.2 --- Histopathology of RCC --- p.14 / Chapter 1.2.3 --- Genetic and epigenetic alterations in RCC --- p.16 / Chapter 1.2.3.1 --- Genetic alterations --- p.17 / Chapter 1.2.3.2 --- Epigenetic alterations --- p.22 / Chapter 1.2.3.2.1 --- Aberrant DNA hypermethylation in RCC --- p.22 / Chapter 1.2.3.2.2 --- Histone and chromatin regulations in RCC --- p.24 / Chapter 1.2.4 --- Signaling pathways associated with RCC --- p.25 / Chapter 1.2.4.1 --- VHL/HIF signaling in RCC --- p.26 / Chapter 1.2.4.2 --- PI3K/AKT/mTOR signaling in RCC --- p.27 / Chapter 1.2.4.3 --- Wnt/β-catenin signaling in RCC --- p.28 / Chapter 1.2.4.4 --- HGF/MET signaling in RCC --- p.31 / Chapter 1.3 --- Transcription factor family of zinc finger proteins --- p.32 / Chapter 1.3.1 --- Zinc Finger Protein 382 (ZNF382) --- p.33 / Chapter 1.3.2 --- B cell CLL/lymphoma 6, member B (BCL6B) --- p.34 / Chapter Chapter 2 --- Aim of Study --- p.36 / Chapter 2.1 --- Identify two zinc finger repressors as TSGs for RCC --- p.37 / Chapter 2.2 --- Study their tumor suppressor roles in RCC --- p.37 / Chapter 2.3 --- Explore the mechanisms of their tumor suppressor function --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.39 / Chapter 3.1 --- Cell lines and tissue samples --- p.39 / Chapter 3.1.1 --- Cell lines, tumors and normal tissue samples --- p.39 / Chapter 3.1.2 --- Maintenance of cell lines --- p.39 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.40 / Chapter 3.1.4 --- Total RNA extraction --- p.40 / Chapter 3.1.5 --- Genomic DNA extraction --- p.41 / Chapter 3.2 --- General techniques --- p.42 / Chapter 3.2.1 --- Agarose gel electrophoresis --- p.42 / Chapter 3.2.2 --- TA cloning --- p.43 / Chapter 3.2.3 --- Transformation of cloning vectors into E. coli competent cells --- p.43 / Chapter 3.2.4 --- Plasmid DNA extraction --- p.44 / Chapter 3.2.4.1 --- Mini-prep of plasmid DNA --- p.44 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.45 / Chapter 3.2.5 --- Measurement of DNA and RNA concentrations --- p.45 / Chapter 3.2.6 --- Preparation of reagents and medium --- p.46 / Chapter 3.2.6.1 --- Reagents for agarose gel electrophoresis --- p.46 / Chapter 3.2.6.2 --- Reagents for mini-prep of plasmid DNA --- p.46 / Chapter 3.2.6.3 --- LB medium and LB plates --- p.46 / Chapter 3.3 --- Semi-quantitative Reverse transcription (RT)-PCR --- p.47 / Chapter 3.3.1 --- Reverse Transcription --- p.47 / Chapter 3.3.2 --- Semi-quantitative PCR --- p.48 / Chapter 3.3.2.1 --- Primer design --- p.48 / Chapter 3.3.2.2 --- PCR reaction --- p.49 / Chapter 3.4 --- Real-time PCR --- p.49 / Chapter 3.5 --- Methylation analysis --- p.50 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.50 / Chapter 3.5.2 --- Bioinformatical analysis of CpG island --- p.51 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.5.3.1 --- Primers design --- p.51 / Chapter 3.5.3.2 --- PCR reaction --- p.53 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.5.4.1 --- Primers design --- p.53 / Chapter 3.5.4.2 --- PCR amplification and TA-cloning --- p.54 / Chapter 3.6 --- Construction of expression plasmids for studied genes --- p.54 / Chapter 3.6.1 --- Construction of the ZNF382-expressing vector --- p.54 / Chapter 3.6.2 --- Construction of the BCL6B-expressing vector --- p.55 / Chapter 3.7 --- Functional Study --- p.56 / Chapter 3.7.1 --- Colony formation assay on monolayer culture --- p.56 / Chapter 3.7.2 --- Wound healing assay --- p.57 / Chapter 3.7.3 --- TUNEL assay --- p.58 / Chapter 3.8 --- Western blot --- p.58 / Chapter 3.9 --- Statistical analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Epigenetic and Functional study of ZNF382 in RCC --- p.60 / Chapter 4.1.1 --- Expression profiling of ZNF382 in human adult tissues --- p.60 / Chapter 4.1.2 --- Expression profiling of ZNF382 in RCC cell lines --- p.61 / Chapter 4.1.3 --- Dense promoter CpG methylation of ZNF382 correlated with its reduced expression in RCC --- p.62 / Chapter 4.1.4 --- Restoration of ZNF382 expression by pharmacologic demethylation --- p.65 / Chapter 4.1.5 --- Frequent methylation of ZNF382 in RCC primary tumors --- p.67 / Chapter 4.1.6 --- Functional study of ZNF382 in RCC --- p.68 / Chapter 4.1.6.1 --- Ectopic expression of ZNF382 inhibits clonogencity of RCC cells --- p.68 / Chapter 4.1.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.71 / Chapter 4.1.7 --- ZNF382 induces apoptosis of RCC cells --- p.72 / Chapter 4.1.8 --- ZNF382 represses the expression of multiple oncogenes and stem cell markers in RCC --- p.73 / Chapter 4.1.9 --- Discussion --- p.76 / Chapter 4.2 --- Epigenetic and Functional study of BCL6B in RCC --- p.82 / Chapter 4.2.1 --- Expression profiling of BCL6B in human adult tissues --- p.82 / Chapter 4.2.2 --- Expression profiling of BCL6B in RCC cell lines --- p.83 / Chapter 4.2.3 --- Correlation of the methylation status of ZNF382 promoter CpG island with its aborted expression in RCC --- p.84 / Chapter 4.2.4 --- Restoration of BCL6B expression by pharmacological demethylation --- p.87 / Chapter 4.2.5 --- Frequent BCL6B methylation in RCC primary tumors --- p.88 / Chapter 4.2.6 --- Functional study of BCL6B in RCC --- p.90 / Chapter 4.2.6.1 --- Ectopic expression of BCL6B inhibits clonogencity of RCC cells --- p.90 / Chapter 4.2.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.92 / Chapter 4.2.7 --- BCL6B induces apoptosis of RCC cells --- p.93 / Chapter 4.2.8 --- BCL6B represses the expression of multiple oncogenes and stem cell markers in RCC --- p.94 / Chapter 4.2.9 --- Discussion --- p.97 / Chapter Chapter 5 --- General discussion --- p.103 / Chapter Chapter 6 --- Summary --- p.106 / Chapter Chapter 7 --- Future Study --- p.108 / Chapter 7.1 --- Identification of key responsive elements in gene promoter --- p.108 / Chapter 7.2 --- Study of genetic alterations leading to gene inactivation --- p.109 / Chapter 7.3 --- Elucidation of the transcription-repressor activity in RCC --- p.109 / Reference List --- p.110

by Tong Hung Man Joanna. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 150-172). / Abstract --- p.i / Acknowledgments --- p.iii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 1. --- Overview of renal cell carcinoma --- p.6 / Chapter 1.1 --- Epidemiology --- p.6 / Chapter 1.2 --- Etiology --- p.6 / Chapter 1.3 --- Clinical features --- p.7 / Chapter 1.4 --- Pathology --- p.9 / Chapter 2. --- The biological cell cycle --- p.15 / Chapter 2.1 --- Cell Cycle --- p.15 / Chapter 2.2 --- Cell cycle control --- p.18 / Chapter 3. --- Overview of DNA ploidy and the relationship with the biological behavior of renal cell carcinoma --- p.18 / Chapter 3.1 --- Overview of DNA ploidy --- p.18 / Chapter 3.2 --- Intratumoral heterogeneity --- p.20 / Chapter 3.3 --- Controversial prognostic value of DNA ploidy analysis in RCC --- p.21 / Chapter 3.4 --- Assessment of DNA ploidy --- p.23 / Chapter 4. --- Cell proliferation and its assessment by immunohistochemical methods --- p.34 / Chapter 4.1 --- Proliferation activity and tumor growth --- p.34 / Chapter 4.2 --- Basic principles of immunohistochemistry (IHC) --- p.34 / Chapter 4.3 --- "Ki 67, a cell proliferation marker" --- p.39 / Chapter 4.4 --- "p27kipl, a cell cycle arrest marker" --- p.41 / Chapter Chapter 3 --- Aims of the study --- p.44 / Chapter Chapter 4 --- Materials and Methods --- p.46 / Chapter 1. --- Tissue samples --- p.47 / Chapter 1.1 --- Sample retrieval --- p.47 / Chapter 1.2 --- Tissue processing --- p.47 / Chapter 1.3 --- Preparation of tissue sections --- p.47 / Chapter 2. --- Methods for quantitative DNA analysis --- p.49 / Chapter 2.1 --- Instrumentation --- p.49 / Chapter 2.2 --- Procedures for quantitative DNA analysis --- p.50 / Chapter 3. --- Immunohistochemical (IHC) studies of proliferation activity of RCC --- p.59 / Chapter 3.1 --- Antibodies used --- p.59 / Chapter 3.2 --- Other reagents --- p.60 / Chapter 3.3 --- Unmasking of antigens --- p.62 / Chapter 3.4 --- ABC method for monoclonal antibodies with a avidin/biotin blocking --- p.62 / Chapter 3.5 --- Interpretation and scoring of immunostaining --- p.64 / Chapter 4. --- Clinical data retrieval --- p.64 / Chapter 5. --- Statistical analysis --- p.65 / Chapter Chapter 5 --- Results --- p.66 / Chapter 1. --- Clinical information --- p.67 / Chapter 2. --- Pathological features --- p.67 / Chapter 2.1 --- Histological subtypes --- p.67 / Chapter 2.2 --- Nuclear grading --- p.68 / Chapter 2.3 --- Clinical stage --- p.68 / Chapter 3. --- DNA ploidy analysis --- p.76 / Chapter 3.1 --- By flow cytometry --- p.76 / Chapter 3.2 --- By static image cytometry using cytospin preparations --- p.82 / Chapter 3.3 --- By static image cytometry using tissue sections --- p.87 / Chapter 4. --- Immunohistochemistry --- p.92 / Chapter 4.1 --- Ki 67 (MIB-1) --- p.92 / Chapter 4.2 --- p27kipl --- p.96 / Chapter 5. --- Statistical analysis --- p.101 / Chapter 5.1 --- DNA ploidy analysis --- p.101 / Chapter 5.2 --- Ki 67 (MIB-1) --- p.108 / Chapter 5.3 --- p27kipl --- p.110 / Chapter 5.4 --- Nuclear grade and nuclear area --- p.112 / Chapter 5.5 --- Stage --- p.115 / Chapter 5.6 --- Survival analysis --- p.117 / Chapter Chapter 6 --- Discussion --- p.118 / Chapter 1. --- DNA ploidy analysis --- p.119 / Chapter 1.1 --- Flow cytometry --- p.119 / Chapter 1.2 --- Image analysis using cytospin preparations --- p.123 / Chapter 1.3 --- Image analysis using tissue sections --- p.126 / Chapter 1.4 --- Intratumoral heterogeneity --- p.130 / Chapter 1.5 --- Comparison of the results from three methods --- p.131 / Chapter 1.6 --- The potential significance of the DNA ploidy status --- p.137 / Chapter 2. --- Proliferation activity of RCC --- p.139 / Chapter 2.1 --- Ki 67 --- p.139 / Chapter 2.2 --- p27kipl --- p.140 / Chapter 3. --- Nuclear grade --- p.142 / Chapter 4. --- Stage --- p.143 / Chapter Chapter 7 --- Conclusion --- p.144 / Chapter Chapter 8 --- Further studies --- p.147 / References --- p.149

The nucleotide sequence of the PRD1 terminal protein gene has been determined. The coding region for PRD1 terminal protein is 777 base pairs long and encodes 259 amino acid residues (29,326 daltons). The deduced amino acid sequence of PRD1 terminal protein reveals no overall homology with other known terminal proteins or related proteins. A closer examination revealed a highly conserved amino acid sequence, YSRLRT, exist among all identified DNA terminal proteins including PRD1, PZA, Nf, φ29 and adenovirus. This is the first conserved amino acid sequence that has been found in all identified DNA terminal proteins. Not only is the YSRLRT sequence conserved, but its spatial location is similar as well. Therefore, the significance of the YSRLRT conserved sequence is suggested by both its conservative spatial location and high degree of homology across species. To study the structure-function relationship of the YSRLRT sequence of PRD1 terminal protein, in vitro site-directed mutagenesis was performed to determine the role of each amino acid in this conserved region. The PRD1 terminal protein and DNA polymerase genes were cloned into phagemid pEMBLex3, and the recombinant plasmid used for constructing mutants. Eleven PRD1 terminal protein mutant clones were examined for their priming complex formation activities. Our results have strongly demonstrated that the positive charge residue of arginine-174 plays an important role for PRD1 terminal protein function. There are 13 tyrosine residues in the predicted PRD1 terminal protein. It was of interest to known which tyrosine is actually linked to terminal nucleotide of the PRD1 DNA. We used a new approach involving replacing the tyrosine residues with phenylalanine residues in the carboxyl terminal portion of the protein. From analyses, the tyrosine-190 has been determined to be the most likely linkage site between terminal protein and PRD1 DNA.

No description available.

This thesis describes methods for constructing nanopatterned surfaces to array DNA. These surfaces enable direct observation of heretofore-unseen single-molecule reactions, eliminating bulk effects and enabling scientists to examine DNA mismatch repair and replication, including the first direct visualization of proteins binding to a target mismatch. This also facilitates directed self-organization of nanoscale features on a patterned substrate using DNA as an assembly tool. To make techniques for single-molecule visualization of biological processes more accessible, we have developed a novel technology called "DNA curtains," in which a combination of fluid lipid bilayers, nanofabricated barriers to lipid diffusion, and hydrodynamic flow can organize lipid-tethered DNA molecules into dened patterns on the surface of a microfluidic sample chamber. Using DNA curtains, aligned DNA molecules can be visualized by total internal reflection fluorescence microscopy, allowing simultaneous observation of hundreds of individual molecules within a field-of-view. Ultimately, this results in a 100X improvement in experimental throughput, and a corresponding increase in statistically signicant amounts of data. We also demonstrate site-specific labeling of DNA using DNA analogues, such as peptide nucleic acid (PNA), locked nucleic acid (LNA), and techniques such as nick-translation. Through PNA invasion, labeled DNA was self-assembled in arrays on surfaces and tagged with gold nanoparticles. In this work, DNA formed a template to self-assemble a nanoparticle in between nanoimprinted AuPd dots. Surface-based self-assembly methods offer potential for DNA employment in bottom-up construction of nanoscale arrays. This offers further proof that DNA can be useful in directed self-assembly of nanoscale architectures.

Chan Ching-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 109-123). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / Abbreviations --- p.vii / Table of contents --- p.xi / List of figures --- p.xiv / List of tables --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature review --- p.3 / Chapter 2.1 --- History --- p.3 / Chapter 2.2 --- The living environment and life cycles of Cordyccps --- p.3 / Chapter 2.3 --- Chemical constituents of Cordyceps --- p.4 / Chapter 2.3.1 --- Determintaion of active ingredients in Cordyceps --- p.6 / Chapter 2.4 --- Therapeutic Functions --- p.8 / Chapter 2.4.1 --- Cardiovascular and circulatory functions --- p.8 / Chapter 2.4.1.1 --- Effects on Cholesterol and lipid metabolism --- p.8 / Chapter 2.4.1.2 --- Dilation of vasculature and cerebolature --- p.8 / Chapter 2.4.2 --- Respiratory functions --- p.9 / Chapter 2.4.3 --- Renai functions --- p.9 / Chapter 2.4.3.1 --- Effccts on chronic renal failure patients --- p.9 / Chapter 2.4.3.2 --- Protective effects on kidney toxicity --- p.9 / Chapter 2.4.4 --- Hepatic functions --- p.10 / Chapter 2.4.4.1 --- Effect on hepatitis B patients --- p.10 / Chapter 2.4.4.2 --- Energy state of liver --- p.10 / Chapter 2.4.5 --- Aging and senescence: Longevity enhancement --- p.11 / Chapter 2.4.5.1 --- Senescence --- p.11 / Chapter 2.4.5.2 --- Antioxidant effects --- p.11 / Chapter 2.4.6 --- Immune functions --- p.12 / Chapter 2.4.6.1 --- Enhancing immune system --- p.12 / Chapter 2.4.6.2 --- Anti-tumor effects --- p.12 / Chapter 2.4.7 --- Reproductive effects --- p.13 / Chapter 2.4.8 --- Hyperglycemic effects --- p.13 / Chapter 2.5 --- Cultivation --- p.15 / Chapter 2.5.1 --- Carbon and nitrogen sources --- p.15 / Chapter 2.5.2 --- Initial pH and temperature --- p.16 / Chapter 2.5.3 --- Bioelements --- p.16 / Chapter 2.5.4 --- Agitation intensity --- p.16 / Chapter 2.5.5 --- Aeration rate --- p.18 / Chapter 2.6 --- Fungal genetics --- p.19 / Chapter 2.6.1 --- EST approach --- p.19 / Chapter 2.7 --- Proteomic studies --- p.21 / Chapter 2.7.1 --- 2D gel electrophoresis --- p.21 / Chapter 2.7.2 --- Mass spectrometry --- p.22 / Chapter 2.7.3 --- Limitations and improvements --- p.22 / Chapter 2.7.4 --- Multiple spots for the same proteins --- p.24 / Chapter 2.7.5 --- Fungal proteomics --- p.24 / Chapter 2.7.5.1 --- Extraction method --- p.24 / Chapter 2.7.5.2 --- Combined uses of EST sequences and amino acid sequences --- p.26 / Chapter 2.7.5.3 --- Glycosylation --- p.26 / Chapter 3. --- Materials and methods --- p.28 / Chapter 3.1 --- Genomic Studies --- p.28 / Chapter 3.1.1 --- Strains and growth conditions --- p.28 / Chapter 3.1.2 --- Total RNA Extraction --- p.28 / Chapter 3.1.3 --- Isolation of mRNA --- p.30 / Chapter 3.1.4 --- cDNA Library Construction --- p.30 / Chapter 3.1.5 --- PCR Screening --- p.31 / Chapter 3.1.6 --- EST sequencing --- p.31 / Chapter 3.1.7 --- EST assembling and annotation --- p.32 / Chapter 3.2 --- Proteomic Studies --- p.33 / Chapter 3.2.1 --- Sample Preparation --- p.33 / Chapter 3.2.2 --- Quantitation --- p.34 / Chapter 3.2.3 --- 2-D PAGE --- p.34 / Chapter 3.2.4 --- In-gel digestion and peptide extraction --- p.36 / Chapter 3.2.5 --- MALDI-TOF MS Analysis --- p.37 / Chapter 3.3 --- Determination of adenosine using RP-HPLC --- p.38 / Chapter 4. --- Result --- p.39 / Chapter 4.1 --- Genomic studies --- p.39 / Chapter 4.1.1 --- cDNA library --- p.39 / Chapter 4.1.2 --- cDNA sequence analysis --- p.39 / Chapter 4.1.3 --- Functional annotation and analysis --- p.42 / Chapter 4.2 --- Proteomic --- p.67 / Chapter 4.2.1 --- 2D analysis and resolution --- p.67 / Chapter 4.2.2 --- Protein identification and annotation --- p.74 / Chapter 4.2.3 --- Image analysis --- p.81 / Chapter 4.3 --- Presence of adenosine (HPLC) --- p.84 / Chapter 5. --- Discussion and conclusion --- p.91 / Chapter 5.1 --- Genomic studies --- p.91 / Chapter 5.2 --- Presence of adenosine --- p.95 / Chapter 5.3 --- Proteomic --- p.96 / Chapter 5.3.1 --- Protein with increasing expression level --- p.97 / Chapter 5.3.1.1 --- MEI5 (Spot1084) --- p.97 / Chapter 5.3.1.2 --- Hsp70 and hsp60 (Spot 894 & 903) --- p.97 / Chapter 5.3.1.3 --- GRP 78 (Spot 1085) --- p.98 / Chapter 5.3.1.4 --- Ubiquitin (Spot1071) --- p.98 / Chapter 5.3.1.5 --- "Serine-tRNA ligase, glutaminyl-tRNA synthase (Spot 1037, 924)" --- p.99 / Chapter 5.3.1.6 --- 2-isopropylmalate synthase (Spot 862) --- p.99 / Chapter 5.3.1.7 --- Acyl-CoA oxidase 3 (Spot 882) --- p.100 / Chapter 5.3.1.8 --- ATP synthase beta chain (Spot 937) --- p.100 / Chapter 5.3.2 --- Proteins with decreasing expression --- p.101 / Chapter 5.3.2.1 --- 14-3-3 protein (spot 1080) --- p.101 / Chapter 5.3.2.2 --- Actin (Spot 945) --- p.101 / Chapter 5.3.2.3 --- GTP binding protein SPI1 (Spot1031) --- p.102 / Chapter 5.3.2.4 --- Hoclp (Spot 972) --- p.102 / Chapter 5.3.2.5 --- Rchl8p (Spot 983) --- p.103 / Chapter 5.3.2.6 --- Formaldehyde dehydrogenase (Spot 958) --- p.103 / Chapter 5.3.2.7 --- V-type ATPase (Spot 961) --- p.104 / Chapter 5.3.2.8 --- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Spot 987) --- p.104 / Chapter 5.3.2.9 --- Idp3p (Spot 929) --- p.105 / References

This thesis examines the evolution, applications, and statistical interpretations of DNA typing as a tool in the field of forensic science as well as in our criminal justice system. The most controversial aspect of DNA typing involves the determination of how likely it is that two people share the same DNA profile. This involves the use of population genetics and databases of allelic frequencies as well as some assumptions about population structuring.