Poon Kin-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-147). / Abstracts in English and Chinese. / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.iii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.3 / Chapter 1.1.1.1 --- Descriptive Epidemiology --- p.4 / Chapter 1.1.1.2 --- Risk Factors --- p.7 / Chapter 1.1.2 --- Pathology --- p.22 / Chapter 1.1.2.1 --- Macroscopic Appearance --- p.22 / Chapter 1.1.2.2 --- Symptoms and Diagnosis --- p.23 / Chapter 1.1.2.3 --- Staging Classification --- p.25 / Chapter 1.1.2.4 --- Histopathology --- p.29 / Chapter 1.2 --- Microsatellite Instability (MSI) --- p.35 / Chapter 1.2.1 --- Microsatellite --- p.35 / Chapter 1.2.2 --- Mismatch Repair --- p.37 / Chapter 1.2.3 --- Microsatellite Instability (MSI) --- p.38 / Chapter 1.2.4 --- MSI in Various Cancers --- p.42 / Chapter 1.2.5 --- The Role of MSI in Carcinogenesis --- p.49 / Chapter 1.2.6 --- MSI as a Diagnostic / Prognostic Tool --- p.50 / Chapter CHAPTER II --- AIMS OF THE STUDY --- p.53 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.1.1 --- Patients and Specimens --- p.56 / Chapter 3.1.2 --- Microsatellite Markers --- p.57 / Chapter 3.2 --- Methods --- p.59 / Chapter 3.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.59 / Chapter 3.2.2 --- Microdissection of Epithelial Cells and Neoplastic Cells from Specimen Sections --- p.60 / Chapter 3.2.3 --- DNA Extraction --- p.60 / Chapter 3.2.3.1 --- Normal Blood --- p.61 / Chapter 3.2.3.2 --- Dissected Cells --- p.62 / Chapter 3.2.4 --- DNA Amplification --- p.64 / Chapter 3.2.4.1 --- End-labeling of Primers --- p.64 / Chapter 3.2.4.2 --- Polymerase Chain Reaction --- p.65 / Chapter 3.2.5 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.66 / Chapter 3.2.6 --- Autoradiography --- p.67 / Chapter 3.2.7 --- Determination of MSI --- p.67 / Chapter 3.2.8 --- HPV Detection --- p.68 / Chapter 3.2.9 --- Statistical Analysis --- p.69 / Chapter CHAPTER IV --- RESULTS --- p.70 / Chapter 4.1 --- Incidence of MSI in Cervix --- p.70 / Chapter 4.1.1 --- Incidence of MSI in Normal Cervix --- p.70 / Chapter 4.1.2 --- Incidence of MSI in CIN --- p.70 / Chapter 4.1.3 --- Incidence of MSI in Cervical Carcinoma --- p.71 / Chapter 4.1.4 --- Correlation of MSI-positive with the Evolution of Cervical Neoplasm --- p.77 / Chapter 4.2 --- Correlation of MSI-positive with Clinicopathological Characteristics in Cervical Carcinoma --- p.77 / Chapter 4.2.1 --- MSI and Age --- p.80 / Chapter 4.2.2 --- MSI and Clinical Stage --- p.80 / Chapter 4.2.3 --- MSI and Histological Grade --- p.80 / Chapter 4.2.4 --- MSI and Clinical Status --- p.81 / Chapter 4.3 --- Comparison between Two Panels of Microsatellite Markers used in MSI Detection --- p.84 / Chapter 4.4 --- Human Papilloma Virus (HPV) Infection in Cervical Neoplasm --- p.89 / Chapter 4.4.1 --- HPV Infection and Typing in CIN and Cervical Carcinoma --- p.89 / Chapter 4.4.2 --- Correlation of MSI-positive with HPV Infection in Cervical Carcinoma --- p.94 / Chapter CHAPTER V --- DISCUSSION --- p.96 / Chapter 5.1 --- MSI Detection --- p.96 / Chapter 5.1.1 --- Techniques in MSI Assays --- p.98 / Chapter 5.1.2 --- Choice of Microsatellite Markers --- p.101 / Chapter 5.1.3 --- Diagnostic Criteria of MSI --- p.105 / Chapter 5.2 --- The Role of MSI in the Carcinogenesis of Cervical Neoplasm --- p.107 / Chapter 5.3 --- The Clinical Significant of MSI in Cervical Carcinoma --- p.111 / Chapter 5.4 --- The Interaction between HPV Infection and MSI in Cervical Carcinoma --- p.113 / Chapter CHAPTER VI --- CONCLUSION --- p.116 / REFERENCES --- p.119

The domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (± 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (± 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set – 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>±2-fold change) and statistically significant (p-value<0.05).

<p>The legally binding international declaration of the Convention on Biological Diversity (signed by over 180 countries) recently acknowledged the importance of conserving genetic diversity within livestock species. This study aimed to help Canada assess molecular diversity in its horse and pony (<i>Equus ferus caballus</i>) genetic resources. Here, 24 populations were examined, with special focus on the native Canadian, Mountain and Moorland, and Nordic pony populations, using two well accepted molecular tools. Additional horse breeds and feral populations were also included in this project as some may have influenced the development of the three equine groups of interest. Altogether, 821 individuals were genotyped at 38 microsatellite loci, and 280 individuals were sequenced using a 421 base pair portion of the mitochondrial displacement Hypervariable Region I.</p> <p>Results from the microsatellite analyses indicated that 13.33% of genetic diversity arose from breed differences, whereas 84.60% and 2.07% of diversity arose from within and among individuals respectively. The New Forest and Welsh breeds were found to be the most diverse while having the highest average effective number of alleles and allelic richness (4.31 and 6.01; 4.33 and 5.87 respectively). The Eriskay and Lac La Croix breeds were found to have the lowest average effective number of alleles and allelic richness (2.51 and 3.98; 2.83 and 4.01 respectively). Expected heterozygosities were lowest in the Lac La Croix (0.61) and highest in the Welsh and New Forest (0.74) breeds, whereas observed heterozygosities were highest in the Kerry Bog (0.77) and lowest in the Exmoor (0.57) breeds. The genetic structure and admixture analyses suggested that the most probable number of unique genetic clusters was 21 as opposed to the 24 predefined populations.</p> <p>Results from the mitochondrial sequence data revealed that there were 36 informative sites producing 62 haplotypes, 20 of which were previously unreported. The Connemara was found to have the highest haplotype diversity of the pony breeds (0.89); however, the Highland pony was found to have the highest nucleotide diversity and pairwise difference (0.16 and 6.73 respectively). In contrast, the Fell pony had the lowest haplotype diversity (0.22), and the feral Sable Island population had the lowest nucleotide diversity and pairwise difference (0.01 and 0.29 respectively). Multiple phylogenetic trees were reconstructed and produced similar topologies. In general, the Mountain and Moorland and Nordic breeds were spread among the clades, whereas native Canadian populations were most frequent in the D and E clades. Interestingly, a large portion of ponies were found within the rare E clade as opposed to horses.</p> <p>Information gathered from this project can be incorporated with other available data into pre-existing conservation/breeding programs currently managed by the various breed societies to ensure that the most optimal and sustainable strategies are in place.</p>

The lake sturgeon (Acipenser fulvescens), is a species with considerable ecological, social and economic value. Unfortunately, over-exploitation and habitat alteration have led to the collapse of lake sturgeon fisheries across North America. Based on conservation concerns, the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) identified eight designatable units (DUs) among Canadian populations of lake sturgeon using the limited information available. These DUs are intended to represent taxonomically, genetically, geographically, or biogeographically distinct units below the species level. In this study, the genetic structuring among 20 lake sturgeon localities was examined using nine microsatellite loci. Lake sturgeon localities conformed to hierarchical partitioning of genetic diversity, with the greatest genetic divergence between localities in the Great Lakes and Hudson Bay drainages. Finally, minimal divergences among the current DUs warrant adjustment of lake sturgeon DU boundaries, to more appropriately reflect the distribution of genetic differentiation among lake sturgeon localities.

Karenia brevis is the major harmful bloom forming dinoflagellate in the Gulf of Mexico. The toxin produced by this dinoflagellate can cause large fish kills, marine mammal mortality, respiratory irritation, and neurotoxic shellfish poisoning in humans. Blooms can occur anywhere in the Gulf of Mexico (hereafter Gulf) but are predominantly observed off the west coast of Florida and the coast of Texas. The west coast of Florida has been hypothesized to be the origin for blooms of K. brevis in other regions within the Gulf based upon the frequent formation of blooms in this region. To investigate this possibility, microsatellite markers were used to determine the population-genetic structure of K. brevis in the Gulf of Mexico. The difficulties of culturing K. brevis required development and use of a single-cell PCR amplification protocol for preserved cells. Lugol's iodine-preserved bloom samples of K. brevis were destained with sodium thiosulfate and subjected to two rounds of PCR amplification. The destaining protocol resulted in the successful, simultaneous amplification of five microsatellite markers from single cells of K. brevis. A total of 18, highly polymorphic microsatellite markers are available for K. brevis. Each marker was amplified from 40 cultures of K. brevis isolated from water samples from Florida and Texas. Observed genetic diversity was high but similar to the genetic diversity observed in other phytoplankton species. No genetic divergence was detected between isolates from Florida and isolates from Texas. Single cells from a total of 38 field samples were analyzed at five microsatellite markers to determine if population-genetic structure was present in K. brevis in the Gulf. Significant genetic divergence between several individual samples was detected, reflecting the high genetic diversity present within the species. Observed genetic divergence was low between blooms from the west coast of Florida and the coast of Texas and supports the hypothesis of a common origin for blooms of K. brevis in the Gulf of Mexico.

The lake sturgeon (Acipenser fulvescens), is a species with considerable ecological, social and economic value. Unfortunately, over-exploitation and habitat alteration have led to the collapse of lake sturgeon fisheries across North America. Based on conservation concerns, the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) identified eight designatable units (DUs) among Canadian populations of lake sturgeon using the limited information available. These DUs are intended to represent taxonomically, genetically, geographically, or biogeographically distinct units below the species level. In this study, the genetic structuring among 20 lake sturgeon localities was examined using nine microsatellite loci. Lake sturgeon localities conformed to hierarchical partitioning of genetic diversity, with the greatest genetic divergence between localities in the Great Lakes and Hudson Bay drainages. Finally, minimal divergences among the current DUs warrant adjustment of lake sturgeon DU boundaries, to more appropriately reflect the distribution of genetic differentiation among lake sturgeon localities.

<p>The legally binding international declaration of the Convention on Biological Diversity (signed by over 180 countries) recently acknowledged the importance of conserving genetic diversity within livestock species. This study aimed to help Canada assess molecular diversity in its horse and pony (<i>Equus ferus caballus</i>) genetic resources. Here, 24 populations were examined, with special focus on the native Canadian, Mountain and Moorland, and Nordic pony populations, using two well accepted molecular tools. Additional horse breeds and feral populations were also included in this project as some may have influenced the development of the three equine groups of interest. Altogether, 821 individuals were genotyped at 38 microsatellite loci, and 280 individuals were sequenced using a 421 base pair portion of the mitochondrial displacement Hypervariable Region I.</p> <p>Results from the microsatellite analyses indicated that 13.33% of genetic diversity arose from breed differences, whereas 84.60% and 2.07% of diversity arose from within and among individuals respectively. The New Forest and Welsh breeds were found to be the most diverse while having the highest average effective number of alleles and allelic richness (4.31 and 6.01; 4.33 and 5.87 respectively). The Eriskay and Lac La Croix breeds were found to have the lowest average effective number of alleles and allelic richness (2.51 and 3.98; 2.83 and 4.01 respectively). Expected heterozygosities were lowest in the Lac La Croix (0.61) and highest in the Welsh and New Forest (0.74) breeds, whereas observed heterozygosities were highest in the Kerry Bog (0.77) and lowest in the Exmoor (0.57) breeds. The genetic structure and admixture analyses suggested that the most probable number of unique genetic clusters was 21 as opposed to the 24 predefined populations.</p> <p>Results from the mitochondrial sequence data revealed that there were 36 informative sites producing 62 haplotypes, 20 of which were previously unreported. The Connemara was found to have the highest haplotype diversity of the pony breeds (0.89); however, the Highland pony was found to have the highest nucleotide diversity and pairwise difference (0.16 and 6.73 respectively). In contrast, the Fell pony had the lowest haplotype diversity (0.22), and the feral Sable Island population had the lowest nucleotide diversity and pairwise difference (0.01 and 0.29 respectively). Multiple phylogenetic trees were reconstructed and produced similar topologies. In general, the Mountain and Moorland and Nordic breeds were spread among the clades, whereas native Canadian populations were most frequent in the D and E clades. Interestingly, a large portion of ponies were found within the rare E clade as opposed to horses.</p> <p>Information gathered from this project can be incorporated with other available data into pre-existing conservation/breeding programs currently managed by the various breed societies to ensure that the most optimal and sustainable strategies are in place.</p>

The domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (± 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (± 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set – 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>±2-fold change) and statistically significant (p-value<0.05).

The wild origin, early breeding history, and diversity of the China Rose group, including R. chinensis and its varieties, cultivars, and hybrids, are largely unknown. The aims of this study were to investigate the genetic diversity and relationships of the China Roses with related species and hybrids, including information in support of, or refuting, the hypothesis that these roses are the hybrid result of the wild R. chinensis var. spontanea and R. odorata var. gigantea. Ninety Rosa accessions, including China Roses, a Miscellaneous Old Garden Rose, Noisettes, early Polyanthas, Bourbons, Teas, and species from Sections Indicae and Synstylae were surveyed using 23 microsatellite primer pairs. The trnH-psbA chloroplast intergenic spacer was also sequenced for the China Roses, Misc. Old Garden Rose, and the species to look specifically at maternal relationships. A total of 291 alleles were scored for the 23 microsatellites, with alleles per locus ranging from 6-22 and averaging 12.65. A dendrogram based on Dice similarity and a three-dimensional Principle Coordinate Analysis (PCoorA) graph were plotted with the data. In the cluster analysis, the similarity coefficients ranged from ~0.15-0.99, with the cultivated roses forming well-defined groups at about 0.45 similarity. These groups generally reflected the American Rose Society horticultural classifications. A large number of sports and synonyms in the China Rose group were identified through this analysis as well. The PCoorA gave a better graphical representation of the relationships of the species and cultivars, and with the inclusion of the chloroplast sequence haplotypes, some maternal relationships could also be identified. This study shows that the cultivated China Roses are a closely related group and identified which accessions were likely Hybrid China Roses. The results also suggest that the China Roses were maternally derived from R. chinensis var. spontanea. Based on the microsatellites and chloroplast sequence haplotypes, the identity of the R. odorata var. gigantea accessions in this study are suspect, but the China Roses may also have this species in their background as the result of natural or artificial hybridization.

Although the Neophocaena is currently thought to be monotypic (¡§Neophocaena phocaenoides¡¨) with three ¡§subspecies¡¨, the taxonomy of this genus still remains uncertainty. The finless porpoise (¡§Neophocaena phocaenoides¡¨) is one of the small cetacean species under threats from human activities. At present, finless porpoise is listed in Appendix I of Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES). For conservation issue, it is important to define appropriate and unambiguous ¡§units¡¨. In this study, I intended to settle the taxonomic status of the specimens of finless porpoise from the southern part of the East China Sea (including the Taiwan Strait) in which the status has been under debate. Results from this study, indicated that they should belong to ¡§N. p. sunameri¡¨. Comparing the genotypes of the microsatellite of additional individuals of the ¡§N. p. asiaeorientalis¡¨ belonging to the VN-type group given in Xia and coworkers report with our data, three distinguished genetic groups were revealed: (1) the group occurring in the Taiwan Strait (i.e., the W-type group, currently recognized as ¡§N. p. phocaenoides¡¨); (2) the group occurred in the Yangtze Rive (i.e., the VN-type group, currently recognized as ¡§N. p. asiaeorientalis¡¨); (3) the group occurred in the Yellow Sea and the Taiwan strait (i.e., the IN-type + UN-type group, currently recognized as ¡§N. p. sunameri¡¨). Population differentiation was absent not only within the W-type group but also within the IN-type +UN-type group. I tried to detect the taxonomy of the two parapatric groups in where the W-type and IN-type +UN-type groups are co-exit sympatrically. No specimens with intermediate character state of the width of the dorsal denticles (i. e., hybrids) were presented in Matsu Islands on the Chinese coast where the W-type and UN-type groups were sympatric. Hybrid individual exhibiting the hybrid states of the 11 microsatellite loci between these two groups was also not found. According to E.O. Wiley¡¦s criteria for recognizing species, these two groups are eligible to be considered separate species. Based on Crandall and his coworkers¡¦ criteria of evolutionarily significant unit (ESU), the three groups, the W-type, IN-type +UN-type and VN-type groups, should be treated as three distinct ESUs.