<p>Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the <i>Kcnq1, Igf2</i> and <i>H19</i> genes, located at the distal end of mouse chromosome 7.</p><p>The <i>H19</i> ICR, located in the 5' flank of the <i>H19</i> gene represses paternal <i>H19</i> and maternal <i>Igf2</i> expression, respectively, but has no effect on <i>Kcnq1</i> expression, which is controlled by another ICR located at the intron 10 of the <i>Kcnq1</i> gene. This thesis demonstrates that the maternal <i>H19</i> ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the <i>H19</i> ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the <i>H19</i> ICR and CTCF. The <i>Kcnq1</i> ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the <i>Kcnq1</i> ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions.</p><p>In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.</p>

Background: Mitochondrial genomes are a valuable source of data for analysing phylogenetic relationships. Besides sequence information, mitochondrial gene order may add phylogenetically useful information, too. Sipuncula are unsegmented marine worms, traditionally placed in their own phylum. Recent molecular and morphological findings suggest a close affinity to the segmented Annelida. Results: The first complete mitochondrial genome of a member of Sipuncula, Sipunculus nudus, is presented. All 37 genes characteristic for metazoan mtDNA were detected and are encoded on the same strand. The mitochondrial gene order (protein-coding and ribosomal RNA genes) resembles that of annelids, but shows several derivations so far found only in Sipuncula. Sequence based phylogenetic analysis of mitochondrial protein-coding genes results in significant bootstrap support for Annelida sensu lato, combining Annelida together with Sipuncula, Echiura, Pogonophora and Myzostomida. Conclusion: The mitochondrial sequence data support a close relationship of Annelida and Sipuncula. Also the most parsimonious explanation of changes in gene order favours a derivation from the annelid gene order. These results complement findings from recent phylogenetic analyses of nuclear encoded genes as well as a report of a segmental neural patterning in Sipuncula.

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin specific manner. Most of the imprinted genes are located in clusters. Genetic evidences suggest that genes in imprinted clusters are regulated by Imprinting Control Regions (ICRs). To elucidate the mechanisms by which the imprinting is maintained in clusters, we have chosen a well characterized cluster at the distal end of mouse chromosome 7. This cluster contains 15 imprinted genes and they have been shown to be regulated by H19 and Kcnq1 ICRs. The mouse H19 ICR, which is shown to have a chromatin insulator function, is implicated in the regulation of H19 and Igf2 genes by interacting with the CTCF protein. It has been documented that CTCF is also involved in the maintenance of differential methylation at the ICR. In this investigation we demonstrated that CTCF maintained differential methylation is lost when we subjected the ICR containing episomal plasmids to de novo methylation machinery of the human choriocarcinoma cell line, JEG3, suggesting that the H19 ICR looses its methylation privilege property under neoplastic conditions. The Kcnq1 ICR has been implicated in the regulation of 11 imprinted genes. The Kcnq1 ICR is methylated on the active maternal allele but unmethylated on the inactive paternal allele and overlaps an oppositely oriented and paternally expressed gene known as Kcnq1ot1. In this investigation, we documented that the Kcnq1 ICR controls the imprinting of neighboring genes by behaving as a bidirectional silencer and that this function is regulated by antisense RNA Kcnq1ot1. Furthermore, we have documented that duration of antisense transcription plays a critical role in the antisense RNA- mediated silencing. In conclusion, this thesis provides more insights into the complex mechanistic aspects by which ICRs, control imprinting of genes in clusters during development and neoplasia.

The genetic diversity and phylogeography of brown bear maternal lineages have been studied extensively over the last two decades. In this study the genetic diversity and maternal phylogeography of non-invasively sampled 35 brown bears, including 5 captive individuals were reported from Turkey. In addition to the optimization of DNA extraction from hair, faeces and old skin samples and their PCRs, Bayesian phylogenetic analyses based on a 269 bp long piece of bear mitochondrial DNA were conducted and 14 novel haplotypes belonging to three major lineages were revealed. The most widespread lineage was found to be the &ldquo / Eastern&rdquo / clade 3a, while geographically more restricted &ldquo / Western&rdquo / and &ldquo / Middle Eastern&rdquo / lineages were reported for the country for the first time. A specimen from the Taurus range (southern Turkey) was shown to be closely related to the presumably extinct bears in Lebanon. Moreover, a unique novel lineage that appears to have split early within the Middle Eastern clade was defined. Despite limited sampling, this study demonstrates a high level of mitochondrial diversity in Turkish brown bears, extends the ranges of both European and Middle Eastern clades into Turkey, and identifies a new divergent lineage of possibly wider historical occurrence while demonstrating the significance of non-invasive genetic sampling for such analysis.

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.

Summarized the results of previous studies, the body size of Niviventer coxingi seemed to be correlated with altitude. For more exact evidence, geographic variations of body lengths and appendage sizes among areas were examined. Moreover, Freckleton et al. (2003) indicated that phylogenetic relationships may affect the results of one¡¦s study while variation of body size is discussed. Therefore, the phylogeographic variation among different areas was also examined in this study. The body lengths of N. coxingi in higher altitude (Fong-gang, 1700 m) were shorter than in lower altitude (Shan-ping 700 m). Body lengths of N. coxingi in Shan-ping were longer than other areas but Wu-shih-kang; and N. coxingi in Fong-gang were longer than those in Wu-tou Mountain; but no significant differences were found among other areas. No significant differences were found in appendage sizes but hind-foot length. Significant differences of the hind-foot length were only found between Shan-ping which had the longest hind-foot lengths in average and those in Wu-tou Mountain which had the shortest. No correlations were found between altitudes and the body length or appendage sizes of N. coxingi. The phylogenetic relationships based on D-loop region of N. coxingi were reconstructed by neighbor-joining, maximum parsimony and maximum-likelihood methods. An N. culturatus was used to be an outgroup. All three trees represented similar patterns. Although some individuals from neighborhood grouped together, some individuals from the same area represented distantly. Moreover, many branches represented in the root of the consensus trees because of the low bootstrap value. The results revealed the geographic variations did not correlated with their phylogenetic relationships and the heat conservation/ dissipation mechanism, which was the traditional explanation of Bergmann¡¦s rule, was not appropriate for N. coxingi, either.

<p>The first method for DNA analysis in forensics was presented in 1985. Since then, the introduction of the polymerase chain reaction (PCR) has rendered possible the analysis of small amounts of DNA and automated sequencing and fragment analysis techniques have facilitated the analyses. In most cases short tandemly repeated regions (STRs) of nuclear DNA are analysed in forensic investigations, but all samples cannot be successfully analysed using this method. For samples containing minute amounts of DNA or degraded DNA, such as shed hairs, analysis of mitochondrial DNA (mtDNA) is generally more successful due to the presence of thousands of copies of mtDNA molecules per cell.</p><p>In Sweden, ~40 % of all households have cats or dogs. With ~9 million humans shedding ~100 scalp hairs per day, and ~1.6 million cats and ~1 million dogs shedding hairs it is not surprising that shed hairs are one of the most common biological evidence found at crime scenes. However, the match probability for domestic dog mtDNA analysis has only been investigated in a few minor studies. Furthermore, although breed –sequence correlations of the noncoding mtDNA control region (CR) have been analysed in a few studies, showing limited correlations, no largescale studies have been performed previously. Thus, there have not been any comprehensive studies of forensic informativity of dog mtDNA. In the two papers presented in this thesis we have tried to lay a foundation for forensic use of analysis of domestic dog mtDNA. In the first paper, CR sequences were analysed and the exclusion capacity was investigated for a number of different populations. This is also the first comprehensive study of the correlation between mtDNA CR type and breed, type, and geographic origin of domestic dogs. Since the exclusion capacity for analysis of domestic dog CR sequences is relatively low, it was investigated in the second paper to what extent the discrimination power is improved by analysis of coding sequence. The exclusion capacity improved considerably when 3,000 base pairs of coding sequences where analysed in addition to CR sequences. This study will hopefully work as a basis for future development of analysis of dog mtDNA for forensic purposes.</p>

Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the Kcnq1, Igf2 and H19 genes, located at the distal end of mouse chromosome 7. The H19 ICR, located in the 5' flank of the H19 gene represses paternal H19 and maternal Igf2 expression, respectively, but has no effect on Kcnq1 expression, which is controlled by another ICR located at the intron 10 of the Kcnq1 gene. This thesis demonstrates that the maternal H19 ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the H19 ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the H19 ICR and CTCF. The Kcnq1 ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the Kcnq1 ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions. In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 125 pages. Includes Vita. Includes bibliographical references.

Orientador: Diogo Teruo Hashimoto / Resumo: A aquicultura brasileira tem passado por um crescimento significativo nos últimos anos. Entretanto, o potencial de produção do país certamente será mais bem aproveitado com a utilização de produtos resultantes de melhoramento genético. O pacu, Piaractus mesopotamicus (Characiformes, Serrasalmidae), é um peixe de ocorrência na bacia do rio Paraná-Paraguai e uma das principais espécies nativas utilizadas para a produção em aquicultura. A utilização de marcadores moleculares na piscicultura tem se mostrado fundamental, pois podem auxiliar no monitoramento genético das populações e em programas de pré-melhoramento. O objetivo deste trabalho foi realizar a análise da variabilidade genética de lotes selvagens e cultivados de Piaractus mesopotamicus, utilizando marcador molecular mitocondrial (Região Controladora - CR). De forma geral, os índices de diversidade se apresentaram com valores moderados (Hdsel = 0,692 e Hdpisc = 0,711; πsel = 0,0045845 e πpisc = 0,00660) e foi observada similaridade entre esses valores nas populações selvagens e cultivadas. Além disso, foi observada alta estruturação genética entre as populações selvagens e cultivadas (FST= 0,14404). Os baixos valores de diversidade encontrados em algumas das populações selvagens juntamente com os desvios de equilíbrio (D Tajima p<0,05) podem estar relacionados a diversos fatores antrópicos que vêm influenciando as populações de pacu ao longo dos anos, tais como pressões de pesca e barramentos para geração de energia elé... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Brazilian aquaculture has experienced significant growth in recent years. However, the national potential of production will certainly be better explored with the use of products resulting from breeding programs. The pacu, Piaractus mesopotamicus (Characiformes, Serrasalmidae), is a fish of occurrence in the Paraná-Paraguay River Basin and it is one of the main native species used for production in aquaculture. The use of molecular markers in fish farming has been shown to be essential, especially for genetic monitoring of populations and pre-breeding programs. The mitochondrial molecular markers (mtDNA) have a higher rate of mutations and exclusively maternal inheritance, being useful for monitoring the genetic variability of a population and generate information about the founder stock. The objective of this study was to analyze the genetic variability of wild and cultivated stocks of Piaractus mesopotamicus using mitochondrial molecular marker (Control Region). In general, the diversity indexes presented moderate values (Hdwild = 0.692 and Hdfarm = 0.711; πwild = 0.0045845 and πfarm = 0.00660) and similarity was observed between these values in the wild and cultivated populations. In addition, high genetic structuring was observed among wild and cultivated populations (FST = 0.14404). The low values of diversity found in some of the wild populations together with the equilibrium deviations (D Tajima p <0.05) may be related to several anthropic factors that have been in... (Complete abstract click electronic access below) / Mestre