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Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 76-86).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Keimzellen entwickeln sich aus den sogenannten Urkeimzellen, die auch als primordiale Keimzellen (PGC) bezeichnet werden. PGC entwickeln sich während der Embryonalentwicklung und wandern dann in die Gonadenanlagen, wo sie sich vermehren und bei getrenntgeschlechtlichen Arten in Spermien in Männchen und Oozyten bei Weibchen differenzieren. Während der Geschlechtsdifferenzierung sind die Gonaden anfällig für den Einfluss externer Faktoren, wie z. B. der Wassertemperatur. Eine Erhöhung der Wassertemperatur von 28°C auf 36°C während der kritischen Phase der Geschlechtsdifferenzierung, vom 10. bis zum 20. Tag nach der Befruchtung kann zu einer Vermännlichung genetisch-weiblicher Tilapien führen. In der ersten Studie führte eine entsprechende Temperaturbehandlung bei genetisch weiblichen Tilapien zu einem Anteil funktioneller Männchen von 37%. Makromorphologisch konnten die Gonaden 90 Tage alter Weibchen aus Kontroll- und Behandlungsgruppen als unreife Ovarien klassifiziert werden. Erste Reifungsprozesse bis hin zur Oogenese begannen 120 Tage nach der Befruchtung. Die Oogenese konnte mikro-morphologisch weiterhin in folgende Stadien eingeteilt werden: Chromatin Nukleolus, Peri-Nukleolus, kortikale Alveolus, Vitellogenese und reife Eizelle. Oozyten in den Phasen des Chromatin Nukleolus und des Peri-Nukleolus, welche auch die primäre Wachstumsphase darstellen, wurden bei weiblichen Fischen aller Altersgruppen festgestellt. Während weiter fortgeschrittenen Oozytenstadien erst ab einem Alter von 120 Tagen festgestellt werden konnten. Oozyten in der Phase des kortikalen Alveolus wurden demnach frühestens am 120. Lebenstag gefunden. In der Vitellogenese befindliche Oozyten traten erst nach 150 Tagen auf. Reife Eizellen wurden ab einem Alter von 180 dpf festgestellt. Es konnten keine signifikanten Unterschiede in der Eizellentwicklungsstadien zwischen Kontroll- und Behandlungstieren festgestellt werden. Das Ziel der zweiten Studie war die Identifikation von Keimbahn-Stammzellen bei Tilapien, die während der Geschlechtsdifferenzierung verschiedenen Aufzuchttemperaturen ausgesetzt waren. Die Identifizierung der Keimbahn-Stammzellen erfolgte anhand von Immunohistochemie mittels Vasa und PCNA (Proliferierendes Cell Nuclear Antigen) Antikörperfärbung. Es wurden zunächst histologische Schnitte von in Paraffin eingebettetem Ovargewebe hergestellt. Keimbahn-Stammzellen wurden im Keimepithel der Ovarien identifiziert, wobei diese in einzelner, isolierter oder in Clusterform vorlagen. Keimbahn-Stammzellen wurden sowohl bei weiblichen Tieren aus Kontroll- als auch Behandlungsgruppen identifiziert. Zusammenfassend, konnten erstmalig Keimbahn-Stammzellen und ihre Lage in den Gonaden genetisch weiblicher Tilapien, aus Kontroll- und Behandlungsgruppen, mittels Vasa und PCNA Antikörperfärbung charakterisiert werden.

"Mémoire présenté à la faculté des études supérieures en vue de l'obtention du grade de maîtrise en droit (LL.M.) option droit, biotechnologies et société" / Le transfert d'ooplasme est une nouvelle technique de reproduction (NTR) qUI bouscule les fondements utilisés pour encadrer les modifications génétiques chez l'humain. Par l'intervention dans le matériel génétique contenu dans les mitochondries des cellules, ce nouveau procédé implique la création d'enfant issus du matériel génétique de trois parents. L'exemple est intéressant en ce qu'il permet à la fois d'analyser une situation spécifique aux enjeux éthiques et sociaux considérables, mais également de poser une réflexion plus générale sur les modes d'encadrement des NTR et leur impact sur la liberté de la recherche scientifique. Les théories sociologiques issues de l'analyse de la technoscience permettent de démontrer d'une part un enthousiasme pour la recherche et d'autre part les craintes de sa dérive. L'hypothèse du pluralisme normatif, issue de ces craintes et de l'incapacité du droit à parvenir à les calmer, permet de mettre en lumière la multiplication des normes destinées à encadrer la recherche scientifique. Cette pléthore de normes est responsable d'une confusion dans l'interprétation des différents principes qui les justifient (dignité humaine, innocuité, bienfait thérapeutique), d'autant plus qu'elles doivent être conciliées entre les niveaux international, régional et national. Cette réflexion éthique sur la limitation de la liberté de la recherche par l'encadrement des NTR permet la démonstration des véritables enjeux qu'impliquent la génétique de la reproduction et propose un regard neuf sur la façon de l'envisager. / Ooplasm transfer is a new reproductive technique that jostles the basis of human gene modification. This new fertility treatment involved the transplantation of genetic material included in mitochondrion, and results in new-born with DNA from three different persons. This technique brings important sociological and ethical dilemmas. It also raises a critical discussion on how new reproductive techniques are regulated and how that regulation limits the freedom of research. Sociological theories about technosciences have shown that there is a great enthusiasm for research in society, but also great concerns on its excess. Those concerns have generated a multiplication of norms in order to control possible abuses of researchers. The multiplication of norms limits not only the freedom of research, but is also responsible for the confusion in interpreting the principles that justify them (human dignity, innocuity, health benefits), especial1y when these principles have to be reconciled at the national, regional and international level. This study is an ethical reflection on limits imposed on the freedom of research in the new reproductive genetics area. By using ooplasmic transfer as an example, this work addresses main issues of reproductive genetic and proposes a new way of understanding and considering genetics in the socio-economical context of technoscientific societies.

Genomic instability underlies diseases of unregulated cell growth that result in cancers and developmental abnormalities in humans. Similar genome destabilizing mechanisms are used to create genetic variety in crops for use in breeding and trait development. Errors that occur during DNA replication may cause mutations if they are not corrected before further cell divisions. DNA mismatch repair (MMR) corrects misinsertions and insertion/deletion DNA loop-outs that arise during DNA replication in plants, animals, prokaryotes, and some archaea, all of which incur mutations at rates 100 to 1,000-fold greater when subjected to inherited or somatic-mismatch repair deficiencies. An understanding of the effects of mismatch repair on somatic and germ-line cells in Arabidopsis thaliana is critical to the development of this plant as a model system for the study of genomic instability. Insertions and deletions of multiples of two base pairs in dinucleotide repeat sequences (microsatellites) occur more frequently in the absence of mismatch repair, and the mismatch-repair status of an individual, tissue, or cell may be inferred on the basis of microsatellite mutation frequency. Single-template PCR analysis measured microsatellite mutation frequencies in leaves and shoot-apical-meristem stem cells, and allowed me to address for the first time an important question: Do plants relax mismatch repair in vegetative tissues relative to meristematic germ-line and floral tissue? Analyses of four microsatellite loci in mismatch repair-deficient and wild type plants surprisingly suggest that there is little difference in mismatch repair activity between leaves and seeds. Mismatch-repair-deficient leaves displayed only two-fold higher microsatellite mutation frequency compared to wild type, and wild-type leaves also displayed a two-fold higher microsatellite mutation frequency compared to shoot-apical- meristems. The high frequency of microsatellite mutation in these wildtype tissues is unexpected, and it suggests that plants relax mismatch repair in differentiated tissues while maintaining genetic fidelity in a small set of stem cells in the shoot apical meristem (SAM). Genome sequencing of msh2⁻/⁻ mutation accumulation A. thaliana lines provides an estimated germ-line mutation rate of 3.9 × 10⁻⁷ in the absence of mismatch repair. Comparison of the rates of base substitution mutation per chromosome in mismatch repair-deficient plants with rates reported for wild-type plants suggests mismatch repair is more efficient on chromosome 5 than on chromosomes 1-4. Bias towards G:C → A:T mutations among transitions is maintained but increased nearly 100-fold in the absence of mismatch repair. / Graduation date: 2012

Le transfert d'ooplasme est une nouvelle technique de reproduction (NTR) qUI bouscule les fondements utilisés pour encadrer les modifications génétiques chez l'humain. Par l'intervention dans le matériel génétique contenu dans les mitochondries des cellules, ce nouveau procédé implique la création d'enfant issus du matériel génétique de trois parents. L'exemple est intéressant en ce qu'il permet à la fois d'analyser une situation spécifique aux enjeux éthiques et sociaux considérables, mais également de poser une réflexion plus générale sur les modes d'encadrement des NTR et leur impact sur la liberté de la recherche scientifique. Les théories sociologiques issues de l'analyse de la technoscience permettent de démontrer d'une part un enthousiasme pour la recherche et d'autre part les craintes de sa dérive. L'hypothèse du pluralisme normatif, issue de ces craintes et de l'incapacité du droit à parvenir à les calmer, permet de mettre en lumière la multiplication des normes destinées à encadrer la recherche scientifique. Cette pléthore de normes est responsable d'une confusion dans l'interprétation des différents principes qui les justifient (dignité humaine, innocuité, bienfait thérapeutique), d'autant plus qu'elles doivent être conciliées entre les niveaux international, régional et national. Cette réflexion éthique sur la limitation de la liberté de la recherche par l'encadrement des NTR permet la démonstration des véritables enjeux qu'impliquent la génétique de la reproduction et propose un regard neuf sur la façon de l'envisager. / Ooplasm transfer is a new reproductive technique that jostles the basis of human gene modification. This new fertility treatment involved the transplantation of genetic material included in mitochondrion, and results in new-born with DNA from three different persons. This technique brings important sociological and ethical dilemmas. It also raises a critical discussion on how new reproductive techniques are regulated and how that regulation limits the freedom of research. Sociological theories about technosciences have shown that there is a great enthusiasm for research in society, but also great concerns on its excess. Those concerns have generated a multiplication of norms in order to control possible abuses of researchers. The multiplication of norms limits not only the freedom of research, but is also responsible for the confusion in interpreting the principles that justify them (human dignity, innocuity, health benefits), especial1y when these principles have to be reconciled at the national, regional and international level. This study is an ethical reflection on limits imposed on the freedom of research in the new reproductive genetics area. By using ooplasmic transfer as an example, this work addresses main issues of reproductive genetic and proposes a new way of understanding and considering genetics in the socio-economical context of technoscientific societies. / "Mémoire présenté à la faculté des études supérieures en vue de l'obtention du grade de maîtrise en droit (LL.M.) option droit, biotechnologies et société"

Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p < 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.

Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.