Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 71-82). Also available for download via the World Wide Web; free to University of Oregon users.

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Life histories unfold within the ecological context of an organism's environment, and thus are intimately linked to organismal fitness. The evolution of alternate life history strategies, either within or between taxa, can profoundly affect ontogeny, ecology, and population dynamics. Many cnidarians (sea anemones, corals, jellyfish, etc.) exhibit complex life histories involving sexual reproduction and multiple modes of asexual reproduction. Sea anemones of the family Edwardsiidae exemplify this complexity, and are therefore an attractive system for studying the developmental and ecological ramifications of life history evolution. I used intra- and interspecific comparisons of two Edwardsiid anemones, Edwardsiella lineata, and Nematostella vectensis to investigate alternative life histories using a multifaceted approach that included field-based ecological surveys, functional genetics, transcriptomics, and phylogenetics. Both anemones are capable of sexual and asexual reproduction. N. vectensis produces a rapidly maturing direct developing larva. By contrast, E. lineata has evolved a new larval stage that parasitizes the ctenophore, Mnemiopsis leidyi. Through fieldwork surveys and laboratory culture, I documented several life history traits, such as a previously un-characterized, pre-parasitic larval stage, and the developmental dynamics of early-stage parasitic infections, that augmented gaps in our knowledge of E. lineata's life history. To better understand how and when E. lineata evolved its novel, parasitic life history, I worked with collaborators in the Finnerty lab to sequence, assemble and annotate the transcriptome. Through a multigene molecular clock approach, enabled by the E. lineata transcriptome assembly, I estimated the divergence date for these two anemones between 215-364 million years ago, thereby establishing an upper bound for the innovation of E. lineata's derived, parasitic life history. Testing a hypothesis that Wnt signaling, which patterns the oral-aboral (OA) axis during embryogenesis, also patterns the OA axis during regeneration, I demonstrated that canonical Wnt signaling is sufficient for oral tissue fate across alternate life histories (embryogenesis and regeneration) of N. vectensis. Taken together, these dissertation research activities constitute an integrative approach to investigating the evolution of life histories, and are a step towards establishing E. lineata and N. vectensis as models for studying the evolutionary developmental mechanisms of parasitism and regeneration.

Made available in DSpace on 2015-07-22T20:49:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27. Added 1 bitstream(s) on 2015-08-11T03:25:42Z : No. of bitstreams: 1 Publico-094.pdf: 903461 bytes, checksum: edf5b6e40862ad70fc99b723417b0669 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivo: Analisar as vias canônica e não-canônicas da família Wnt no ovário normal e na neoplasia benigna e maligna do ovário. Métodos: Obtiveram-se tecidos ovarianos no período entre 1993 e 2004. As pacientes foram divididas em três grupos: Grupo A, neoplasia ovariana epitelial maligna (N = 38); Grupo B, neoplasia ovariana epitelial benigna (N = 28) e Grupo C, ovários normais (N = 26). A imunoexpressão para Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) e Betacatenina foi avaliada em cada grupo. Resultados: A proporção de pacientes Wnt1-positivas no grupo A (29,4%) foi significantemente maior do que nos grupos B (4,3%) e C (9,1%) (p = 0,020). A proporção de mulheres FZD1-positivas no grupo C (54,5%) foi significantemente menor do que nos grupos A (97,1%) e B (90,0%) (p < 0,001). A proporção de pacientes wnt5apositivas foi significantemente maior no grupo A (80,0%) comparado aos grupos B (25,0%) e C (27,3%) (p<0,001). A proporção de pacientes Beta-catenina-positivas no grupo C (95,8%) foi significantemente maior do que no grupo B (52,4%) (p = 0,004). A comparação nas curvas de sobrevida no grupo A relacionada à expressão de Wnt5a mostrou diferença significante entre as pacientes positivas e negativas, sendo que as pacientes Wnt5a-positivas apresentaram resultados piores (p=0,050). Conclusão: Os achados demonstram que as vias relacionadas ao Wnt5a têm papel relevante na neoplasia ovariana maligna. Outrossim, a imunoexpressão do Wnt5a revelou ser marcador de mau prognóstico para câncer de ovário. / Objectives: To analize the canonical and noncanonical Wnt pathway in normal ovary, benign ovarian tumor and ovarian cancer. Methods: Ovarian specimens were obtained from surgeries performed between 1993 and 2004. The patients were divided in three groups: Group A, epithelial ovarian cancer (N = 38); Group B, benign epithelial neoplasia (N = 27) and Group C, normal ovaries (N = 26). Immunoreactivity for Wnt1, FZD1, Wnt5a, FZD5 and -catenin was scored for each group. Results: The proportion of Wnt1 positive women at the group A (29.4%) was significantly higher than the group B (4.3%) and C (9.1%) (p = 0.020). The proportion of FZD1 positive patients in group C (54.5%) was significantly lower than the group A (97.1%) and B (90.0%) (p < 0.001). The proportion of Wnt5a positive women was significantly higher for group A (80.0%) compared to the group B (25.0%) and C (27.3%) (p<0.001). The proportion of ß-catenin positive patients in the group C (95.8%) was significantly higher than the group B (52.4%) (p = 0.004). Comparison of the survival curves in group A according to Wnt5a expression showed a significant difference between positive and negative patients, whereas the Wnt5a positive women showed worse results (p=0.050). Conclusion: Our findings suggest that the pathways related to Wnt5a have an important role in ovarian malignant neoplasia. Furthermore, Wnt5a was found to be a predictor of poor prognosis for ovarian cancer. / FAPESP: 06/51401-1 / TEDE / BV UNIFESP: Teses e dissertações

Doctor of Philosophy / Division of Biology / Susan J. Brown / A segmented body plan is conserved in vertebrates and arthropods. Comparative studies implicate a conserved mode of A-P axis patterning and segmentation among vertebrates: Wnt signaling is involved in fate map patterning along the A-P axis and in segmentation in the posterior region of the embryo. On the other hand, comparative studies in arthropods have revealed distinct modes of development between long and short germ insects, which differ both morphologically and genetically. In the short germ insect Tribolium, a Wnt activity gradient contributes to A-P axis patterning and generates a posterior Tc-cad expression gradient that regulates segmentation through a pair-rule gene clock, forming segments sequentially as in vertebrates. In contrast, instead of Wnt activity, a hierarchy of regulatory genes regionalizes the blastoderm and defines segments simultaneously in the long germ insect Drosophila. In Tribolium, Tc-zen-1, Tc-otd-1 and Tc-cad play key roles in patterning serosa, head and trunk regions, respectively, of the blastoderm fate map, which are impacted by changes in Wnt activity levels. However, interactions between these genetic factors have not been described. My work revealed that cross talk between the Wnt and Dpp signaling pathways regulates the expression of Tc-zen-1 to determine the serosa. Furthermore, mutually repression between Tcotd-1 and Tc-cad defines the head and trunk regions while mutual repression between Tc-zen-1 and cad determines the posterior extent of the dorsal serosa. Analysis of target genes of the posterior Tc-cad gradient indicates that the Tc-cad gradient regulates the serial expression of gap genes, which are predominately regulators of Hox genes. Thus the posterior Tc-cad gradient determines segment formation through regulation of pair-rule genes in the insect segmentation clock, and defines segmental identity through regulation of gap genes. Given its effect on Tc-zen-1 and Tc-cad, the Wnt activity gradient is a key organizer of the Tribolium blastoderm fate map. Since homologs of these genes as well as the Wnt signaling pathway have also been identified in several other short germ band insects, and affect cell fates along the A-P body axis, this work provides a new paradigm for the short germ mode of development to guide studies in other arthropods.

A via canônica Wnt/beta-catenina regula várias funções em vertebrados, incluindo diferenciação de células T, bem como a proliferação, sobrevivência, morfogênese e migração de vários tipos celulares. As células T CD4+ é fundamental para a competência imunológica. Foi observado pelo nosso grupo que células T CD4+ humanas apresentam ativação da via Wnt/beta-catenina após tratamento com sais de lítio ou outros agonistas da via. A ativação desta via induziu a proliferação de células T CD4+ naive e de memória central. Em conjunto, estes dados sugerem um importante papel da via Wnt/beta-catenina na homeostase de células T CD4+ humanas. Seria importante avaliar o papel da via Wnt/beta-catenina nas células do sistema imune no modelo murino, já que pouco se sabe sobre seu efeito na homeostase de células T CD4+ murinas. A ativação da via Wnt/beta-catenina pode ser induzida com inibidores da proteína Glicogênio sintase quinase 3beta (GSK3beta), por exemplo, os sais de lítio (LiCl e Li2CO3) e inibidores específicos (SB, CHIR) em vários tipos celulares. Neste trabalho, avaliamos o efeito de inibidores de GSK3? na ativação da via Wnt/beta-catenina canônica em esplenócitos e células T CD4+, através da realização de experimentos in vivo e in vitro, avaliando a expressão de seus genes alvo HIG2, Bcl-xL, Ciclina D1 e c-myc. Verificou-se que o tratamento in vivo agudo (2-12 h após a administração) ou crônico (administração diária por 30 dias) de camundongos não é capaz de ativar a via Wnt/beta-catenina in vivo em células esplênicas e células T CD4+, embora o mesmo tratamento induza a expressão dos genes alvo da via no tecido cerebral (córtex e hipocampo). Além disso, também não foi possível verificar ativação da via em esplenócitos e células T CD4+ após tratamento in vitro das mesmas com LiCl ou os inibidores específicos de GSK3beta testados(CHIR99021, SB-216763), embora essa ativação tenha sido observada na linhagem celular HEK293. Nossos resultados sugerem que a via Wnt/beta-catenina (canônica) não é induzível em células T CD4+ murinas maduras, com os agonistas testados. Isso pode ter implicações fisiológicas, por exemplo sobre a homeostase de células T CD4+, já que a proliferação homeostática de células T, influenciada em humanos pela via Wnt/beta-catenina, é menos importante em camundongos / The Wnt/beta-catenin pathway regulates many functions in vertebrates, including T cell differentiation, as well as proliferation, morphogenesis and migration in different cell types. CD4+ T cells play is fundamental for immunological competence. Our group has observed that human CD4+ T cells present activation of the Wnt/beta-catenin pathway after treatment with lithium salts or other pathway agonists. The activation of this pathway induced proliferation in naive and central memory CD4+ T cells. Together, these results suggest an important role for the Wnt/beta-catenin pathway in the homeostasis of human CD4+ T cells. It would be very important to evaluate the role of the Wnt/beta-catenin pathway in T cells in the mouse model, since little is known about its effect in mice CD4+ T cell homeostasis. The activation of the Wnt/beta-catenin pathway may be induced with Glycogen Synthase Kinase 3B (GSK3beta) inhibitors, i.e., lithium salts as mentioned above, and specific GSK3beta inhibitors (SB, CHIR) in different cell types. In this work, we evaluated the effect of GSK3beta inhibitors in the activation of the canonical Wnt/beta-catenin in splenocytes and CD4+ T cells, by conducting experiments in vivo and in vitro, evaluating the expression of its target genes HIG2, Bcl-xL, Cyclin D1 and c-myc. We verified that acute (2-12 hours after administration) or chronic (daily administration for 30 days) treatment of mice with lithium salts is not able to activate the Wnt/beta-catenin pathway in splenocytes and CD4+ T cells, although we could observe activation in brain tissues (cortex and hypothalamus). Besides, no activation of the Wnt/beta-catenin pathway was observed in these cell types after in vitro treatment with LiCl or the specific inhibitors of GSK3beta (CHIR99021, SB-216763), while the pathway was activated by the same treatments in HEK293 cells. Our results suggest that the Wnt/beta-catenin pathway is not inducible in murine mature CD4+ T cells with the tested agonists. This may have physiological implications, for instance on the homeostasis of CD4+ T cells, where homeostatic proliferation - influenced the Wnt/beta-catenin pathway in human T cells - is less important in the maintenance of the murine peripheral T cell pool

Abstract Secreted signalling molecules are important for the regulation of developmental cell responses. In the developing kidney, signalling occurs between epithelial ureteric bud and metanephric mesenchyme and in between their derivatives. Wnt6 gene activity was localized to the ureteric bud and newly formed branches of the ureteric tree during early stages of kidney development. In a classic organ culture system, Wnt6 signalling induced the activation of marker genes for early nephrogenesis. The metanephric mesenchymes isolated from the Wnt4 deficient embryos were also induced, and the Wnt4 gene became activated in the presence of a Wnt6 signalling source. We propose that Wnt-6 is involved as a metanephric inducer and controls nephrogenesis. Wnt4 is essential for nephrogenesis in mouse and we indicate an additional role for Wnt4 in the control of periureteric stromal differentiation. A failure in vascular development was also found. Bmp4 expression in the medullar stroma of the Wnt4-deficient kidneys was absent concomitantly with a loss of expression of the smooth muscle marker, α-SMA. In vitro Wnt4 signalling induced Bmp4 expression and local α-SMA production. Hence, we conclude that lack of Wnt4 signalling leads to a loss of the periureteric smooth muscle cells, and Wnt4 may locally regulate this cell population in normal kidneys via regulation of Bmp4 signalling. The pluripotent neural crest cells are proposed to play regulatory roles in the early metanephros. Here, the use of transgenic animals allowed visualisation of the lumbo-sacral neural crest (NC) cells in close proximity to the early metanephros. The NC cells, however, disappeared in most part of the kidney by E12.5. The Splotch embryos lack the NCs from the early urogenital region. A developmental defect in the kidneys of Splotch embryos was not observed in vivo or in vitro. The results suggest that the neural crest is not essential for early embryonic kidney development. In sum, the work presented indicates an important role for Wnt6 in the induction of kidney tubules in vitro, for Wnt4 in the specification of kidney smooth muscle cells and for endothelial development in kidney. The neural crest cells apparently have no active morphogenetic role in early kidney development.

Die Regulation des Wnt/β-catenin Signalwegs ist nicht nur entscheidend für alle Stadien der kardialen Entwicklung, sondern auch für die Homöostase im adulten Herzen. Tatsächlich ist die Aktivierung des Wnt/β-catenin Signalwegs mit dem pathologischen Herz-Remodeling assoziiert. Ein besseres Verständnis der Regulation des Wnt/β-catenin Signalwegs bei Herzinsuffizienz könnte die Identifikation potentieller Faktoren zum Blockieren des pathologischen Herz-Remodelings und/oder das Aktivieren der endogenen Regeneration ermöglichen. Diese Arbeit konzentriert sich auf die Identifikation von gewebespezifischen Regulatoren des Wnt/β-catenin Signalwegs im Herzen. Frühere Arbeiten unserer Gruppe identifizierten basic leucine zipper and W2 domains containing protein (BZW) 2 als einen kardialen Interaktionspartner von β-catenin und KLF15. Die Rolle von BZW2 im Wnt/β-catenin Signalweg bei der Kardiogenese und der Homöostase des adulten Herzens war das Thema dieser Studie. Eine Analyse des BZW2-Proteins zeigte, dass die mutmaßliche ZIP und bZIP umfassende Domäne wichtig für die nukleare Lokalisation ist. Die Expression von BZW2 hat sich als bedeutsam im adulten Herzen, aber auch während der embryonalen Kardiogenese erwiesen. Eine niedrige BZW2-Expression ist für die effektive Bildung des kardialen Mesoderms notwendig, denn es kam unter BZW2-Überexpression zu einem Abbruch der Kardiomyozytenbildung in einem Modell der in vitro Kardiogenese. Dennoch war die BZW2-Expression nicht entscheidend für die Embryogenese, was auf eine kompensatorische Funktion verwandter Proteine hindeuten könnte. Interessanterweise war BZW2 für den Erhalt der normalen Herzfunktion und während der Reaktion auf Stress notwendig. Obwohl BZW2 die Wnt-Transkriptionsaktivität in vitro nicht signifikant inhibiert hat, resultierte das Fehlen von BZW2 in de novo Synthese von β-catenin spezifisch im adulten Herzgewebe in vivo. Außerdem wurde BZW2 selbst durch den Wnt/β-catenin Signalweg reguliert, was auf seine Rolle als regulatorischer Rückkopplungsfaktor in vivo hindeutet. Zusammenfassend identifizierte diese Arbeit einen neuen herzgewebespezifischen Faktor des Wnt/β-catenin Signalwegs auf einer neuen Regulationsebene und demonstrierte seine Relevanz für die normale Herzhomöostase. Angesichts der ubiquitären Expression und der vielfältigen Funktionen von β-catenin könnte die gewebespezifische Modulation neue Therapieoptionen darstellen.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.