Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 188-204).

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2008. / Includes bibliographical references (p. 38-40).

Thesis (Ph. D.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 109-113).

Techn. Univ., Diss., 2005--Darmstadt

GOMES, K. N. Avaliação do efeito da Doxiciclina associada ou não à decametasone no modelo de reparo ósseo alveolar em ratos. 2015. 94 f. Tese (Doutorado em Farmacologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-09-28T15:45:53Z No. of bitstreams: 1 2015_tese_kngomes.pdf: 1745792 bytes, checksum: 93bc1957b422226ecef0a412b0c9758b (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-09-28T15:46:04Z (GMT) No. of bitstreams: 1 2015_tese_kngomes.pdf: 1745792 bytes, checksum: 93bc1957b422226ecef0a412b0c9758b (MD5) / Made available in DSpace on 2016-09-28T15:46:04Z (GMT). No. of bitstreams: 1 2015_tese_kngomes.pdf: 1745792 bytes, checksum: 93bc1957b422226ecef0a412b0c9758b (MD5) Previous issue date: 2015-11-27 / The alveolar bone repair model, through dental excision in rats, allows the longitudinal study of local and systemic parameters. This allows learning the pathophysiology in inflammatory bone remodeling process. The objective of the research was to investigate the effect of doxycycline associated or not with dexamethasone on the events that follow the bone reabsorption process in alveolar bone repair in rats. The animals (190-200 g) were treated daily (per os) with saline solution Control (SSC), doxycycline (DOX 10 and 25 mg/kg), Dexamethasone (DEXA 1 mg/kg) (IP), and sacrificed according to experimental group (7, 14 and 21 days) to remove their jaws, which were used in the qualitative analysis of microstructural parameters, histomorphometry, and immunohistochemistry for Wnt10b and Dkk-1. It was observed that DOX (10 and 25 mg / kg ) improve some parameters such as the number of osteoblasts increase, reduction in the number of osteoblasts and % increase in bone tissue compared to the CTL group. The results also showed an increase in the expression of Wnt 10b, and Dkk-1 reduction, during the process of bone repair in animals that received DOX (10 and 25 mg/kg), compared to groups CTL. These findings suggest that DOX offers osteoinductive and osteoprotector effects on bone tissue during the alveolar bone remodeling process. It is possible that, in addition to anti-inflammatory effects recognized in DOX, this Tetracycline can influence the expression of Wnt 10b, favoring bone neoformation, and modulate the expression of Dkk-1, inhibiting tissue resorption in the alveolus of the animals. / O modelo de reparo ósseo alveolar através de exérese dentária em ratos permite que parâmetros locais e sistêmicos possam ser estudados longitudinalmente possibilitando o conhecimento da fisiopatogênese no processo de reabsorção óssea inflamatória. O objetivo da pesquisa foi investigar o efeito de doxiciclina associada ou não à dexametasona sobre os eventos que sucedem o processo de remodelamento ósseo em modelo de reparo ósseo alveolar em ratos. Os animais (190-200 g) foram tratados diariamente (v.o.) com solução salina Controle (CTL), doxiciclina (DOX 10mg/kg e 25 mg/kg), Dexametasona (DEXA 1 mg/kg)(i.p.) e sacrificados de acordo com grupo experimental (7, 14 e 21 dias) para remoção de suas maxilas utilizadas na análise qualitativa de parâmetros microestruturais, histomorfometria e imunohistoquímica para Wnt 10b e Dkk-1. Foi observado que DOX (10 e 25 mg/kg) melhoram alguns parâmetros como: aumento do número de osteoblastos, redução no número de osteoblastos e % de tecido ósseo comparados ao grupo CTL. Os resultados também mostraram um aumento na expressão de WNT 10b e redução de Dkk-1 durante o processo de reparo ósseo nos animais que receberam DOX (10 e 25 mg/kg) quando comparados aos grupos CTL. Esses achados sugerem que DOX oferece um efeitos osteoindutor e osteoprotetor do tecido ósseo durante o processo de remodelamento ósseo alveolar. É possível que além dos efeitos antiiflamatórios reconhecidos em DOX, essa Tetraciclina pode influenciar a expressão de WNT 10b, favorecendo a neoformação óssea ou modular a expressão de Dkk-1, inibindo a reabsorção tecidual nos alvéolo dos animais.

Obesity-related comorbidities, including type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) have become a major public health concerns. These complications primarily arise in response to cellular changes in white adipose tissue (WAT). In particular, when a majority of fat cells become hypertrophic it promotes a metabolically unhealthy phenotype, which is characterized by chronic low-grade inflammation, insulin resistance, and ectopic lipid accumulation. Research has implicated synthetic (i.e., Bisdehydrodoisynolic acid, BDDA) and natural (i.e., genistein and daidzein) xenoestrogens in the protection against obesity-related pathologies. Bisdehydrodoisynolic acid (BDDA) reduced weight gain and adiposity, as well as improved lipid homeostasis in obese rodents. Alternatively, phytoestrogens, such as genistein and daidzein were reported to induce adipocyte differentiation through potential interactions with PPARγs, canonical WNT proteins, and estrogen receptors (ER) signaling. The current investigation was conducted to test the effects of synthetic and natural estrogenic compounds (BDDA, daidzein, and genistein) for their effects on the induction of adipogenic signaling, which may potentially improve WAT morphology by reducing adipocyte hypertrophy.

WNT lipoglycoproteins (WNTs) modulate a plethora of cellular functions through the activation of the family of frizzled receptors (FZDs). Deregulation in components of the WNT signalling pathways is often observed in human cancers and associated with uncontrolled proliferation and metastasis. Frizzled receptor 6 (Fzd6), one of the ten human FZDs, is frequently overexpressed in cancer, but its role in tumorigenesis is still unclear. In this study we investigated the role Fzd6 in breast cancer. We found that expression of Fzd6 predicts distant relapse in patients with localised breast cancers, particularly in those bearing the triple negative subtype. Using a loss of function approach, we demonstrated that Fzd6 is important to regulate motility and invasion of breast cancer cells in vitro and in vivo. Indeed, Fzd6 regulates the tropism of breast cancer cells the bone, liver and heart of mice. Mechanistically, we found that Fzd6 signalling activates the small GTPase Rho and is important in the organisation of the fibronectin matrix. Both Rho and fibronectin have been previously implicated in the development of metastasis in different systems. All together, these results demonstrate that Fzd6 is an important driver of metastatic spread and a predictive marker of metastatic relapse in breast cancer patients. Fzd6 could therefore be used as a biomarker and target in metastatic breast cancer.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

The development of complex metazoans depends on the integration of a handful of signalling pathways that eventually modulate precise patterns of gene expression. The fact that just a few pathways are involved in the generation of such complexity in different organisms, suggests that these are highly regulated and conserved processes. The accurate spatio-temporal coordination of the signalling pathways controls the assignation of different cell fates and their patterning into tissues and organs. The source of diversity relies on the different possible interactions between signalling pathways, such as, the combination of signals and the order in which they are received by the cell or crosstalk. Due to their importance in development, abnormal signalling through these pathways has been strongly associated with developmental disorders, cancers and other diseases. The Notch and Wnt signalling pathways are key components of the intricate network that controls gene expression during development, and genetic analysis in Drosophila has highlighted that interactions between these two signalling pathways are important during this process.This thesis investigates the cross-regulatory interactions between Notch and Wnt signalling pathways in mammals. Using transcriptional reporter assays and biochemical analysis, I have found two molecular mechanisms underlying the inhibitory crosstalk between Notch and β-catenin, the effector of Wnt signalling pathway, in mammalian cells. At the membrane Notch inhibits β-catenin transcriptional activity through Deltex mediated endocytosis of Notch and a component required for β-catenin activation. This is similar to results observed in Drosophila. In the nucleus, I have identified a novel mechanism by which NICD-dependent transcription of Hes/Hey family of transcription factors prevents the activation of Wnt signalling pathway. This mechanism involves the formation of a physical complex between Hey1 and β-catenin/TCF, which allows Hey to block Wnt transcriptional activation. Additionally, I have found that these two mechanisms are conserved across vertebrates.Together the findings of this thesis improve our understanding of the molecular mechanism underlying the Notch/Wnt crosstalk. In turn, this will give an insight into unravelling how a handful of signalling pathways can generate sufficient diversity in signalling output to specify the hundreds of different cell fates generated to make a mammal. Elucidating these signalling networks will also contribute to our understanding of diseases, both their aetiology, by knowing how changes in one signal can influence another, and their treatment as mimicking points of crosstalk is likely to generate very specific therapeutic agents.

The primary aim of my PhD thesis was to identify and characterise novel modulators of intracellular signalling during early vertebrate development. The first phase of my thesis was to design and execute a large-scale gain of function screen in order to identify novel modulators of various important signal transduction pathways during early Xenopus development. From this screen I identified twenty novel of growth factor signalling. In the second phase of my PhD study, I concentrated on the characterization and mode of action of one of the genes I identified in the screen; namely fezf2. I showed that Fezf2 regulates neurogenesis in the diencephalon by locally promoting Wnt signalling through repression of lhx2 and lhx9. Notably, this investigation on the function of fezf2 not only revealed the previously undiscovered role of fezf2-mediated Wnt regulatory mechanism during diencephalon development, but also confirmed our in vivo screening approach in identifying potential regulators of signalling pathways. To the end, my PhD project has provided me with a fruitful journey of discovery, which started with the design and execution of a large-scale screen, ending with the detailed characterization of a factor involved in the modulation of signalling and forebrain development. This study is has broadened our understanding of how intracellular and extracellular signals are integrated during embryonic development process, which forms an interactive network ultimately resulting in appropriate cell differentiation, organ formation, and regional patterning.