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Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 111-140).

Student Number : 9105022E - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / SNAMA is the Drosophila melanogaster homologue of a group of proteins that are known to bind p53 and the retinoblastoma protein (Rb). This multi domain protein consists of a conserved N-terminal domain called Domain With No Name (DWNN), a zinc finger, a cysteine rich RING finger-like domain, a probable p53 binding region, and a glutamic acid-rich and lysine-rich region. These associated domains indicate that SNAMA plays an important regulatory role in the cell and may function in RNA processing and in apoptosis. The DWNN domain was first identified in Cytotoxic T-cell resistant Chinese hamster ovary (CHO) cells using promoter trap mutagenesis to screen for genes involved in apoptosis. Subsequently, this domain was identified in other eukaryotic organisms including animals and plants. The SNAMA transcriptional unit consists of 9 exons and 8 introns that code for a 1231 amino acid protein with the 76 residue N-terminal DWNN domain. The DWNN domain has a 23.5% sequence identity to the ubiquitin protein and a predicted folded structure similar to ubiquitin. Western blots identified multiple bands indicative of ubiquitin tagged proteins. Taken together this suggests a role in the ubiquitin pathway either as an ubiquitin domain protein or the DWNN domain of SNAMA tagging other proteins. The cysteine rich RING finger-like domain has a histidine to serine substitution at the fourth position of the putative RING finger and represents a distinct class of RING finger-like proteins that could have ubiquitin ligase activity. Northern blot analysis identified a single 4.6 kbp transcript expressed abundantly throughout development early in embryogenesis but reduced in older embryos and in adult male and females. SNAMA probably interacts with Dmp53 as a suppressor of apoptosis or a negative regulator of an activator of apoptosis. It is a vital gene required for development, as the mutant P-element insertion line in which the Pelement is inserted in the first intron of SNAMA is lethal when homozygous. Acridine orange staining of these mutant flies showed a direct correlation between the presence of SNAMA and apoptosis. An increase in the levels of apoptosis occurred in embryos with relatively low levels of SNAMA expression. The mode of this action is either direct, or via other proteins that are involved in the apoptotic pathway.

Mais estudos sobre ações neuromoduladoras das angiotensinas no Sistema Renina Angiotensina (SRA) poderiam melhorar a compreensão de muitas funções e distúrbios do sistema nervoso central. A via de sinalização de PTEN/PI3K/AKT/mTOR que pode ser ativada pela Angiotensina-(1-7) desempenha um papel essencial na regulação do crescimento, metabolismo e sobrevivência das células. Este estudo teve como objetivo identificar e descrever a(s) via(s) metabólica(s) angiotensinérgica(s) principal(is) no hipocampo de camundongos (Mus musculus); Estudar a regulação da cascata de sinalização PTEN/PI3K/Akt/mTOR a partir da ativação com a Ang-(1-7) e realizar ensaios de inibição com o antagonista do receptor Mas. Analisamos também a expressão das enzimas chave da cascata de mTOR, com foco na enzima PTEN. Inicialmente foi realizada a identificação da via metabólica angiotensinérgica no hipocampo de camundongos através do estudo da atividade proteolítica angiotensinérgica com o extrato hipocampal de camundongos Swiss para identificar as enzimas e peptídeos formados a partir de AngI e/ou inibidores em HPLC. Para o estudo da regulação da cascata de sinalização PTEN/PI3K/Akt/mTOR a partir da ativação/inibição do SRA os experimentos foram realizados em células imortalizadas de neuroblastoma denominadas SH-SY5Y e western blotting para determinação dos níveis de PTEN e S6k (total e fosforilada). Nossos resultados mostraram que a caracterização do perfil do sistema angiotensinérgico hipocampal gerou resultados preliminares, em que a partir de AngI, o principal peptídeo formado foi a Ang-(1-7) e, em menor quantidade, a Ang II. Concluímos então que a AngII pode estar sendo formada por carboxipeptidades de forma geral e a Ang-(1-7) pode estar sendo formada por endopeptidases. Além disso, a Ang-(1-7) provoca a diminuição da expressão da PTEN total, bem como, a diminuição da fosforilação da PTEN, e nesse caso, a diminuição da fosforilação significa geração de mais proteína desfosforilada e aumento da atividade fosfatase resultando em efeitos neuroprotetores. Esses resultados nos permitiram observar que em células SH-S5SY, o mecanismo pelo qual a Ang-(1-7) ativa mecanismos neuroprotetores pode estar relacionado com a ativação da enzima PTEN. / Further studies on neuromodulatory actions of angiotensins in the Renin Angiotensin System (RAS) could improve understanding of many central nervous system functions and disorders. The PTEN/PI3K/AKT/mTOR signaling pathway that can be activated by Angiotensin-(1-7) plays an essential role in regulating cell growth, metabolism and survival. The aim of this study was to identify and describe the main angiotensinergic metabolic pathway(s) in the hippocampus of mice (Mus musculus); Study the regulation of the PTEN/PI3K/Akt/mTOR signaling cascade from activation with Ang-(1-7) and perform inhibition assays with the Mas receptor antagonist. We also analyzed the expression of the key enzymes of the mTOR cascade, focusing on the enzyme PTEN. Identification was initially performed of the angiotensinergic metabolic pathway in the mouse hippocampus was performed by studying the angiotensinergic proteolytic activity with the hippocampal extract of Swiss mice to identify the enzymes and peptides formed from AngI and/or inhibitors in HPLC. For the study of PTEN/PI3K/Akt/mTOR signaling cascade regulation from the activation/inhibition of RAS the experiments were performed on immortalized neuroblastoma cells called SH-SY5Y and western blotting for determination of PTEN and S6k levels (total and phosphorylated). Our results showed that the characterization of the profile of the hippocampal angiotensinergic system generated preliminary results, in which from AngI, the main peptide formed was Ang-(1-7) and, to a lesser extent, Ang II. We conclude that AngII may be formed by carboxypeptids in general and Ang-(1-7) may be formed by endopeptidases. In addition, Ang-(1-7) causes a decrease in total PTEN expression, as well as a decrease in PTEN phosphorylation, in which case the decrease in phosphorylation means generation of more dephosphorylated protein and increased phosphatase activity resulting In neuroprotective effects. These results allowed us to observe that in SH-S5SY cells, the mechanism by which Ang-(1-7) activates neuroprotective mechanisms may be related to the activation of the PTEN enzyme. / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.

Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.

Open Access Article

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

O câncer de esôfago é a sexta neoplasia maligna mais comum no mundo. No Rio Grande do Sul, Brasil, o carcinoma epidermóide de esôfago apresenta coeficientes de mortalidade elevados e com tendência ascendente com, pelo menos, o dobro dos coeficientes padronizados de mortalidade encontrados em outros estados brasileiros ou em países do cone sul da América latina. O diagnóstico tardio parece ser o principal responsável pelo mau prognóstico. Nos últimos anos, diversos estudos têm demonstrado a possibilidade de identificação das lesões precursoras do câncer esofágico, mas sem repercussão no prognóstico, até o momento. Considera-se, atualmente, que a carcinogênese esofágica está relacionada a uma interação entre fatores ambientais e anormalidades genéticas Recentemente, estudos em biologia molecular têm demonstrado a influência dos fatores reguladores do ciclo celular no prognóstico de diversas moléstias, inclusive o câncer. O Rb é um gene supressor tumoral envolvido no mecanismo de controle do ciclo celular, cuja expressão tem sido demonstrada no câncer do esôfago. O objetivo deste estudo foi determinar a prevalência da perda da expressão da proteína pRb na mucosa esofágica de indivíduos sob risco para o carcinoma epidermóide de esôfago, bem como relacionar esta expressão com o consumo de tabaco, álcool e chimarrão, achados histopatológicos e cromoscopia com lugol. Foram estudados 170 casos e 20 controles através de reação imunohistoquímica utilizando anticorpo monoclonal anti-pRb em amostras teciduais fixadas em formalina e armazenadas em parafina. Um total de 33 casos demonstrou perda da expressão imunohistoquímica da proteína pRb, determinando uma prevalência de 19,4% na amostra estudada. Não houve associação estatisticamente significativa entre a perda da expressão da proteína pRb e as variáveis idade, raça, exposição ao fumo, álcool e chimarrão, bem como a cromoendoscopia com lugol. Foi demonstrada uma associação significativa entre a perda da expressão da pRb com a história de câncer na família. Da mesma forma, foi demonstrada uma associação linear significativa entre a perda da pRb e o grau histopatológico das lesões. Estes resultados demonstraram uma influência da proteína pRb na evolução da carcinogênese esofágica e permitem sugerir que os indivíduos expostos aos fatores de risco estudados sejam candidatos a uma maior vigilância.