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<p>mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.</p> / Dissertation

Glioblastomas continue to carry poor prognoses for patients despite advances in surgical, chemotherapeutic, and radiation regimens. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and elevated expression levels of hypoxia-inducible factor-1 á (HIF-1á). HIF-1á expression allows metabolic adaptation to low oxygen availability, partly through upregulation of vascular endothelial growth factor (VEGF) and increased tumor angiogenesis as well as induction of anaerobic glycolysis. In this study, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) by hypoxia in glioblastoma cells. AEG-1 has the capacity to promote anchorage-independent growth and cooperates with Ha-ras in malignant transformation. In addition, AEG-1 was recently demonstrated to serve as an oncogene and can induce angiogenesis and autophagy in glioblastoma. Results from in vitro studies show that hypoxic induction of AEG-1 is dependent on HIF-1á stabilization during hypoxia and that phosphatidylinositol 3-kinase (PI3K) inhibition abrogates AEG-1 induction during hypoxia through loss of HIF-1á stability. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) production prevents this induction. Additionally, AEG-1 knockdown results in increased ROS production and increased glucose deprivation-induced cytotoxicity, whereas AEG-1 overexpression prevents ROS production and decreases glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of cell stress. From studies examining the expression of enzymes involved in glucose metabolism, we demonstrate that AEG-1 alters the tumor metabolic profile in a partially 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner. Moreover, glycolytic inhibition modulates the metabolic effects induced by AEG-1, and AEG-1 knockdown reduces the growth and alters the metabolic phenotype of glioblastoma subcutaneous xenografts. These observations link AEG-1 overexpression observed in glioblastoma with hypoxia and glucose metabolic signaling, and targeting these physiological pathways may lead to therapeutic advances in the treatment of glioblastoma in the future. Recent studies have reported the detection of the human neurotropic virus, JC Virus (JCV), in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as â-catenin and IRS-1, play a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells that endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by AMPK, a critical sensor of the AMP/ATP ratio in cells. We have found that AMPK activation inhibits T-antigen expression, whereas AMPK inhibition prevents glucose deprivation-mediated T-antigen suppression. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen-expressing medulloblastoma cells, as compared to those which do not express T-antigen, exhibit less G1 arrest and an increased percentage of cells in the G2 phase of the cell cycle during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on ROS production during glucose deprivation. Additionally, studies indicate that T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. We have also found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamde (6-AN) and oxythiamine (OT). Enzyme expression studies also indicate that T-antigen upregulates the expression of the pentose phosphate enzyme, transaldolase-1 (TALDO1), demonstrating a potential link between T-antigen and glucose metabolic regulation. These studies highlight the potential involvement of JCV T-antigen in the proliferation and metabolic phenotype of medulloblastoma and may enhance our understanding of the role of viral proteins in tumor glycolytic metabolism, thus implicating these proteins as potential targets for the treatment of virus-associated tumors. / Biomedical Neuroscience

Submitted by Luis Eduardo Wanderley (luis.wanderley@fgv.br) on 2018-01-02T15:56:18Z No. of bitstreams: 1 Dissertação Wanderley, Luis Eduardo T L - MEX 2016.pdf: 605486 bytes, checksum: 743045e4541c798bd60dc4f255407bcc (MD5) / Approved for entry into archive by Janete de Oliveira Feitosa (janete.feitosa@fgv.br) on 2018-01-02T19:00:06Z (GMT) No. of bitstreams: 1 Dissertação Wanderley, Luis Eduardo T L - MEX 2016.pdf: 605486 bytes, checksum: 743045e4541c798bd60dc4f255407bcc (MD5) / Made available in DSpace on 2018-01-09T16:33:06Z (GMT). No. of bitstreams: 1 Dissertação Wanderley, Luis Eduardo T L - MEX 2016.pdf: 605486 bytes, checksum: 743045e4541c798bd60dc4f255407bcc (MD5) Previous issue date: 2017-12-05 / Audit Expectation Gaps (AEG) are a major challenge for auditors to debate. Besideshaving the potential to undermine auditors´ credibility, AEGs increase the risks of financiallosses to firms and auditors as they may influence the outcome of lawsuits. This study examinesthe existence of AEG in Brazil and evaluates the relevance of actions aimed at improving thequality of audits.The research included the analysis of the previous studies onthe subject and their findings and the empirical investigation of the AEG in Brazil, through theapplication of a questionnaire survey. 97 questionnaire replies were obtained, of which 31 wereauditors and 66 were non-auditors.The evidence obtained suggests that AEGs are perceived in Brazil and that the gapis mainly a result of unreasonable expectations from society and of information needs in relationto the audit work and the audited companies. In addition, the study reveals that the respondentsconsidered relevant the adoption of proposed measures aiming the increase of quality andregulation of the audit activity.An understanding of the AEGs in Brazil can facilitate the discussionof the topic in academia, in the professional environment - independent auditors, accountants,regulatory boards, judiciary, capital market entities and press vehicles - and in the society. / Objetivo: As diferenças de expectativas em relação ao trabalho dos auditores independentes (Audit Expectation Gaps - AEG) constituem um importante desafio a ser debatido pelos auditores. Além de terem potencial de minar a credibilidade dos auditores perante os usuários do relatório de auditoria, os AEG trazem às firmas e aos auditores riscos de impactos financeiros, à medida que podem influenciar o resultado de processos judiciais que questionem a responsabilidade dos auditores em casos de fraudes e outras distorções relevantes não detectadas. O presente estudo objetiva apurar a existência de AEG no ambiente brasileiro e avaliar a relevância de ações visando a melhoria da qualidade das auditorias. Metodologia: A pesquisa envolveu a revisão da bibliografia sobre o tema, com o levantamento dos principais achados reportados, e a investigação empírica dos AEG, no Brasil, através da aplicação de questionários eletrônicos. Foram obtidas 97 respostas ao questionário, sendo 31 de auditores e 66 de não auditores. Resultados: As evidências obtidas sugerem que os AEG também são percebidos no Brasil e decorrem, principalmente, de expectativas não razoáveis dos usuários das demonstrações contábeis e de uma maior demanda por informações em relação ao trabalho de auditoria. Adicionalmente, o estudo aponta que os respondentes consideraram relevantes as medidas sugeridas na pesquisa visando aumentar a qualidade e a regulação da atividade de auditoria. Aplicabilidade: A compreensão das diferenças de expectativas sobre o trabalho dos auditores no Brasil pode facilitar o debate do tema no meio acadêmico, no meio profissional – auditores independentes, contadores, órgãos reguladores, poder judiciário, entes do mercado de capitais e veículos de imprensa – e na sociedade em geral.

The discounted cash flow model and relative valuation models are ever-increasingly prevalent in today’s investment-heavy environment. In other words, theoretically inferior models are used in practice. It is this paradox that has lead us to compare the discounted cash flow model (DCFM), discounted dividend model (DDM), residual income-based model (RIVM) and the abnormal earnings growth model (AEGM) and their relative accuracy to observed stockprices. Adding to previous research, we investigate their performance in relation to the OMX30 index. What is more, we test how the performance of each model is affected by an extension of the forecast horizon. The study finds that AEGM outperforms the other models, both before and after extending the horizon. Our analysis was conducted by looking at accuracy, spread and the inherent speculative nature of each model. Taking all this into account, RIVM outperforms the other models. In this sense, one can question the rationale behind investor’s decision to primarily use the discounted cash flow model in equity valuation.

La neurotoxine β-N-méthyl-amino-l-alanine (BMAA) et ses isomères, notamment la N-(2- aminoéthyl glycine) (AEG), la β-amino-N-méthyl alanine (BAMA) et l'acide 2,4- diaminobutyrique (DAB), ont été détectés précédemment dans des échantillons de cyanobactéries. Cependant, il existe des rapports contradictoires concernant leur présence dans les eaux de surface. Dans cette étude, nous avons évalué l'impact de l'acide trichloracétique (TCA 0,1M) sur la détection des isomères de BMAA, par rapport aux protocoles préexistants. Une méthode instrumentale sensible a été utilisée pour l'étude, avec des limites de détection de l'ordre de 5-10 ng L-1. Des meilleures limites de détection plus élevés et des niveaux significativement plus importants (test des rangs signés de Wilcoxon appariés, p < 0,001) d'isomères de BMAA ont été observés dans les échantillons traités par le TCA, avec des augmentations relatives allant jusqu'à +725 % pour l'AEG et +1450 % pour le DAB, et des augmentations de concentration absolue allant jusqu'à +15 000 ng L-1 pour l'AEG et +650 ng L-1 pour le DAB. Nous avons également documenté les tendances de la présence des isomères de BMAA dans plusieurs lacs de différents pays tels que le Brésil, le Canada, la France, le Mexique et le Royaume-Uni. Les données obtenues au cours de cette étude (n = 390 provenant de 45 sites d'échantillonnage) indiquent des détections fréquentes des isomères AEG et DAB, avec des taux de détection de 30 % et 43 % et des niveaux maximums de 19 000 ng L-1 et 1 100 ng L-1, respectivement. En revanche, le BAMA a été trouvé dans moins de 8 % des échantillons d'eau, et la BMAA n'a été trouvée dans aucun échantillon. Ces résultats appuient les analyses des cyanobactéries libres, dans lesquelles la BMAA a souvent été détectée avec des concentrations inférieures de 2 à 4 ordres de grandeur à celles de l'AEG et du DAB. Les mesures saisonnières effectuées dans deux lacs impactés par des efflorescences ont indiqué des corrélations limitées entre les isomères de la BMAA et les microcystines totales ou la chlorophylle-a, ce qui mériterait une étude plus approfondie. / The neurotoxic alkaloid β-N-methyl-amino-l-alanine (BMAA) and related isomers, including N-(2-aminoethyl glycine) (AEG), β-amino-N-methyl alanine (BAMA) and 2,4-diaminobutyric acid (DAB), have been reported previously in cyanobacterial samples. However, there are conflicting reports regarding their occurrence in surface waters. In this study, we evaluated the impact of amending lake water samples with trichloroacetic acid (0.1M TCA) on the detection of BMAA isomers, compared with pre-existing protocols. A sensitive instrumental method was enlisted for the survey, with limits of detection in the range of 5-10 ng L-1. Higher detection limits ans significantly greater levels (paired Wilcoxon’s signed-rank tests, p < 0.001) of BMAA isomers were observed TCA-amended samples (method B) compared to samples without TCA (method A). The overall range of B/A ratios was 0.67-8.25 for AEG (up to +725 %) and 0.69-15.5 for DAB (up to +1450 %), with absolute concentration increases TCA-amended samples up to +15,000 ng L-1 for AEG and +650 ng L-1 for DAB. We also documented the trends in the occurrence of BMAA isomers for a large breadth of field-collected lakes from Brazil, Canada, France, Mexico, and the United Kingdom. Data gathered during this overarching campaign (overall n = 390 within 45 lake sampling sites) indicate frequent detections of AEG and DAB isomers, with detection rates of 30 % and 43 % and maximum levels of 19,000 ng L-1 and 1,100 ng L- 1, respectively. In contrast, BAMA was found in less than 8 % of the water samples, and BMAA not found in any sample. These results support analyses of free-living cyanobacteria, wherein BMAA was often reported at concentrations 2-4 orders of magnitude lower than AEG and DAB. Seasonal measurements conducted at two bloom-impacted lakes indicated limited correlations of BMAA isomers with total microcystins or chlorophyll-a, which deserves further investigation.

First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.