The transcription factor p53 (also known as TP53) guards against tumour and virus replication and is inactivated in almost all cancers. p53-activated transcription of target genes is thought to be ...synonymous with the stabilization of p53 in response to oncogenes and DNA damage. During adenovirus replication, the degradation of p53 by E1B-55k is considered essential for p53 inactivation, and is the basis for p53-selective viral cancer therapies. Here we reveal a dominant epigenetic mechanism that silences p53-activated transcription, irrespective of p53 phosphorylation and stabilization. We show that another adenoviral protein, E4-ORF3, inactivates p53 independently of E1B-55k by forming a nuclear structure that induces de novo H3K9me3 heterochromatin formation at p53 target promoters, preventing p53-DNA binding. This suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. These findings are important for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumour-suppressor loci. Our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53-selective oncolytic viral therapies.
Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown.
To elucidate ...molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program.
The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls.
DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV
(representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay.
DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.
ONYX-015 is an adenovirus that lacks the E1B-55K gene product for p53 degradation. Thus, ONYX-015 was conceived as an oncolytic virus that would selectively replicate in p53-defective tumor cells. ...Here we show that loss of E1B-55K leads to the induction, but not the activation, of p53 in ONYX-015-infected primary cells. We use a novel adenovirus mutant, ONYX-053, to demonstrate that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells. In contrast, we show that tumor cells that support ONYX-015 replication provide the RNA export function of E1B-55K. These data reveal that tumor cells have altered mechanisms for RNA export and resolve the controversial role of p53 in governing ONYX-015 oncolytic selectivity.
ONYX-015 is an E1B-55K-deleted adenovirus that has promising clinical activity as a cancer therapy. However, many tumor cells fail to support ONYX-015 oncolytic replication. E1B-55K functions include ...p53 degradation, RNA export, and host protein shutoff. Here, we show that resistant tumor cell lines fail to provide the RNA export functions of E1B-55K necessary for ONYX-015 replication; viral 100K mRNA export is necessary for host protein shutoff. However, heat shock rescues late viral RNA export and renders refractory tumor cells permissive to ONYX-015. These data indicate that heat shock and late adenoviral RNAs may converge upon a common mechanism for their export. Moreover, these data suggest that the concomitant induction of a heat shock response could significantly improve ONYX-015 cancer therapy.
The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in ...non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community.
Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant ...functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central β core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.
Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra‐ and extracellular factors that normally control it. Here we show that adenovirus ...encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor‐limiting conditions. E4‐ORF1 mimics growth factor signaling by activating PI3‐kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4‐ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4‐ORF4 protein phosphatase 2A‐binding domain. We also show that mTOR activation is required for efficient S‐phase entry, independently of E2F activation, in adenovirus‐infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.
Dissertação (mestrado)—Universidade de Brasília, Faculdade de Educação, Programa de Pós-Graduação, 2012.
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Este trabalho investiga as relações estabelecidas entre os fundamentos das Ecologias Humana e Profunda nas atividades desenvolvidas na Escola da Natureza situada na cidade de Brasília. Faz um levantamento das visões que os professores e a direção da mesma têm quanto às implicações dessas ecologias em suas práticas pedagógicas. Realiza também um analise documental sobre elementos presentes nos dois últimos Projetos Político Pedagógicos da Escola. A metodologia tem como procedimento um estudo de caso abordado a partir dos referenciais da etnopesquisa. A observação participante teve como foco as atividades pedagógicas desenvolvidas pela Escola, nos atendimentos a estudantes e cursos de formação ;
de professores da rede pública e reuniões de coordenação e eventos pedagógicos realizados. Durante a investigação percebemos que a educação ambiental é realizada na perspectiva da ecopedagogia e a visão tácita de ecologia humana aproxima-se dos princípios da ecologia profunda. Nas práticas educativas observou-se, ainda, a aproximação dessas práticas com os ;
fundamentos de Educação Integral, que propõem um visão integrada dos aspectos físicos, vitais, mentais e psíquico-espirituais dos educandos e educadores. No programa Parque-Escola, nota-se a referência à proposta educacional de Anísio Teixeira para uma formação humana mais completa e às propostas discutidas no país sobre o sentido mais abrangente e ;
complexo da Educação Integral. A análise dos dados mostrou o entrelaçamemto de diversas ;
perspectivas da ecologia humana e a ligação com elementos teóricos e práticos da Educação Ambiental Crítica em uma abordagem Ecopedagógica. ______________________________________________________________________________ RESUMEN
Este trabajo investiga las relaciones establecidas entre los fundamentos de las Ecologías Humana y Profunda en las actividades desarrolladas en la Escuela de la Natureza situada en la ciudad de Brasilia. Hace un levantamento de las visiones que los profesores y la dirección de la misma en cuanto a las implicaciones de esas ecologías en sus prácticas pedagógicas. Realiza también un análisis documental sobre elementos presentes en los dos últimos Proyectos Político Pedagógicos de la Escuela. La metodología tiene como procedimiento un estudio de caso abordado a partir de los referenciales de la etnoinvestigación. La observación ;
participante tuvo como foco las actividades pedagógicas desarrolladas por la Escuela, en los atendimientos a estudiantes y cursos de formación de profesores de la red pública y reuniones de coordinación y eventos pedagógicos realizados. Durante la investigación percebimos que la educación ambiental es realizada en la perspectiva de la ecopedagogia y la visión tácita de ecología humana se aproxima de los principios de la ecología profunda.En las ;
prácticas educativas se observó también la aproximación de esas prácticas con los ;
fundamentos de Educación Integral que propome una visión integrada de los aspectos físicos, vitales, mentales y psíquico espirituales de los educandos y educadores. En el programa Parque-Escuela se nota la referencia a la propuesta educacional de Anísio Teixeira para una ;
formación humana más completa y las propuestas discutidas en el país sobre el sentido más amplio y complejo de la Educación Integral. El análisis de los datos mostró el entrelazamiento de diversas perspectivas de la ecologia humana y la conexión con elementos teóricos y prácticos de la Educaçión Ambiental Crítica en un abordaje Ecopedagógico.
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