In recent years, LIM domains-containing protein 1 (LIMD1) has been identified as a critical component in microRNA (miRNA)-induced silencing complex (miRISC) to regulate miRNA-mediated gene silencing. Human Argonaute (AGO) 2 with its family members (AGO1-4) are critical for the biogenesis of miRNA and thus miRNA-mediated gene silencing. In this study, we have investigated the direct interaction interfaces between LIMD1 and AGO2. A distinct interface within LIMD1, amino acid (a.a) 140-166, is identified to be responsible for the binding to AGO2 and other members of AGO family. Furthermore, the Linker-2 (L2) domain within AGO2 is identified to be responsible for LIMD1 binding and its dependency on the phosphorylation at serine 387 (S387) residue within the L2 domain of AGO2. The phospho-mimic mutant (S387E) enhances the binding of AGO2 to LIMD1, whereas the phospho-deficient mutant (S387A) attenuates AGO2-LIMD1 interaction. In addition, the association of LIMD1 with other AGOs is also dependent on the phosphorylation at the equivalent conserved serine residue within the L2 domain on other AGOs. In addition to the above aspects, LIMD1 is a tumour suppressor gene frequently down-regulated in more than 75% human lung tumours. Because of their loss of expressions or functions, it is of the inherent difficulty in targeting tumour suppressor genes to treat cancers. In this study, the concept of synthetic lethality was used to identify possible protein kinases, the ablation of which are synthetically lethal to LIMD1 negative cancer cell lines. As a result, drugs that target these kinases may represent novel targeted therapies for LIMD1 negative lung tumours. ACVR2B and STK39 are validated to be synthetically lethal with LIMD1 loss. Additionally, the complete loss of LIMD1 expression causes a dramatic increase of STK39 expression due to miRNA-mediated gene silencing pathway. The inverse relationship between LIMD1 and STK39 may represent a conserved and fundamental signalling response and may be a predictive marker for STK39-targeted therapy.

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2016-08-23T12:17:22Z No. of bitstreams: 1 Karina Acosta Camargo.pdf: 1412678 bytes, checksum: 2f59e0e0b546f1e40f86fc7ec293bbc2 (MD5) / Made available in DSpace on 2016-08-23T12:17:22Z (GMT). No. of bitstreams: 1 Karina Acosta Camargo.pdf: 1412678 bytes, checksum: 2f59e0e0b546f1e40f86fc7ec293bbc2 (MD5) Previous issue date: 2016-03-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Child sexual abuse – a cartography starts from the author’s body fissures, presenting her own lacerations due to sexual abuses experienced during childhood. This path goes through the issues of silencing, testimony, resentment, forgetfulness, and, lastly, lead – like a birth made through the womb of the earth – to the amor fati. It is a research that aims to overcome the victim-aggressor dichotomy, in order to discuss the complex web that invisibly establishes itself and enables the incitement and the continuity of the sexual abuses, mainly in the family scope. Furthermore, it highlights ways of life invention from the unbearable, excessive and cruel, and art as a possibility of resistance and creation of new ways of existence / Abuso sexual infantil – uma cartografia parte das fissuras do corpo da autora, apresentando seus próprios dilaceramentos, decorrentes de abusos sexuais vividos durante a infância. Este percurso atravessa as questões do silenciamento, do testemunho, do ressentimento, do esquecimento e, por último, leva – como um nascimento que se faz através do útero da terra – ao amor fati. É uma pesquisa que busca ir além da dicotomia vítima-agressor, para pensar a complexa trama que se estabelece invisivelmente e possibilita a incitação e a continuidade dos abusos sexuais, principalmente no âmbito familiar. Além disso, ressalta as modalidades de invenção vital a partir do insuportável, excessivo e cruel, e a arte como possibilidade de resistência e criação de novos modos de existência

Eukaryotic nuclear DNA is folded with histone and non-histone proteins into chromatin, a nucleoprotein structure regulated by histone post-translational modifications and substitution with histone variants. Chromatin mediates processes such as DNA damage repair, cell differentiation, gene silencing, and centromere specification. Mistaken inheritance of chromatin-mediated gene silencing, for instance, can cause both aberrant development and cancer. Gene silencing at pericentromeres and centromeres, which can be attained through obstruction of transcription as well as through recruitment of specific RNA-degrading proteins, is essential for centromere specification. However, the molecular mechanisms of these processes are not yet thoroughly understood, and therefore they will be the focus of this thesis. A structure termed heterochromatin, for which the essential hallmark is histone H3 lysine 9 methylation (H3K9me), preferentially assembles at repetitive DNA such as pericentric regions, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNA interference (RNAi) machinery is required for heterochromatin assembly over DNA repeats in diverse organisms by targeting histone-modifying activities. Surprisingly, RNA splicing factors are also required for this process. A widely-held model derived from studies in fission yeast is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, we discovered the requirement of four non-essential splicing factors for pericentric heterochromatin assembly, allowing us to more clearly address the role of splicing in heterochromatin assembly. Sequencing total cellular RNA from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors, which correspond to strong reduction in levels of a central RNAi protein, Argonaute. Moreover, introducing cDNA versions of RNAi factors significantly restores pericentric heterochromatin in splicing mutants. We also found that mutation of splicing factors affects telomeric heterochromatin, and replacement of mis-spliced factor tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres. In addition to post-translational modifications, chromatin silencing can be regulated by histone variants such as H2A.Z. The incorporation of H2A.Z into chromatin regulates chromatin structure and gene expression. The Swr1 chromatin remodeling complex deposits H2A.Z in budding yeast and mammals. Here we characterize a novel component of the fission yeast Swr1 complex, Msc1, which is a Jumonji domain protein frequently associated with histone demethylation. We found that Msc1 is required for Swr1-mediated incorporation of H2A.Z into chromatin at gene promoters. We demonstrated that H2A.Z is required for the expression of CENP-C, which in turn regulates centromere silencing and chromosome segregation. Together, these results show that chromatin silencing at pericentromeres and centromeres is mediated by splicing factors and H2A.Z, respectively, to promote proper regulation of other chromatin factors, thus ensuring faithful chromosome segregation.

Friedreich ataxia (FRDA) is a neurodegenerative disorder that is inherited in an autosomal recessive pattern. The most common FRDA mutation is hyperexpansion of a GAA triplet repeat sequence in the first intron of the affected gene, frataxin (FXN), resulting in decreased frataxin protein expression. The hyperexpanded GAA repeats can adopt unusual DNA structures and induce aberrant epigenetic changes leading to heterochromatin mediated gene silencing. Several epigenetic changes, including increased levels of DNA methylation, histone modifications, repressive chromatin formation and elevated levels of non-coding RNA have been reported in FRDA. It has been reported that a novel FXN antisense transcript (FAST-1), is present at higher levels in FRDA patient-derived fibroblasts and its overexpression is associated with the depletion of CTCF, a chromatin insulator protein, and heterochromatin formation involving the critical +1 nucleosome. Previously, characteristics of FAST-1 were investigated in our lab and a full-length FAST-1 transcript containing a poly (A) tail was identified. To investigate any possible effects of FAST-1 on FXN expression, I first overexpressed this FAST-1 transcript in three different non-FRDA cell lines and a consistent decrease of FXN expression was observed in each cell type compared to control cells. I also identified that FAST-1 copy number is positively correlated with increased FAST-1 expression, which in turn is negatively correlated with FXN expression in FAST-1 overexpressing cells. Additionally, we found that FAST-1 overexpression is associated with increased levels of DNA methylation at CpG sites U6 and U11 of the FXN upstream GAA repeat region, together with CTCF depletion and heterochromatin formation at the 5'UTR of the FXN gene. To further investigate the role of FAST-1 in FXN gene silencing, I used a small hairpin RNA (shRNA) strategy to knock down FAST-1 expression in FRDA fibroblast cells. I found that knocking down FAST-1 increases FXN expression, but not to the level of control cells. Lastly, I investigated the pattern of FAST-1 expression and histone modifications at the FXN transgene in our new FRDA mouse model, designated YG8LR. The YG8LR mice showed decreased levels of FXN expression and H3K9ac and increased levels of FAST-1 expression and H3K9me3. Our data suggest that since FAST-1 is associated with FXN gene silencing, inhibition of FAST-1 may be an approach for FRDA therapy.

Existem alguns assuntos que mesmo tendo relevância do ponto de vista jornalístico muitas vezes não fazem parte dos noticiários. Por interesses ideológicos, econômicos ou políticos ocorre um processo de silenciamento sobre temas que passam a não figurar em reportagens ou mesmo em pequenos textos noticiosos nas páginas dos jornais. Por meio desta pesquisa de dissertação, procurou-se investigar especificamente os conteúdos relacionados ao sistema prisional brasileiro publicados nas primeiras páginas dos jornais Folha de S. Paulo e O Globo. Durante um período de três anos consecutivos, todas as capas foram analisadas, bem como suas respectivas matérias jornalísticas das páginas internas que faziam referência ao esse sistema. A partir daí foi possível identificar ausências, lacunas ou omissões de tópicos importantes para a compreensão e contextualização da complexidade do universo que compõe os procedimentos legais de investigação, julgamento e execução penal, tanto quanto as condições em que isso é realizado. Tal identificação se deu através do cruzamento das informações contidas nas matérias analisadas com as informações, na íntegra, das fontes de domínio público que eventualmente fizeram parte desses textos. / There are some issues that even having a journalistic relevance are often not part of the news. By ideological, economic or political interests, a process of silencing takes place on subjects that do not appear in reports or even in small news texts on the pages of newspapers. Through this dissertation research, we sought to investigate specifically the contents related to the Brazilian prison system published in the first pages of the newspapers Folha de S. Paulo and O Globo. During a period of three consecutive years, all the covers were analyzed, as well as their respective journalistic articles of the internal pages that made reference to this system. From that point on, it was possible to identify absences, or omissions of important topics for understanding and contextualizing the complexity of the universe that makes up the legal procedures for investigation, prosecution and criminal execution, as well as the conditions in which this is done. Such identification took place through the crossing of the information contained in the analyzed material with the information of the sources of public domain that occasionally became part of these journalistic texts.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.