The STS movement has as a main objective the democratization of decision-making on social issues involving science-technology (ST). Also, this arises in contexts in which the supposed neutrality of ST began to be questioned. Neutrality understood as legitimizing technocratic decision models. In the educational field, I seek curriculum geared toward to the creation of a culture of participation, based on reference Freire-STS and LATSTS (Latin American Thought in Science Technology Society). As well, my research problem was: Historic Buildings, called myths, in other words, the supposed neutrality / superiority of technocratic decision model, the Salvationist perspective / redeeming attributed to ST and scientific-technological determinism, sustained and feeding back the supposed neutrality have been worked on educational practices related to the Science-Technology-Society? Which referrals have been given? The objectives that guided the research were: i) identify, in educational practices presented in the literature of the area, the approaches given to the three historic buildings; ii) analyze and deepen the featured aspects in regard to the three historic buildings, in a vision of non-neutrality of ST; and iii) signal parameters to Science Education in order to support proposals for educational practices. Methodologically, consisted of a qualitative research, bibliographic nature, being the corpus of analysis consisting of six articles published in journals in the field of Science Education. I used the Textual Discourse Analysis, which resulted in three categories of analysis: a) Silencing on the design of ST may be signaling an endorsement to a close understanding of the scientific-technological determinism; b) Silencing on dimensions other natures, beyond the scientific-technological in decision-making processes; and, c) Silencing about the values internalized in the scientific-technological product. / O movimento Ciência-Tecnologia-Sociedade (CTS) tem como um dos objetivos centrais à democratização de processos decisórios sobre temas sociais envolvendo ciência-tecnologia (CT). Também, este surge em contextos nos quais a suposta neutralidade da CT passou a ser questionada. Neutralidade entendida como legitimadora de modelos decisórios tecnocráticos. A pesquisa está inserida na busca de currículos voltados para a constituição de uma cultura de participação, apoiada nos referenciais Freire-CTS e no Pensamento Latino Americano em Ciência-Tecnologia-Sociedade (PLACTS). Assim, o problema de pesquisa foi: Construções históricas, denominadas de mitos, ou seja, a suposta neutralidade/superioridade do modelo de decisões tecnocráticas, a perspectiva salvacionista/redentora atribuída à CT e o determinismo científico-tecnológico, sustentadas e realimentando a suposta neutralidade, têm sido trabalhadas em práticas educativas relacionadas à CTS? Quais encaminhamentos têm sido dados? Os objetivos que guiaram a investigação foram: i) identificar, em práticas educativas, apresentadas em literaturas da área, as abordagens dadas a estas três construções históricas; ii) analisar e aprofundar os aspectos caracterizados no que tange as três construções históricas, em uma visão de não neutralidade da CT; e, iii) sinalizar parâmetros para a Educação em Ciências, fundamentando propostas de práticas educativas. Metodologicamente, constituiu-se de uma pesquisa qualitativa, de cunho bibliográfico, sendo o corpus de análise constituído de artigos publicados em seis periódicos da área da Educação em Ciências. Utilizou-se a Análise Textual Discursiva, da qual resultaram três categorias de análise: a) Silenciamento sobre a origem, sobre a concepção de CT pode estar sinalizando o endosso a uma compreensão próxima do determinismo científico-tecnológico; b) Silenciamento sobre dimensões de outras naturezas, além da científico-tecnológica, em processos decisórios; e, c) Silenciamento quanto aos valores internalizados no produto científico-tecnológico. Estas categorias, sintetizando os resultados da pesquisa, sinalizam desafios para a educação em ciências, particularmente para a constituição de uma cultura de participação.

Teachers’ beliefs are central to determining children’s optimal educational experiences. However, some studies related to teachers’ beliefs yielded findings that rendered beliefs and practices incongruous. Although the principles of developmentally appropriate practices that synthesize theoretical and empirical research on child development have been adapted to various contexts other than its original United States of America (USA) context, developmentally appropriate practices remain contentious as to its relevance in these contexts. What is appropriate for children’s education seems debatable, largely determined by social expectations of childhood and children. Cultural diversity seems to be the new dispensation in this discourse. Fundamentally, research on preschool teachers’ beliefs about developmentally appropriate educational practices, adds to literature about cultural context variables in preschool provision from different contexts. Purpose: This study examined how teachers’ practical experiences framed their beliefs and understanding of children’s educational experiences within a developmentally appropriate framework and a bioecological systems theory. Paradigm/Design/Methods: A constructivist paradigm within the qualitative approach guided this study. Video and photographs became the basis to elicit teachers’ beliefs about children’s educational experiences. Children’s educational experiences were analysed along five constructs related to the concept of DAP; teaching strategy, use of materials, scheduling of children’s learning, assessment, and consideration of children’s individuality. Findings: Teachers’ beliefs corroborated the DAP framework, but their practices that were more teacher-directed, contrasted the DAP principles. They used formally structured teaching approaches, as materials in three-out-of-four classes observed remained ‘silenced’. The schedules were formally structured, subject-based, with limited flexibility, as assessment for children’s learning focused on limited aspects of the cognitive domain. Conclusion: Teachers’ beliefs seemed to support educational practices that embrace the principles of DAP. However, some context-related factors, which include pressure from parents, competitive school environments, preparation for the interview, different transition requirements, peer pressure, and perceived lack of time limited their use of DAP. I extrapolate these factors to the bioecological systems theory, to understand the dynamics of early childhood education provision in Kenya. Practical implications/Originality/Value: This study adds to literature on teachers’ beliefs about children’s educational experiences from a developing country context, as well as adding to studies that have used visually elicited interviews. It also provides the details of children’s educational experiences, in part, to appreciate the current conversation on the status and the nature of focus on standards or skills-based dynamics in preschool provision. Besides, it might be the first study in Kenya to embrace the DAP framework and the bioecological systems theory. The seesaw model advanced in this study synthesizes the originality of the study by conceptualizing the theoretical as well as empirical literature on developmentally appropriate educational practices, as a valuable framework to understand and interpret competing priorities that might affect preschool provision. The seesaw model is also valuable in locating and extending the conversation about different stakeholders’ priorities, not only in Kenya, but also in other societies. / Thesis (PhD)--University of Pretoria, 2010. / Educational Psychology / unrestricted

Argonaute proteins are the core component of an RNA silencing complex. The human genome encodes four Argonaute paralogs –Ago1, Ago2, Ago3 and Ago4– proteins that are guided to target mRNAs by microRNAs. More than 500 miRNAs are conserved between mammals, and each microRNA can repress hundreds of genes, regulating almost every cellular process. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression. Although we understand many aspects of microRNA biogenesis and formation of the RNA-induced silencing complex, much less is known about the subsequent steps leading to target mRNA regulation. Mammalian microRNAs rarely have complete complementarity to their target mRNAs so, instead of endonucleolytic cleavage by Ago2, microRNAs destabilize or repress translation of target mRNAs. Here I explored the functional limits of Argonaute proteins bound to their targets directly and indirectly through microRNAs in mammalian cells. I revealed the different abilities for Argonaute proteins bound at multiple sites in a target to generate cooperativity in silencing based on the extent of pairing between the microRNA and target mRNA. Further, I harnessed the endogenous microRNA silencing mechanism to repress an mRNA that is not a direct target of the microRNA by tethering the RNA-induced silencing complex to the 3´ UTR of an mRNA. This strategy allows tissue-specific gene silencing due to the limited endogenous expression profile of the recruited microRNA. Efforts made herein further our mechanistic knowledge of microRNA-induced gene silencing in mammalian cells and advance microRNA-based strategies toward treating human disease.

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation, and 4) Disrupted nucleocytoplasmic trafficking. Due to lack of treatments for this disease, we have pursued an AAV-RNAi dependent gene therapy approach, using an artificial microRNA (amiR) packaged in a recombinant adeno-associated virus (rAAV). After validating our in vitro results, we advanced to in vivo experiments using transgenic mice that recapitulate the major histopathological features seen in human ALS/FTD patients. Adult and neonate mice were injected through clinically relevant routes and our results indicate that AAV9-mediated amiR silencing not only reduced mRNA and protein levels of C9ORF72 but also the expansion derived toxic GP dipeptides. Although our amiR is not targeting the expansion itself but exon 3, we illustrate here that the evident dipeptide decrease is achievable due to the presence of aberrant transcripts in the cytoplasm containing miss-spliced Intron-HRE-C9ORF72 species. These encouraging results have led to the continued testing of this treatment as a therapeutic option for C9ORF72 - ALS patients.

Argonaute proteins are small DNA/RNA-guided endonucleases found in all domains of life. In animals, small RNAs of length 21–35 nucleotides direct the PIWI-clade of Argonautes to silence complementary target RNAs; these are called PIWI-interacting RNAs (piRNAs). During spermatogenesis in mice, piRNA-guided PIWI proteins, MIWI2, MILI, and MIWI, silence transposons, regulate expression of protein-coding genes and are necessary for fertility. A working endonuclease activity of MIWI and MILI is essential to complete spermatogenesis. Yet, both MIWI and MILI produce weak and slow target cleavage in vitro, thwarting biochemical examination of the silencing step. Here, we find that PIWI proteins require an auxiliary protein to efficiently cleave their targets, unlike any other known Argonaute. Gametocyte Specific Factor 1 (GTSF1) is a conserved zinc-finger protein essential for fertility and piRNA-directed silencing. We show GTSF1 accelerates the pre-steady-state rate of target cleavage by MIWI and MILI; this role of GTSF1 is also preserved in insects. A critical step in GTSF1 mechanism entails binding RNA. GTSF1 allowed detailed kinetic analyses of catalytic PIWIs: they require extensive 3′ complementarity between the guide and target to efficiently cleave them, but this base-pairing also limits turnover. Interestingly, within a species, different PIWI proteins have unique kinetic properties. In sum, our findings provide molecular mechanisms of GTSF1 function and target silencing by PIWIs as well as a useful method for future studies.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

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Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

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[pt] Após uma longa ditadura civil-militar e uma transição política controlada, o processo de acerto de contas do Brasil com seu passado autoritário tem sido conturbado e disputado. Esse é o tema sobre o qual essa tese se debruça. Por meio de entrevistas produzidas pela metodologia da História Oral com formuladores e executores de políticas públicas, bem como com atingidos, foram identificadas três perspectivas diferentes para o tratamento desse passado: a dos conservadores, a dos atingidos e a dos negociadores. Cada uma delas representa a maneira como atores políticos se organizaram e articularam no decorrer da redemocratização a fim de orientar o debate político. Tendo isso em vista, a hipótese central deste trabalho é que, sob a justificativa de evitar uma nova ruptura institucional e virar a página da história, a perspectiva conservadora predominou em um processo deliberado de silenciamento da memória dos atingidos. Trata-se, portanto, do que reconhecemos como política pública de silenciamento. Embora reconheçamos no decorrer do trabalho que houve uma movimentação do Estado em prol de políticas e medidas de memória entre 2007 e 2014, estas permaneceram circunscritas aos grupos diretamente afetados, como familiares de mortos e desaparecidos, ex-perseguidos políticos e movimentos de defesa de direitos humanos, de modo que a sociedade como um todo não se percebe lesionada pelas arbitrariedades do autoritarismo, tampouco os seus efeitos. Para fundamentar nossa análise, refletimos sobre o uso político da Lei de Anistia de 1979 como instrumento garantidor do silenciamento até a atualidade. Ainda, apontamos a opção por priorizar a reparação como uma estratégia que gerou um efeito individualizante, mantendo a sociedade distanciada da pauta. Por fim, analisamos as disputas de narrativa em torno da Comissão Nacional da Verdade e seu impacto disruptivo na política pública de silenciamento, o que gerou uma intensa reação de grupos conservadores nas mais diferentes esferas políticas. / [en] Following a long civil-military dictatorship and a controlled political transition, the Brazilian process of reckoning with its authoritarian past has been turbulent and disputed. This is the theme this thesis focuses on. Based on interviews produced by the Oral History methodology with policymakers, as well as with the people who were affected by the authoritarian regime, we identified three different perspectives on the treatment of this past: that of the conservatives, that of the groups affected, and that of the negotiators. Each one of them represents how political actors organized and articulated themselves during the redemocratization process in order to shape the political debate. Considering this, the central hypothesis of this research is that, under the justification of avoiding a new institutional rupture and turning the page on history, the conservative perspective predominated in a deliberate process of silencing the memory of the affected people. This is, therefore, what we define as a public policy of silencing. Although we recognize that there was a movement of the State in favor of memory policies and measures between 2007 and 2014, these remained circumscribed to directly affected groups, such as family members of the dead and disappeared, former political prisoners, and movements for the defense of human rights, so that society does not perceive itself as having been harmed by the arbitrariness of authoritarianism, nor its effects. To support our analysis, we reflect on the political use of the 1979 Amnesty Law as an instrument to guarantee silencing until today. Furthermore, we point to the priority given to reparations as a strategy that produced an individualizing effect, keeping society at a distance from the agenda. Finally, we analyze the narrative disputes around the National Truth Commission and its disruptive impact on the public policy of silencing, which generated an intense reaction from conservative groups in different political spheres.