Transcription is an inherently mutagenic process that requires tight surveillance mechanisms to guarantee the preservation of genomic integrity. During transcription, the nascent RNA molecule can hybridize with the template DNA and form a DNA:RNA hybrid, displacing the single-stranded DNA (ssDNA). Although these triple-stranded structures, called R-loops, are physiologically relevant intermediates of several cellular processes, such as immunoglobulin class-switch recombination and gene expression, non-scheduled or persistent R-loops constitute an important source of DNA damage, namely DNA double-strand breaks (DSBs). To preserve genome integrity, cells possess diverse mechanisms to prevent the formation of R-loops or to resolve them. R-loop formation is restricted by RNA-binding proteins and topoisomerases, whereas R-loops are removed by helicases and ribonucleases, such as ribonuclease H enzymes RNase H1 and RNase H2, which degrade R-loops by digesting the RNA strand of the DNA:RNA hybrid. The concerted action of several R-loop resolving enzymes at different stages of the transcription cycle and in distinct physiological contexts is extremely important to maintain gene expression homeostasis and to prevent transcription-dependent DNA damage. Intrinsic features of the transcribed DNA influence the propensity to form R-loops. An asymmetrical distribution of guanines (G) and cytosines (C) nucleotides in the DNA duplex, with an excess of Cs in the template DNA strand (positive G:C skew), favors R-loop formation, which is further stabilized by the establishment of G quadruplexes in the G-rich coding strand. Additionally, the negative DNA supercoiling accumulating upstream of a transcribing RNA Polymerase II (RNA Pol II) creates a local DNA unwinding that provides a window of opportunity for nascent RNA to hybridize with the template DNA, hence promoting R-loop formation. R-loops can drive chromatin modifications that are critical for transcription regulation. Promoter-proximal R-loops enhance the recruitment of the Tip60–p400 histone acetyltransferase complex and inhibit the binding of polycomb repressive complex 2 and histone H3 lysine-27 methylation. Also, R-loops formed over G-rich terminator elements promote histone H3 lysine-9 dimethylation, a repressive mark that reinforces RNA Pol II pausing during transcription termination. Besides affecting histone modifications, R-loops act as barriers against DNA methylation spreading into active genes. DNA methylation, namely 5-methylcytosine (5mC), results from the covalent addition of a methyl group to the carbon 5 of a C attached to a G through a phosphodiester bond (CpG). The activity of DNA methyltransferase (DNMT) enzymes makes 5mC widespread across the mammalian genome, where it plays major roles in imprinting, retrotransposon silencing, and gene expression regulation. More than 70% of all human gene promoters contain stretches of CpG dinucleotides, termed CpG islands (CGIs), whose transcriptional activity is repressed by CpG methylation. R-loops positioned near promoters of active genes maintain CGIs in an unmethylated state, likely by reducing the affinity of DNMT1 binding to DNA, or by recruiting active DNA demethylation machinery: the ten-eleven translocation (TET) methylcytosine dioxygenases. In mammals, TET family comprises TET1, TET2 and TET3, which share the ability to oxidize 5mC to 5-hydroxymethylcytosine (5hmC), a relatively rare DNA modification found across the genome much less frequently than 5mC. TETs can further oxidize 5hmC to 5-formylcytosine (5fC) and to 5-carboxylcytosine (5caC), which engage in different pathways that lead to their replacement by native cytosine. Genome-wide, 5hmC is more abundant at regulatory regions such as promoters and exons, consistent with its role in gene expression regulation. The levels of 5hmC are enriched at active promoter regions, as observed upon activation of neuronal function-related genes in neural progenitors and neurons. Interestingly, 5hmC has the potential to modify the DNA helix structure by favoring DNA-end breathing motion, a dynamic feature of the protein–DNA complexes thought to control DNA accessibility. Moreover, 5hmC weakens the interaction between DNA and nucleosomal H2A-H2B dimers, facilitating transient nucleosome unfolding to accommodate the passage of RNA Pol II during transcription elongation. Also, 5hmC diminishes the thermodynamic stability of the DNA duplex: while 5mC increases the melting temperature, 5hmC reduces the amount of energy needed to separate the two strands of the DNA double helix. Molecular dynamics simulations revealed that the highest amplitude of GC DNA base-pair fluctuations is observed in the presence of 5hmC, whereas 5mC yielded GC base-pairs with the lower amplitude values. 5hmC destabilizes GC pairing by alleviating steric constraints through an increase in molecular polarity, rendering the DNA more flexible and less rigid. Because features that destabilize the DNA duplex, such as supercoiling or G-quadruplexes, are known to facilitate nascent RNA annealing with the template DNA strand, we reasoned that 5hmC may favor R-loop formation. We used an in vitro transcription model to show that the presence of 5hmC in the transcribed DNA promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. This hypothesis was further tested in vivo by editing 5hmC density and assessing the consequent impact on R-loops. Indeed, depletion of the three TET enzymes, which significantly reduced cellular 5hmC levels, caused a pronounced decrease in endogenous R-loops in mouse embryonic stem (ES) and fibroblast cells. Interestingly, the results obtained with individual depletion of each single TET were much milder, suggesting that there is a partial redundancy in the activity of the three TET enzymes. Additionally, CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The above described effects occurred independently of changes in transcription rate, firmly pointing towards a direct impact of 5hmC on DNA:RNA annealing. Collectively, these data suggest that editing 5hmC density by changing the expression levels or the genomic distribution of TET enzymes influences R-loop formation in cells. The interplay between 5hmC and R-loops was further strengthened by genome-wide analysis revealing a strong overlap, detected in half of all active genes, between both structures, which was validated through single-molecule co-localization techniques. Metagene plots of 5hmC and R-loops density show that overlapping of 5hmC and R-loops peaks at the transcription termination site (TTS), suggesting a dedicated role of TET activity in transcription termination by guiding the formation of R-loops. Strikingly, TET abrogation leads to significantly higher levels of readthrough transcripts genome-wide, a characteristic of defective termination. These data support a model whereby TET enzymes act upstream of R-loop formation during efficient transcription termination. Owing to the effect of 5hmC as R-loop facilitator, we also described a positive correlation between 5hmC-rich regions and DNA damage response markers, positioning 5hmC decorated loci as genomic instability hotspots. Finally, we wanted to explore the functional impact of R-loops formed in 5hmC-rich regions. Since TETs drive the developmental DNA methylome reprogramming, the role of 5hmC on stem cell differentiation and development has been widely acknowledged. As such, we used transcriptomic data from ES cells with global R-loop suppression. Gene expression analysis revealed that depletion of R-loops specifically positioned in 5hmC-rich loci impinges most significantly on pathways that control the proliferation/ dormancy balance in ES cells. Therefore, this study discloses a putative role for R-loops, instructed by controlled 5hmC deposition, as mediators in the activation of ES cells gene expression programs.

This thesis is designed to explore, describe and analyze the characteristics of the Petrobras an Eletrobrás cultures in regard to their adaptabilities and conclude which of them could favor the adaptability of these companies. This dissertation has been developed from bibliografical research which identify the theoretical cultural characteristics of organizational adaptability, trom bibliografical research of the principal public policies that have influenced these companies and through interviews, in regard to their organizational cultures, whith persons attached to them.

DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.

OBJECTIVE: The effect of chronic ethanol exposure on chemoreflexes has not been extensively studied in experimental animals. Therefore, this study tested the hypothesis that known ethanol-induced autonomic, neuroendocrine and cardiovascular changes coincide with increased chemoreflex sensitivity, as indicated by increased ventilatory responses to hypoxia and hypercapnia. METHODS: Male Wistar rats were subjected to increasing ethanol concentrations in their drinking water (first week: 5% v/v, second week: 10% v/v, third and fourth weeks: 20% v/v). At the end of each week of ethanol exposure, ventilatory parameters were measured under basal conditions and in response to hypoxia (evaluation of peripheral chemoreflex sensitivity) and hypercapnia (evaluation of central chemoreflex sensitivity). RESULTS: Decreased respiratory frequency was observed in rats exposed to ethanol from the first until the fourth week, whereas minute ventilation remained unchanged. Moreover, we observed an increased tidal volume in the second through the fourth week of exposure. The minute ventilation responses to hypoxia were attenuated in the first through the third week but remained unchanged during the last week. The respiratory frequency responses to hypoxia in ethanol-exposed rats were attenuated in the second through the third week but remained unchanged in the first and fourth weeks. There was no significant change in tidal volume responses to hypoxia. With regard to hypercapnic responses, no significant changes in ventilatory parameters were observed. CONCLUSIONS: Our data are consistent with the notion that chronic ethanol exposure does not increase peripheral or central chemoreflex sensitivity.

OBJECTIVE: Chemoreceptors play an important role in the autonomic modulation of circulatory and ventilatory responses to changes in arterial O2 and/or CO2. However, studies evaluating hemodynamic responses to hypoxia and hypercapnia in rats have shown inconsistent results. Our aim was to evaluate hemodynamic and respiratory responses to different levels of hypoxia and hypercapnia in conscious intact or carotid body-denervated rats. METHODS: Male Wistar rats were submitted to bilateral ligature of carotid body arteries (or sham-operation) and received catheters into the left femoral artery and vein. After two days, each animal was placed into a plethysmographic chamber and, after baseline measurements of respiratory parameters and arterial pressure, each animal was subjected to three levels of hypoxia (15, 10 and 6% O2) and hypercapnia (10% CO2). RESULTS: The results indicated that 15% O2 decreased the mean arterial pressure and increased the heart rate (HR) in both intact (n = 8) and carotid body-denervated (n = 7) rats. In contrast, 10% O2did not change the mean arterial pressure but still increased the HR in intact rats, and it decreased the mean arterial pressure and increased the heart rate in carotid body-denervated rats. Furthermore, 6% O2 increased the mean arterial pressure and decreased the HR in intact rats, but it decreased the mean arterial pressure and did not change the HR in carotid body-denervated rats. The 3 levels of hypoxia increased pulmonary ventilation in both groups, with attenuated responses in carotid body-denervated rats. Hypercapnia with 10% CO2 increased the mean arterial pressure and decreased HR similarly in both groups. Hypercapnia also increased pulmonary ventilation in both groups to the same extent. CONCLUSION: This study demonstrates that the hemodynamic and ventilatory responses varied according to the level of hypoxia. Nevertheless, the hemodynamic and ventilatory responses to hypercapnia did not depend on the activation of the peripheral carotid chemoreceptors.

O trabalho apresentado nesta tese foi realizado no âmbito do projeto de atualização do calorímetro TileCal do detetor ATLAS, mais concretamente, na substituição das placas HV-Opto por uma versão melhorada (HV-Remote) que controlam a alta tensão fornecida aos fotomultiplicadores (PMTs) do detetor. Atualmente, a placa HV-Opto encontra-se em operação há mais de 10 anos, e uma vez que o equipamento se encontra no interior do detetor, está sujeita a elevadas doses de radiação. Outro problema prende-se com a necessidade de substituir equipamento avariado, o que, devido à radiação, só é possível quando se realizam paragens técnicas do LHC, algo que acontece esporadicamente. Assim, uma das propostas de atualização daquele sistema consiste em mover a eletrónica de controlo do TileCal atualmente no detetor, para um local na caverna USA15, num ambiente virtualmente sem radiação pois encontra-se a 100 m do detetor. A atualização proposta pelo grupo de investigação do LIP (Laboratório de Instrumentação e Física Experimental de Partículas) e de outras instituições participantes no projeto está dividida em três partes: conceção e produção de uma carta-protótipo (HV-Remote), realização de testes com o protótipo e criação do sistema de controlo da placa HV-Remote. O trabalho realizado nesta tese incide no sistema de controlo da placa, e teve como objetivo a criação de uma interface que permite ao utilizador gerir os valores de tensão que são aplicados aos PMTs do detetor. Adquiriu-se uma placa de teste que contém um módulo programável em linguagem C e/ou BASIC. Este módulo (da TIBBO), caso o seu desempenho cumpra os requisitos, será colocado na versão final da HV-Remote e terá como função direcionar a informação enviada pela interface de utilizador para os subsistemas, ou componentes, da placa, para que a respetiva parametrização seja efetuada. A interface de utilizador foi desenvolvida em linguagem C++, com recurso ao programa WinCC atualmente utilizado no controlo do TileCal. Esta interface permitirá ao utilizador escrever e ler valores de tensão aplicados aos PMTs, bem como controlar ou monitorizar outras variáveis, tais como a temperatura, que são cruciais para o normal funcionamento de todo o sistema. A ligação entre a interface e a placa HV-Remote será feita com Sockets, através de uma ligação via Ethernet, utilizando o protocolo de comunicação SPI para o envio de dados. No trabalho, foram alcançadas com sucesso as seguintes metas: - Teste do módulo programável que se encontra anexado a uma placa de testes. - Teste do canal SSI do módulo (em configuração de Master) com Arduinos Uno (em configuração de Slave) e comunicação por Sockets sobre Ethernet. - Teste do módulo TIBBO com os vários componentes que constituem a placa HV-Remote. - Planificação e conceção do painel (interface de utilizador) para o DCS. - Programação dos vários botões do painel. - Testes finais com o módulo TIBBO, o painel DCS e os componentes da placa HV-Remote.

ABSTRACT Platycodon grandiflorus is an ornamental species that can be marketed as a potted flower; however, it has fragile and bending floral stems. Plant growth regulators, such as paclobutrazol, are compounds that reduce stem elongation, enabling the production of plants with a more compact formation. This study aimed to evaluate the effects of paclobutrazol on platycodon growth, in a greenhouse. A completely randomized blocks experimental design was used in a 4 × 5 factorial scheme, with four replicates. The treatments consisted of four varieties of ‘Astra Semi-Double’ platycodon (Blue, Lavender, Pink and White) and five paclobutrazol doses (0, 1.25, 2.5, 3.75 and 5.0 mg pot-1). The paclobutrazol application did not affect the stem diameter, number of leaves, days until anthesis, number of flowers or flower buds, and flower diameter. The shoot length, leaf area and shoot dry mass decreased with higher doses of the growth regulator. Lavender showed the highest values for shoot length, number of leaves, leaf area and shoot dry mass. Pink had the earliest anthesis, followed by Blue and White, whereas Lavender had the latest anthesis. The dose of 3.75 mg pot-1 of paclobutrazol efficiently controlled the growth and improved the visual quality of all the platycodon varieties.