DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.

Apicomplexan parasites are leading causes of human and livestock diseases such as toxoplasmosis and malaria caused by Toxoplasma gondii and Plasmodium falciparum respectively. These organisms are varied in their morphologies and astoundingly complex on their life cycles that include infections of more than one host organism, differentiation through several morphologically distinct forms, and both sexual and asexual replication. What we and others have initially proposed was that the control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. The initial hypothesis had to be re-assessed in light of the discovery in Apicomplexa of an expanded family of plant-like transcription factors (TFs) harbouring APETALA2 (AP2)-like domains. Yet, a growing body of evidence tends to favor epigenetic as one of the main contributor to parasite developmental programs and adjustments to fluctuant environment. One way to examine dynamic changes in post-translations modifications (PTMs) patterns is to alter the histone code writing. We therefore took advantage of HDAC inhibitors and showed that specific inhibition of TgHDAC3 by the cyclopeptide FR235222 disrupts the genome wide steady-state level of histone H4 acetylation inducing derepression of stage-specific genes. Yet, many questions about TgHDAC3 modus operandi remain unanswered. During my thesis, I uncovered the TgHDAC3-regulated proteome-wide acetylome typified by the presence of non-histone proteins including AP2 TFs and novel PTMs, e.g. the acetylation at Lys31 within the globular domain of histone H4. H4K31ac promotes a relaxed chromatin state at the promoter of active genes through nucleosome disassembly in both parasites. We identified TgGCN5B and TgHDAC3 as two antagonist enzymes regulating H4K31 acetylation in T. gondii. In contrast, H4K31monomethylation is enriched throughout the gene body of T. gondii active genes and contributes to transcription, whereas it is enriched at transcriptionally inactive pericentromeric heterochromatin regions in P. falciparum, a region that is lacking H3K9me3 and heterochromatin protein 1 in this parasite. We also showed that treating T. gondii cystogenic strains with a low dose of FR235222 induces the levels of proteins known to be expressed exclusively in cat (sporozoite and merozoite) or in murine chronic stage (bradyzoite). Lastly, we determined the specific interactome of TgHDAC3 and found as partners a MORC protein (CR230), several AP2 TFs, and ELM2 domain-containing scaffolding proteins. Collectively, these data established TgHDAC3 family as a central regulator of gene expression and stage conversion in T. gondii and, likely, other Apicomplexa. / Apicomplexan genome architecture is typified by a binary chromatin structure, with a major fraction of the bulk genome packaged as transcriptionally permissive euchromatin while few loci are embedded in silenced heterochromatin. There is evidence that histone modifications occurring at the lateral surface of the nucleosome play a substantial role in shaping chromatin structure, yet our understanding of the exact mechanism of action is poor. Here, we address how versatile modifications at Lys31 within the globular domain of histone H4 contribute to genome organization and expression in Apicomplexa. H4K31 acetylation was found at the promoter of active genes. The residue lies where the DNA wraps around the histone and its acetylation may enhance nucleosome disassembly, thereby favoring a more relaxed, open chromatin state. This residue tends also to be monomethylated and depending of the parasite examined different patterns were found. H4K31me1 was enriched in the core body of Toxoplasma active genes, yet its occupancy was inversely correlated with transcripts levels likely because the mark by reducing histone turnover impedes RNA polymerase progression across transcribed units. In contrast to the methylation of H3, it is the first time that a methylated residue of H4 has been clearly associated with transcriptional regulation. In Plasmodium, H4K31me1 was exclusively enriched at transcriptionally inactive genomic regions and peculiarly at pericentromeric heterochromatin, likely to replace the missing H3K9me3 that commonly decorated pericentric nucleosomes in other species.

Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to the role of ethanol metabolism in oxidative stress and alcohol liver injury (ALI). During ethanol metabolism, both the cytochrome bc1 and NADH dehydrogenase complexes within the mitochondria have been demonstrated to be major contributors to ROS formation and “leak” free radicals [11-13]. As a result, the free radicals superoxide (O2-) and hydrogen peroxide (H2O2) is diffused into the cytoplasm where they can react with other molecules, proteins and DNA and cause tissue injury [4, 14]. Chapter 1 aims to introduce the link between ethanol metabolism and histone post-translational modifications (PTM) such as tyrosine nitration and lysine acetylation using proteomics techniques. Chapter 2 uses a global proteomic study to identify links between gender and ALI. A 10-day chronic-binge mouse model was employed in order to identify gender-specific proteins that may influence the development of ALD. It has previously been established that females are more susceptible to developing ALD, however, the cause is still unknown. This study identifies gender differences in the family of cytochrome P450 proteins using a mouse model for chronic-binge alcohol exposure. The cytochrome P450 family of proteins are important in the metabolism of toxic compounds, such as acetaldehyde, a byproduct of ethanol metabolism. Interestingly, I also identified that female mice expressed naturally higher levels of histone acetylation prior to alcohol exposure when compared to males. Following alcohol exposure, the female mice did not show much change in acetylation, whereas male acetylation levels were raised to similar levels of the female mice. These acetylation changes raised the question, how does alcohol influence epigenetic marks on histone proteins? Recently, new evidence has emerged that supports the role of epigenetics in the pathophysiology of ALD [4, 14-27]. Ethanol metabolism will promote shifts in redox potential and mitochondrial dysfunction, the result is the formation of reactive oxygen and/or nitrogen species (ROS/RNS) [4, 5, 7, 10, 14, 28]. As ethanol is metabolized, the accumulation of ROS/RNS species such as NO- and O2- can induce the post-translational modification nitrotyrosine. Shifts in redox potential will cause the electron transport chain to “leak” the free radical O2-. Another free radical known as nitric oxide (NO-) has been shown to be elevated during times of ethanol consumption [29, 30]. Traditionally, NO has a protective role within the cell at low concentrations, however, in surplus can lead to tissue damage. Ethanol-induced increases in NO- and O2- can instigate to peroxynitrite (ONOO-) formation; a potent oxidant and nitrating agent of tyrosine residues [29, 31-34]. Chapter 3 examines the indirect effect of alcohol metabolism and ROS/RNS formation on histone tyrosine nitration. This project used mass-spectrometry to identify novel targets of histone tyrosine nitration using a mouse-model of chronic-binge alcohol exposure. Interestingly, histone H3 was found to be nitrated on the hinge-region of the N-terminal tail at tyrosine 41. Molecular dynamics of the nitrated and unmodified proteoforms revealed that the DNA prefers a change in conformation upon H3Y41 nitration. Further studies using an antibody synthesized against the nitrated H3y41 region of the protein revealed potential targets within the genome important in fatty acid synthesis and metabolism. Chapter 4 looks at the direct influence of alcohol metabolism and its contribution to histone acetylation via acetate production and acetyl-CoA. Alcohol metabolism has traditionally been thought influence acetylation through the sirtuin family of deacetylase proteins. Sirtuin deacetylases are NAD+-dependent and have been shown to be a regulate protein acetylation within the mitochondria, cytoplasm, and nucleus during times of ethanol exposure [35-37]. Shifts in redox potential attributed to ethanol metabolism can inhibit sirtuin deacetylase activity by out-competing the enzymes for available NAD+, ultimately leading to mitochondrial and nuclear hyperacetylation [17, 28, 38-42]. Currently, there is evidence that ethanol increases acetylation of histone 3 lysine 9, which then targets activation of the alcohol dehydrogenase gene (ADH) [17, 18, 43]. Moreover, Shukla et.al. (2008) support the idea that ethanol can alter epigenetic transcriptional activation based on which modification is selected for a site during times of stress when it can be occupied by more than one modification [22]. Chapter 4 demonstrates the use of mass-spectrometry to metabolically trace 13C2-labeled ethanol in vivo. These new data show clear evidence of 13C2 heavy-labeled ethanol being incorporated into known sites of acetylation on the N-terminal tails of histone H3 and H4. Incorporation of heavy-label was calculated using extracted ion chromatograms (XIC) for the double and singly acetylated and unmodified peptides belonging to H3K9-R17 and H3K18-R23. Total change in acetylation was also assessed for each peptide using the ratio of ratios of total acetylation to unmodified peptide over the fold change in ethanol- to control-fed groups. An interesting observation was observed in that the incorporation of heavy-label suggests site-selectivity of lysine residues over time. Histone 4 contains multiple sites of acetylation on the peptide H4K5-R17, making it hard to quantify manually. MaxQuant evidence files in conjunction with R were used to calculate the 13C2 incorporation on the multiple H4 acetyl-sites over 24-hours. Ethanol-heavy label incorporation at multiple acetyl-sites occurred as a mixture suggesting a role in transcriptional regulation. These new data establish a link between alcohol metabolism and known epigenetic marks on histone proteins. These studies have now established that alcohol metabolism is indirectly linked to histone tyrosine nitration through increased ROS/RNS and directly through acetate production. Understanding how these epigenetic marks fluctuate as ALD progresses will provide potential targets for the development of new drug therapies. The epigenetic marks identified in these studies have previously been established to be important activators in transcription. These data provide novel techniques using proteomics-based metabolic tracing in vivo. Future studies will assess how these marks change after chronic ethanol exposure and whether the changes in epigenetics are heritable. Understanding hereditary of alcoholism will provide insight to those predisposed to the disease.

Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

No description available.

A esterigmatocistina (ST) é uma micotoxina policetônica produzida por diferentes espécies de Aspergillus e outros gêneros fúngicos como Bipolaris e Chaetomium. Esta toxina é caracterizada como uma bifuranoxantona com fórmula molecular C18H12O6, freqüentemente encontrada como contaminante de diversos produtos alimentícios como sementes oleaginosas e cereais. Possui propriedades carcinogênicas, toxigênicas, mutagênicas e teratogênicas, entretanto, ela é menos tóxica do que a aflatoxina. O fungo Emericella nidulans (Eidam) Vuillemin, cujo anamorfo é o Aspergillus nidulans (Eidam) Winter, foi usado como modelo genético para a investigação de genes envolvidos na via biossintética da esterigmatocistina. Linhagens estoque de Utrecht (originárias de Glasgow) foram analisadas quanto à produção de ST e, entre elas, somente a linhagem UT196 [yA2 (I); metA17 (II); piroA4 (IV)] apresentou produção de 4 ppm de ST (stc+), enquanto que as linhagens UT448 e UT184 mostraram-se não produtoras dessa micotoxina (stc). Embora a linhagem UT196 seja muito bem caracterizada geneticamente, este foi o primeiro relato da produção de ST nessa linhagem. Os índices de segregação alélica e todas as freqüências de recombinação entre os marcadores genéticos ligados e não ligados foram determinados tanto pelo cruzamento meiótico UT448 (stc) x UT196 (stc+) como pelo cruzamento mitótico UT448//UT184. 175 segregantes meióticos e 140 segregantes mitóticos foram analisados quanto à produção de ST, aos marcadores de auxotrofia e de resistência a acriflavina. Essas linhagens cruzadas apresentavam vários marcadores em heterozigose e isso permitiu o mapeamento de um gene estrutural da ST (stcZ+), localizado no braço esquerdo do cromossomo I, 4% distante do gene da riboflavina (riboA1). Como um subproduto deste trabalho, foi detectado um pigmento vermelho, de Rf = 0,90, em todos os segregantes meióticos e mitóticos, produtores ou não de ST, indicando tratar-se, provavelmente, de um pigmento policetônico produzido pelos ascosporos do fungo. A baixa expressão da produção de ST em 13 segregantes meióticos e a alta expressão dessa toxina nos diplóides UT448//UT196 e UT448//UT184 (40 ppm) permitiram concluir a existência de um fator regulador, compreendido na região w-met do cromossomo II, responsável pela expressão do gene estrutural stcZ+. A análise desses genes através do ciclo parassexual sugeriu um comportamento epigenético típico envolvendo esses genes. Com base nos dados obtidos, hipóteses para explicar o controle da expressão desses genes e suas inter-relações foram aqui apresentadas. / Sterigmatocystin (ST) is a polyketide produced by different species of Aspergillus as well as by other fungus genera such as Bipolaris and Chaetomium. This toxin is characterized as a bifuranoxanthone, whose molecular formula is C18H12O6 and which is frequently found as a contaminant in several food products such as oil-seed grains and cereals. It has carcinogenic, toxigenic, mutagenic and teratogenic properties; however, it is less toxic than aflatoxin. The fungus Emericella nidulans (Eidam) Vuillemin, whose anamorph is Aspergillus nidulans (Eidam) Winter, was used as a genetic model to investigate the genes involved in the sterigmatocystin biosynthetic pathway. Strains from Utrecht stocks (originally from Glasgow) were analyzed in order to detect ST production and, among them, only the UT196 strain [yA2 (I); metA17 (II); piroA4 (IV)] showed the production of 4 ppm of ST (stc+), whereas the UT448 and UT184 strains showed to be nonproducers of such toxin (stc). Although the UT196 strain is very well characterized genetically, this has been the first report on its production of ST. The allelic segregation rates and all the recombination frequencies between linked and non-linked genetic markers were determined by both the meiotic crossing UT448 (stc) x UT196 (stc+) and mitotic crossing UT448//UT184. 175 meiotic segregants and 140 mitotic segregants were analyzed as to ST production, auxotrophy markers and resistance to acriflavine. These crossed strains presented various markers in heterozygous configuration, which allowed to map a structural gene of ST (stcZ+) located on the left arm of chromosome I, 4% distant from the riboflavin gene (riboA1). As a byproduct of this work, a red pigment of 0.90 Rf was detected in all meiotic and mitotic segregants, whether they were ST producers or not, which indicated that was probably a polyketide produced by the fungus ascopores. The low expression of ST production in 13 meiotic segregants and the high expression of such toxin in the UT448//UT196 and UT448//UT184 diploids (40 ppm) allowed to conclude that a regulating factor existed in the w-met region of chromosome II, which is responsible for the expression of the structural gene stcZ+. The analyses of those genes through the parasexual cycle suggested a typical epigenetic behavior which involved them. Based on the data obtained, hypotheses to explain the expression control of these genes as well as their inter-relationships were here presented.

The composition and diversity of the gastrointestinal microbiota and its interaction with human cells have been frequently associated with immune system functions and disease development, including autoimmunity and allergy. This is believed to be mediated in part through epigenetic modifications, mainly as DNA methylation. Several studies have collectively supported the beneficial effects of probiotics for the prevention of allergic disease. However, there have been few studies addressing the possibility for probiotic supplementation to induce epigenetic changes and its importance for allergy development. This study aims to investigate whether probiotic treatment with Lactobacillus reuteri, distributed during and after the pregnancy period, leads to epigenetic changes in the offspring and if this have any effect on the development of allergic disease. DNA methylation data received from a clinical allergy prevention study was analysed through a set of bioinformatics methods and basic network analysis. The obtained results suggests that supplementation with L. reuteri indeed induces some significant changes in DNA methylation. These changes did not exhibit any significant correlation with allergy outcome of the children. Furthermore, the methylation changes were found at positions located in genes not enriched for any allergy-related biological pathways. However, when taking the genes interactions with other genes into account an interconnected gene interaction module could be identified that showed enrichment for biological processes involved in the T cell receptor signaling pathway, central for immune response transduction. Further analyses did not fit into the time-frame of this thesis, but the obtained results gives a first informative view of the effects of L. reuteri on methylation patterns, and points out directions for the continuing project work.

Estudos recentes têm revelado que uma fração significativa do transcriptoma de eucariotos é composta por RNAs não codificadores longos (lncRNAs). Este trabalho investigou o padrão de expressão de um conjunto de lncRNAs originados a partir de regiões intrônicas de genes codificadores de proteínas em três linhagens celulares tumorais humanas utilizando microarranjos de DNA customizados. Realizamos uma série de análises in silico com a perspectiva de identificar propriedades globais desses transcritos, tais como a abundância relativa em diferentes tecidos, características evolutivas, estruturais e regulatórias, além de possíveis funções celulares. Avaliamos também a contribuição da metilação do DNA, um mecanismo de silenciamento epigenético da expressão de genes codificadores de proteínas, na regulação da expressão de lncRNAs intrônicos. Observamos que uma fração dos lncRNAs intrônicos detectados nas linhagens estudadas são conservados evolutivamente, tem padrão de expressão tecido específico, e está enriquecida em elementos regulatórios na sua extremidade 5\'. Foram identificados subconjuntos de lncRNAs intrônicos possivelmente atuando sobre genes associados a vias regulatórias importantes para o controle do desenvolvimento de organismos e ciclo celular. Comparativamente a mRNAs, uma menor proporção de lncRNAs intrônicos possui ilhas CpGs (CGIs) na vizinhança de seu início de transcrição. Apesar disso, observamos que um subconjunto desses transcritos teve sua expressão sensível ao tratamento com o agente desmetilante de DNA 5-AZA, demonstrando que lncRNAs intrônicos transcritos podem estar sujeitos a regulação transcricional mediada por metilação do DNA. Dentre os lncRNAs intrônicos regulados por metilação do DNA, destaca-se o lncRNA AS-APP, cuja expressão aumentou em 25 a 80 vezes nas linhagens celulares DU-145 e HEK293, respectivamente, após tratamento com 5-AZA. Este lncRNA possui uma CGI metilada e um promotor ativo a cerca de 4 kb de distância do seu início de transcrição conhecido. O aumento da transcrição do lncRNA AS-APP após desmetilação do DNA correlacionou-se a uma diminuição significativa dos níveis de expressão do mRNA do gene APP. Este resultado sugere uma possível ação regulatória em cis do lncRNA AS-APP no locus APP, um importante gene envolvido na doença de Alzheimer e com expressão associada ao prognóstico de alguns tipos de câncer. Os resultados obtidos neste trabalho reforçam a ideia de que lncRNAs intrônicos constituem unidades transcricionais independentes que se encontram sobre controle regulatório nos diferentes tipos celulares. Foi gerado também um catálogo de lncRNAs intrônicos regulados por metilação que permitirá a seleção de candidatos com maior potencial de relevância funcional para caracterização detalhada. / Recent studies have revealed that a significant fraction of the eukaryotic transcriptome is composed of long noncoding RNAs (lncRNAs). This work investigated the expression pattern in three human tumor cell lines of a set of lncRNAs originated from intronic regions of protein coding RNAs, using custom DNA oligoarrays. In silico analyses were performed to identify global properties of these transcripts such as relative abundance in different human tissues, regulatory, evolutionary and structural aspects, as well as their possible cellular functions. In addition, we evaluated the contribution of DNA methylation, an important epigenetic mechanism that control the expression of protein coding genes, in the regulation of intronic lncRNAs expression. We found that a fraction of the intronic lncRNAs detected in the cell lines are evolutionarily conserved, show a tissue specific expression pattern, and is enriched in regulatory elements at their 5\' end region. Subsets of intronic lncRNAs possibly acting on genes associated to important regulatory pathways controlling organism development and cell cycle were identified. A smaller proportion of intronic lncRNAs relative to mRNAs displayed CpG islands (CGI) in the vicinity of the transcription start site. Notwithstanding, we observed that a subset of these transcripts responded to treatment with the DNA demethylation agent 5-AZA, demonstrating that intronic lncRNAs may be under transcriptional regulation mediated by DNA methylation. Among intronic lncRNAs regulated by DNA demethylation, stands out AS-APP lncRNA, which was up regulated 25 to 80 times in DU-145 and HEK293 cell lines following 5-AZA treatment, respectively,. This lncRNAs has a methylated CGI and an active promoter at 4-kb upstream from its known transcription start site. Increased AS-APP lncRNA transcription following DNA demethylation correlated with a significant decrease of APP gene messenger RNA levels. This finding suggests a possible cis-regulatory action of the lncRNA AS-APP in the APP locus, an important gene involved in Alzheimer disease and whose expression is associated with prognosis of different cancer types. The results obtained in this study reinforce the idea that intronic lncRNAs constitute independent transcriptional units under regulatory control in the different cell types. It was generated a catalog of intronic lncRNAs regulated by DNA methylation that will allow the selection of candidates with higher potential of functional relevance for detailed characterization

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-02T14:54:35Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO - Alinny R Isaac - 2016.pdf: 2520039 bytes, checksum: 294508e64fe5208cfd8d2b48f9465be5 (MD5) / Made available in DSpace on 2016-09-02T14:54:35Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO - Alinny R Isaac - 2016.pdf: 2520039 bytes, checksum: 294508e64fe5208cfd8d2b48f9465be5 (MD5) Previous issue date: 2016-02-16 / CNPq / Ácidos graxos essenciais da família ômega-3 são cruciais durante o desenvolvimento cerebral atuando na transcrição gênica e sobre mecanismos epigenéticos, influenciando a gênese e diferenciação de neurônios e astrócitos. Por outro lado, evidências têm mostrado que a desnutrição materno-proteica pode modificar negativamente padrões epigenéticos do genoma fetal. O presente estudo investigou se uma razão ômega-6/ômega-3 (n-6/n-3) favorável na dieta materna pode influenciar alterações pós-traducionais em histonas de células neurais da prole, mesmo em uma condição de deficiência em proteínas e outros micronutrientes. Ratas Wistar foram mantidas em uma dieta com deficiência multifatorial (Dieta Básica Regional – DBR) possuindo, entretanto, uma baixa razão n-6/n-3 em relação a dieta controle, 30 dias antes do acasalamento e durante a gestação. Embriões de 16 dias de gestação e neonatos de 0 a 3 dias pós-natais foram utilizados para a realização de culturas corticais de neurônios e astrócitos, respectivamente. Acetilação de H3K9 e dimetilação de H3K4 e H3K27 foram avaliados por imunocitoquímica e citometria de fluxo. Análises por densitometria ótica mostraram aumento nos níveis de H3K4me2 nos astrócitos do grupo malnutrido (Mn) (27115±9892 P < 0.005) em relação ao grupo nutrido (N) (22583±6194) e nenhuma alteração nos níveis de H3K9Ac (Mn: 27401±10248 vs. N: 30034±10423) ou H3K27me2 (Mn: 16693±4842 vs. N: 16187 ±3378). Tratamento das culturas com um inibidor de histona acetiltransferase (HAT), promoveu uma redução da acetilação de maneira dose dependente menor nos astrócitos do grupo malnutrido em relação ao nutrido. Através de análises por citometria de fluxo não observaram-se diferenças entre as grupos (H3K9Ac - Mn: 13217± 6821 vs. N:14620± 6515 / H3K4me2 - Mn: 22917± 14938 vs. N: 25956± 14258). Por outro lado, o tratamento dos astrócitos por 24 horas com 100μM de DHA promoveu um aumento de 60% na acetilação de H3K9 do grupo malnutrido. Análise de western blot para GFAP mostrou que ambos os grupos apresentam a expressão predominante da isoforma fosforilada desta proteína com 50kDa. Imunocitoquímica para H3K9Ac em neurônios mostrou um aumento nos níveis de fluorescência no grupo malnutrido (Mn: 30107±6789 vs. N: 25257±7562, P = 0.0075), e nenhuma diferença entre os grupos em relação à H3K4me2 (Mn: 23203±7059 vs. N: 22654± 5376). Não houve ativação do fator de transcrição gênica NK-kB nestas células. O perfil de ácidos graxos do leite materno no estômago dos neonatos foi avaliado por cromatografia gasosa, mostrando que a razão ácido linoleico/ácido alfa-linolênico da DBR é mantida. H3K9Ac e H3K4me2 estão envolvidos no aumento da transcrição de genes relacionados à gênese e diferenciação de astrócitos e neurônios. Nossos resultados indicam uma possível relação entre a razão favorável de n-6/n-3 presente na DBR sobre a promoção da transcrição gênica, mesmo em um contexto de baixa proteína. Além disso, sugere-se que o DHA esteja atuando sobre a acetilação, mantendo-a estável, em função da sua influência sobre as enzimas que atuam nesse processo. / Omega-3 essential fatty acids play key roles during brain development acting on gene transcription and epigenetic mechanisms influencing the genesis and differentiation of neurons and astrocytes. On the other hand, evidence has shown that maternal protein malnutrition may negatively change epigenetic patterns of the fetal genome. The present study investigated whether a low and favorable ômega-6/ômega-3 (n-6/n-3) ratio on maternal diet may induce histone post-translational changes in neural cells of the offspring, even in a condition of protein and micronutrients deficiency. Wistar rats were maintained on a diet with multifactorial deficiency (Regional Basic Diet-RBD) containing, however, a low n-6/n-3 ratio compared to the control diet, 30 days prior to mating and during gestation. Embryos of 16 days and newborns from 0 to 3 postnatal days were used to obtain cortical neuron and astrocyte primary cultures, respectively. Acetylation of H3K9 and dimethylation of H3K4 and H3K27 were evaluated by flow cytometry and immunocytochemistry. The results of optical density showed increased levels of H3K4me2 in astrocytes of the malnourished group (Mn) (27115 ± 9892; P <0.005) when compared to the nourished group (N) (22583 ± 6194). On the other hand, no change in the H3K9Ac (Mn: 27401 ± 10248 vs. N: 30034 ± 10423) or H3K27me2 (Mn: 16693 ± 4842 vs. N: 16187 ± 3378) were observed. Treatment of astrocyte cultures with an inhibitor of histone acetyltransferase (HAT), was able to decrease acetylation in a dose-dependent manner; however, this decay was less intense in astrocytes of the malnourished group compared to the nourished. Through analysis by flow cytometry no intergroup differences (H3K9Ac - Mn: 13217± 6821 vs. N: 14620 ± 6515 / H3K4me2 - Mn: 22917± 14938 vs. N: 25956± 14258). On the other hand, the treatment of astrocytes by 24 h with 100 μM DHA increased ~60% H3K9 acetylation levels of astrocytes in the malnourished group. Western blot analysis for GFAP showed in both groups the predominant expression of the phosphorylated isoform with 50 kDa. Immunocytochemistry for H3K9Ac and H3K4me2 in neurons indicated an increase in the levels of H3K9Ac fluorescence in the malnourished group (Mn: 30107 ± 6789 vs. N: 25257 ± 7562, P=0.0075) and no intergroup difference in the H3K4me2 (Mn: 23203 ± 7059 vs. N: 22654 ± 5376). No activation of the transcription factor NK-kB gene was found in these cells. The fatty acid profile of the breast milk in the stomach of newborns was evaluated by gas chromatography, showing that the linoleic/alpha-linolenic ratio present in the RBD is maintained. H3K9ac and H3K4me2 are involved in the increased transcription of genes related to the genesis and differentiation of astrocytes and neurons. In this way, the results obtained in this study points to a possible contribution of low n-6/n-3 present in the RBD on promotion of gene transcription, even in a context of low protein. In addition, it is suggested that DHA is acting on the acetylation, keeping it stable, probably due a potential influence on the enzymes involved in this process.

As bifenilas policloradas (PCBs) são um grupo de compostos hidrocarbonetos halogenados aromáticos, bioacumulativos em organismos vivos e persistente no ambiente. Além da atividade disruptora endócrina, os PCBs podem aumentar os níveis de espécies reativas de oxigênio (ROS), levando ao estresse oxidativo e alteração da metilação de DNA que são fatores importantes nas etiologias da hepatotoxicidade, infertilidade masculina e doença renal. Estes agentes tóxicos podem causar disfunção mitocondrial e distúrbios que afetam a produção de ATP, ROS e morte celular, ocasionando danos à saúde humana. O presente trabalho tem como objetivo investigar possíveis alterações genotóxicas e epigenéticas causadas pelo aroclor 1254 em fígado, rim e testículo, além de verificar a indução de estresse oxidativo e disrupção dos metabólitos intermediários do ciclo de Krebs nos referidos tecidos. Camundongos machos C57/BL6 foram expostos ao Aroclor 1254 em diferentes doses (5, 50, 500 e 1000 ug/kg) por gavagem, uma vez a cada três dias, durante 50 dias. Após a exposição, os animais foram eutanasiados, os órgãos coletados e espermatozoides obtidos a partir dos epidídimos. A peroxidação lipídica em plasma e tecidos foi avaliada pela quantificação de malonaldeído (MDA) por HPLC/DAD. Os níveis de intermediários da via glicolítica, do ciclo de Krebs, de alguns nucleotídeos e aminoácidos, marcas epigenéticas (5-mC e 5-hmC) e adutos de DNA (8-oxodG e CEdG) foram quantificados por HPLC-ESI-MS/MS. A abordagem de benchmark dose (BMD) foi utilizada para a modelagem dose resposta. Após exposição, não foram observadas diferenças significativas da variação da massa corporal, e a razão do peso testicular, fígado e rim por massa corporal. No tecido hepático, foi observado aumento da peroxidação lipídica. Houve redução significativa dos níveis de ATP, ADP, razão NADP+/NADPH, piruvato, malato, fumarato e glutamato. Observou-se redução significativa dos níveis de 5-mC e 5-hmC no DNA nuclear (nDNA), enquanto não foram observadas alterações dos níveis dos adutos. Em DNA mitocondrial (mtDNA) não foram observadas alterações nas marcas epigenéticas, no entanto foi obtido aumento significativo no aduto 8-oxodG após exposição ao Aroclor 1254. No tecido renal foi observado aumento significativo de MDA. Houve aumento significativo dos níveis de lactato e malato e reduções de ATP, ADP, glutamina, NAD+. Foi observada a hipohidroximetilação do mtDNA. As marcas 5-mC de mtDNA, 5mC de nDNA e adutos de DNA nuclear e mitocondrial não apresentaram diferenças após exposição a PCBs. Nos testículos foi verificada redução significativa dos níveis de glutamato, malato, succinato, fumarato e razão NADH/NAD+, hipohidroximetilção em mtDNA e hipermetilação em nDNA. Não foram observadas alterações de 5-mC em mtDNA e 5hmC em nDNA. Não foram verificadas alterações dos níveis de MDA e adutos em nDNA. Adicionando, foi observada redução dos níveis de 5-mC em DNA global de espermatozoide. Os limites inferiores do intervalo de confiança da BMD foram estimados para que estes marcadores possam ser usados na avaliação de riscos de PCBs. Os dados obtidos apontam o Aroclor 1254 como indutor de alterações do metabolismo intermediário, das marcas epigenéticas e estresse oxidativo. Essas alterações podem afetar vias celulares, levando à morte ou transformação, e aumentando o risco de doenças. / Polychlorinated biphenyls (PCBs) are a group of aromatic halogenated hydrocarbon compounds, which bioaccumulate in living organisms and is persistent in the environment. Besides their endocrine disrupting activity, PCBs may increase the levels of reactive oxygen species (ROS), leading to oxidative stress and alter DNA methylation that are important factors in the etiology of liver toxicity, male infertility, and kidney disease. These toxic agents can cause mitochondrial dysfunction and disorders that affect the production of ATP, ROS and cell death, thereby leading to health-related problems. The present work aimed at investigating possible genotoxic and epigenetic changes caused by aroclor 1254 in the liver, kidney and testis, as well as determine the induction of oxidative stress and disruption of intermediate metabolites in these tissues. Male C57/BL6 mice were exposed to Aroclor 1254 at different doses (5, 50, 500 and 1000 &#181;g/kg) by gavage, once every three days, for 50 days. After the exposure period, the animals were euthanized, organs collected, and sperms obtained from the epididymis. Lipid peroxidation in plasma and tissues was determined by quantification of malonaldehyde (MDA) using HPLC/DAD. The levels of intermediate metabolites, epigenetic marks (5-mC and 5-hmC) and DNA adducts (8-oxodG and CEdG) were quantified by HPLC-ESI-MS/MS. The Benchmark dose approach (BMD) was used for dose response modeling. No significant differences in body weight variation, testicular, liver and kidney weight to body weight ratio were observed after exposure. However, in hepatic tissues, an increase in lipid peroxidation was observed. There were significant decreases in the intermediate metabolites including the levels of ATP, ADP, pyruvate, NADP+/NADPH ratio, malate and fumarate, as well as glutamate. Significant reduction of 5-mC and 5-hmC levels in nuclear DNA (nDNA) were observed, whereas no changes were observed in DNA adducts. The epigenetic marks in mitochondrial DNA (mtDNA) were not changed; however, a significant increase was observed in 8-oxodG adduct after exposure to Aroclor 1254. In renal tissues, data showed a significant increase in MDA, while for the intermediate metabolites, the levels of lactate and malate were significantly elevated, whereas significant reductions were recorded for ATP, ADP, glutamine, and NAD+. Hypohydroxymethylation was observed in mtDNA. The 5-mC of mtDNA, 5mC of nDNA and nuclear and mtDNA adducts did not show differences after PCBs exposure. For the testicles, significant reductions in the levels of glutamate, malate, succinate, fumarate and NADH/NAD+ ratio were observed. The PCBs also induced hypohydroxymethylation in mtDNA and hypermethylation in nDNA, but there were no changes of 5-mC in mtDNA and 5-hmC in nDNA. A reduction of nDNA adducts 8-oxodG was observed. No changes were observed in the level of MDA and DNA adducts of nDNA. However, after PCBs exposure there was a significant decrease of 5-mC in global DNA of spermatozoa. The lower bound confidence interval on BMD, which were estimated for these markers can be used in the risk assessment of PCBs. Collectively, the data obtained in this study indicate that Aroclor 1254 induces alteration of intermediate metabolites, epigenetic marks and oxidative stress. These changes can adversely affect cells and cellular pathways, therefore increase the risk of cell death or transformation.