Orientador: Maria Luiza Silveira Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T03:24:19Z (GMT). No. of bitstreams: 1 Alvarenga_EleniceMonte_M.pdf: 2829254 bytes, checksum: 964b17aaf1c7f50d4471a7be569aaa6d (MD5) Previous issue date: 2011 / Resumo: A cromatina pode existir em núcleos interfásicos em dois estados distintos: como eucromatina e como heterocromatina, podendo ser esta constitutiva ou facultativa. Em células somáticas do final da fase ninfal dos hemípteros reduviídeos Triatoma infestans e Panstrongylus megistus há núcleos grandes, poliploides, nos quais a heterocromatina apresenta-se como corpos conspícuos (cromocentros), daí tais células apresentarem-se como um bom modelo para investigação de características morfológicas e funcionais da cromatina. Em estudos sobre a constituição cromatínica, a composição em bases do DNA é algo muito explorado, dado o conteúdo informativo dos achados. Já quando se objetiva a investigação da funcionalidade da cromatina, mais recentemente, tem-se feito uso da abordagem epigenética. Neste trabalho buscou-se investigar a composição em bases do DNA destas células, associando-a aos domínios cromatínicos aí existentes e também à presença das NORs. Por meio de colorações fluorescentes com Cromomicina A3 (CMA3)/Distamicina e 4',6-diamidino-2-fenilindol (DAPI)/Actinomicina D concluiuse que o DNA dos cromocentros de T. infestans e P. megistus são ricos em sequências AT e pobres em GC. Isto foi ainda confirmado por imunodetecção de 5-metilcitosina, que ocorreu somente na eucromatina, e tratamento de ninfas de T. infestans com 5-aza-2'-deoxicitidina (agente demetilante), seguido da análise dos fenótipos nucleares e análise de imagem, em que se observou expansão somente da área eucromática. Com o método de AgNOR evidenciou-se que a região rica em bases GC ao redor do cromocentro coincide com um acúmulo de proteínas argirofílicas, o que sugere associação com NORs. A presença de modificações epigenéticas nas caudas das histonas na cromatina destes insetos foi investigada por meio do uso de anticorpos contra marcadores epigenéticos específicos, permitindo identificar a participação diferencial dos mesmos na composição e na estrutura dos territórios heterocromáticos. Assim, observou-se hipoacetilação e hipermetilação de histonas na região do corpo heterocromático, o que indicaria uma possível ação da modificação de histonas na manutenção da estrutura heterocromática nas células somáticas de ambas as espécies de reduviídeos. Por meio da avaliação da ação de drogas inibidoras de deacetilases de histonas sobre a cromatina dos insetos percebeu-se que, quando ninfas de T. infestans e P. megistus foram tratadas com as drogas, houve aumento na frequência de necroses e, no caso específico do tratamento com tricostatina A (TSA) e butirato de sódio (NaBt), ocorreu descompactação da heterocromatina. Sugere-se que o tratamento com TSA e NaBt afete o processo de deacetilação de histonas, o qual seria, então, um fator importante na estruturação dos cromocentros. A observação da ocorrência de mudas e da sobrevivência de ninfas de T. infestans, realizada a fim de se avaliar a ação do ácido valproico (VPA) sobre o desenvolvimento dos insetos, mostrou que a droga, assim como a injeção de solução salina, reduziu seu período de sobrevivência, além de afetar a ocorrência de mudas / Abstract: Chromatin in interphase cell nuclei can be present in two distinct states: euchromatin and heterochromatin, which may be constitutive or facultative. In somatic cells at the end of the nymphal stage of Triatoma infestans and Panstrongylus megistus there are large nuclei, in which heterochromatin is presented as conspicuous bodies (chromocenters). These cells are an appropriate model for investigation of morphological and functional characteristics of the chromatin. In studies about chromatin constitution, the base DNA composition is explored due to the informational content of the findings. If the objective is to investigate the chromatin functionality, recently has been used the epigenetic approach. In the current study, the aim was to investigate the DNA base composition in these cells, associating with the chromatin domains therein and also the presence of NORs. Through fluorescent stains with Chromomycin A3 (CMA3)/Distamycin and 4',6-diamidino-2-fenilindole (DAPI)/Actinomycin D was found that the chromocenters DNA of T. infestans and P. megistus were AT-rich and GC-poor. This was also confirmed by immunodetection of 5-methylcytidine, which occurred only in the euchromatin, and by T. infestans nymphs treatment with 5-aza-2'- deoxycytidine (demethylating agent), followed by nuclear phenotypes analysis and image analysis, in which expansion was observed only in the euchromatic area. AgNOR test evidenced that the GC-rich region around the chromocenter coincides with an accumulation of argyrophilic proteins, suggesting association with NORs. Epigenetic modifications on histone tails in chromatin of these insects were investigated by using antibodies against specific epigenetic markers, in order to identify their differential participation in the composition and structure of these heterochromatic regions. It was observed hypoacetylation and hypermethylation in heterochromatic body area, suggesting a possible action of histones modification in the maintenance of the heterochromatic structure in somatic cells of both species of reduviids. Through evaluation of histones deacetylases inhibitors action on the chromatin, it was observed that when T. infestans and P. megistus nymphs were treated with these drugs there was an increase in the frequencies of necrosis, and in the specific case of Trichostatin A (TSA) and sodium butyrate (NaBt), occurred heterochromatin decondensation. It is suggested that treatments with TSA and NaBt could affect the histones deacetilation process, which would be an important factor in chromocenters structuring. Observations of the molting occurrence and survival of T. infestans nymphs, carried out in order to evaluate the action of valproic acid (VPA) on the development of insects, showed that this drug, as well as injection of saline, reduced the survival period, besides affecting the molting occurrence / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.