Os programas de melhoramento de figueira (Ficus carica L.) por métodos convencionais tais como cruzamentos dirigidos, para a obtenção de novos cultivares, são inviáveis em muitos países, como no Brasil, principalmente pela pequena variabilidade genética encontrada, e pela dificuldade de obtenção de plantas originadas pela fusão de gametas, uma vez que a vespa Blastophaga psenes, responsável pela polinização natural, não existe no país. Desse modo, o melhoramento genético, com o uso de mutagênicos, se torna uma linha de pesquisa importante para a melhoria da cultura, sendo necessário reunir informações sobre essa espécie, principalmente, em relação à sua variabilidade genética, para que projetos de propagação e manejo adequados sejam realizados. Diante do exposto, o objetivo do presente trabalho foi verificar a existência de variabilidade epigenética devido à metilação do DNA em seleções irradiadas de figueiras, entre si e quando comparadas ao principal cultivar comercial, Roxo-de-Valinhos, utilizando as técnicas de MSAP e ELISA, e posterior sequenciamento do DNA, tratado com bissulfito de sódio, para detecção do posicionamento das regiões polimórficas, analisado por ferramentas de bioinformática. Amostras do DNA genômico foram duplamente digeridas com a enzima HpaII (sensível à metilação) ou com o seu isoesquizomero MspI (não sensível à metilação), juntamente com a enzima EcoRI. Foram testadas 14 combinações de primers e obteve-se 87.9%, 10.1% e 2.0%, respectivamente, de regiões não metiladas CCGG, regiões metiladas CmCGG e regiões hemimetiladas hmCCGG, de um total de 553 produtos de amplificação, demostrando que a técnica MSAP é eficiente para detecção de sítios diferencialmente metilados no material genômico estudado, evidenciando sua divergência epigenética. Com o sequenciamento do DNA isolado desses sítios diferencialmente metilados, foi possível verificar diferentes padrões de metilação nos mesmos pelo sequenciamento dos DNAs tratados com bissulfito de sódio, em regiões codificadoras de genes regulatórios do desenvolvimento e amadurecimento dos frutos, além de terem sido encontrados no DNA mitocondrial dos tratamentos, o qual regula o fornecimento de energia em forma de ATP para as plantas, estando intimamente relacionado com o desenvolvimento das mesmas, justificando os diferentes fenótipos encontrados, tanto nos frutos, quanto no crescimento das plantas que sofreram estresse devido à exposição à radiação gama. Pela técnica de imunoquímica (ELISA), utilizando anticorpos anti 5-mC, foram observadas diferenças significativas pelo teste de Tukey, a 95 % de confiabilidade, no conteúdo global de metilação dos DNAs dos tratamentos, indicando que este fator abiótico foi responsável pelas alterações no epigenoma das plantas. Como o material utilizado como controle se encontrou também metilado, uma possível desmetilação dos materiais genômicos pode ser a responsável pela variação fenotípica entre os tratamentos. Diante disso, o estudo futuro da expressão gênica entre os tratamentos torna-se uma estratégia de extrema importância para o entendimento dos complexos sistemas regulatórios, levando à identificação de genes de interesse agronômico para a cultura da figueira, possibilitando a sua manipulação subsequente e propagação de cultivares melhorados para fins comerciais. / The fig tree (Ficus carica L.) breeding programs by conventional methods such as directed crosses, in order to obtain new cultivars, are unworkable in many countries, as in Brazil, mainly by small genetic variability found, and by the difficulty for obtaining plants originated from the fusion of gametes, since the wasp Blastophaga psenes, responsible for natural pollination, doesn`t exist in the country. In this way, the genetic breeding, with the use of mutagenic, becomes an important research line for the improvement of culture, being necessary to gather information about this species, mainly in relation to its genetic variability, for perform propagation projects and appropriate management. Given the above, The objective of this study was to verify the existence of epigenetic variability due to DNA methylation in irradiated fig selections, with each other and when compared to the main commercial cultivar, Roxo-de-Valinhos, using MSAP and ELISA techniques, and subsequent DNA sequencing, treated with sodium bisulfite, for detection of the position of the polymorphic regions, analyzed by bioinformatics tools. Samples of genomic DNA were double-digested with the HpaII enzyme (sensitive to methylation) with its isoschizomer MspI (insensitive to methylation), together with the EcoRI enzyme. Fourteen primer combinations were tested and it was obtained 87.9%, 10.1% e 2.0%, respectively, unmethylated CCGG, methylated CmCGG and hemimethylated regions hmCCGG, from a total of 553 amplification products, displaying, the MSAP technique, efficient for detection of differentially methylated sites in the genomic material studied, demonstrating their epigenetic divergence. With the sequencing of DNA isolated of these differentially methylated sites, it was possible to verify different patterns of methylation in them by sequencing the DNA treated with sodium bisulfite, in coding regions of regulatory genes of the development and fruits ripening, besides they have been found in the mitochondrial DNA of treatments, which regulates the supply of energy in ATP form for the plants, being closely related to their development, justifying the different phenotypes found in both fruits and plant growth that suffered stress due to exposure to gamma radiation. By the technique of immunochemistry (ELISA), using 5-mC antibodies, significant differences were observed by Tukey test, at 95% of trustworthiness, in the global content methylation of the treatments DNA, indicating that this abiotic factor was responsible for the changes in the epigenome of the plants. Since the material used as control was found also methylated, a supposed demethylation of the genomic material may be responsible for phenotypic variation among treatments. Considering this, future study of gene expression between treatments becomes an extreme important strategy for understanding the complex regulatory systems, leading to the identification of genes with agronomic interest for the fig culture, allowing its subsequent manipulation and propagation of improved cultivars for commercial purposes.

Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-10-05T15:40:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇAO NATALIA MOYSES.pdf: 1041310 bytes, checksum: de2100786e43db8c2d408683c6cadf90 (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-10-05T15:40:32Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇAO NATALIA MOYSES.pdf: 1041310 bytes, checksum: de2100786e43db8c2d408683c6cadf90 (MD5) / Made available in DSpace on 2017-10-05T15:40:32Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇAO NATALIA MOYSES.pdf: 1041310 bytes, checksum: de2100786e43db8c2d408683c6cadf90 (MD5) Previous issue date: 2011 / Universidade Federal Fluminense. Centro de Ciências Médicas. Instituto Biomédico / O silenciamento do gene pINK4a por hipermetilação tem sido sugerido como cofactor importante envolvido na carcinogênese cervical. O objetivo deste estudo foi investigar o padrão de metilação do gene pINK4a no epitélio cervical e avaliar uma possível associação com a infecção pelos papilomavírus humanos (HPV) e o genótipo viral. Nesse estudo transversal retrospectivo foram analisados 141 esfregaços cervicais, classificados pelo Sistema Bethesda como normal (28), lesão intraepitelial de baixo grau (35), lesão intraepitelial de alto grau (49) e câncer invasivo (29). A detecção e genotipagem de HPV foram feitas pela técnica da reação em cadeia da polimerase (PCR). A hipermetilação foi avaliada através de Nested-PCR metilação específica. Para analisar a associação entre presença de metilação e variáveis como: presença de HPV, genótipo viral, hábito de fumar e idade, foi feita análise multivariada por regressão logística. Mais de 60% dos casos apresentaram infecção por HPV e 44,6% apresentaram o gene pINK4a hipermetilado. Houve um aumento significativo da freqüência da metilação de acordo com o grau da lesão cervical (p<0,001). A análise multivariada mostrou associação entre a presença de HPV de alto risco e a metilação (p=0,01). Encontramos também correlação entre metilação e resultados da citologia classificados como lesão intraepitelial de alto grau (p=0.007) e câncer (p<0.0001). Nossos resultados indicam que a metilação do gene pINK4a pode contribuir para o surgimento de lesões pré-malignas e transformação neoplásica do epitélio cervical, juntamente com a infecção por HPV de alto risco / pINK4a gene silencing through hypermethylation have been suggested as a cofactor involved in cervical carcinogenesis. We aimed to investigate its methylation status in cervical epithelia and evaluate an association with HPV infection and genotype. This retrospective cross-sectional study was performed with 141 cervical exfoliated cell samples, classified through Bethesda System as Normal (28), low grade intraepithelial lesion (35), high grade intraepithelial lesion (49) and Invasive cancer (29). HPV detection and genotyping was performed through polymerase chain reaction (PCR). Hypermethylation was assessed with nested-methylation specific PCR. To evaluate an association between pINK4a methylation variables such as HPV infection, viral genotyping, tobacco exposure and age a multivariate analysis was performed. HPV positivity was detected in 62% of the samples and 44.6% showed pINK4a hypermethylation. An upward trend was observed according to lesion severity (p<0.001). Multivariate analysis showed an association between high-risk HPV infection and methylated pINK4a profile (p=0.01). We found a correlation between high grade intraepithelial lesion (p=0.007) and cancer (p<0.0001) cytology results and the presence of methylation. Our results point out that pINK4a methylation may contribute to the establishment of premalignant lesions and neoplastic transformation of cervical epithelium along with hr-HPV infection

L’héritage épigénétique transgénérationnelle est un phénomène très controversé, selon lequel un phénotype non-génétiquement déterminé peut être transmis à la génération suivante. Jusqu'à présent, ce mode de transmission a été décrit dans quelques cas et il a été suggéré que les composants de la chromatine peuvent être impliqués, y compris des protéines du groupe Polycomb, qui agissent comme des répresseurs de gènes clés du développement et coordonnent la différenciation cellulaire et la prolifération. Les mécanismes moléculaires à la base du rôle de la répression génique Polycomb-dépendante à hérédité épigénétique transgénérationnelle sont loin d'être compris. Par conséquent, j’ai développé un système expérimental chez Drosophila melanogaster pour induire un héritage épigénétique transgénérationnelle stable, dans lequel des états d'expression génique alternatifs peuvent être transmis en présence de la même séquence d'ADN. A partir de ces « épilignes » stables, j’ai pu disséquer certaines des propriétés génétiques des épiallèles induits, tels que leur héritage quantitatif et leur capacité à communiquer à longue distance. En outre, les épiallèles montrent une synergie dans leur expression et transmission héréditaire. L'une des signatures moléculaires des épiallèles est une différence de répression médiée par les complexes Polycomb et par leur marque d’histone caractéristique. Cette distribution différente est indépendante de l’activité transcriptionnelles des gènes en aval, au moins dans un stade de développement précoce, et pourrait influer l'organisation tridimensionnelle du locus impliqué. Curieusement Ago2, un composant de la voie ARNi, a été montré interagir avec les épiallèles génétiquement et la protéine Ago2 se fixe directement à leur chromatine, ce qui indique un rôle possible pour le ncRNAs dans l'expression des épiallèles et éventuellement dans leur transmission. Ces résultats plaident en faveur e l’existence d’une hérédité épigénétique transgénérationnelle stable chez les métazoaires et fournissent un modèle qui se prête à une dissection moléculaire de ce phénomène. / Transgenerational epigenetic inheritance is a hotly debated phenomenon whereby a non-genetically determined phenotype can be transmitted to the next generation. So far, this mode of inheritance has been described in few cases and it was suggested that chromatin components might be involved, including Polycomb group proteins, which act as repressors of key developmental genes and coordinate cell differentiation and proliferation. The molecular mechanisms linking Polycomb-mediated silencing to transgenerational epigenetic inheritance are far from being understood. Therefore, I developed an experimental system in Drosophila melanogaster to induce stable transgenerational epigenetic inheritance, in which alternative gene expression states can be transmitted in the presence of the same DNA sequence. Starting from these highly stable “epilines”, I could dissect some of the genetic properties of the induced epialleles, such as their quantitative inheritance and their ability to trans-communicate. Moreover, the epialleles displayed synergy in their expression and transmission. One of the molecular signatures of the epialleles is the differential presence of the Polycomb repressive complexes and their related epigenetic marks. This different distribution is independent of the transcriptional activity of the downstream genes, at least in an early developmental stage, and could influence the three-dimensional organization of the locus involved. Intriguingly Ago2, an RNAi pathway component, has been found to genetically interact with the epialleles and to be directly bound on their chromatin, indicating a possible role for the ncRNAs in the expression of the epialleles and possibly in their transmission. These results make a case for strong and stable transgenerational epigenetic inheritance in metazoan and provide a model that is amenable for the molecular dissection of this phenomenon.

Le cancer colorectal représente la deuxième cause de mortalité par cancer en France. Dans l’intestin, l’initiation et la progression tumorale sont corrélées à l’accumulation séquentielle de mutations génétiques et épigénétiques au niveau du compartiment de renouvellement de l’épithélium. Ces altérations ont pour conséquence une croissance incontrôlée de l’épithélium et, à terme, la formation de lésions cancéreuses. En tenant compte du nombre croissant de cas de cancers colorectaux, la découverte de facteurs de prédisposition de cette pathologie reste d’un intérêt majeur. Les données de séquençage du génome humain ne suffisant pas à expliquer la prévalence de la maladie à l’échelle de la population, nous nous intéressons aux mécanismes permettant le contrôle de l’expression de certains gènes : les mécanismes épigénétiques. Dans notre équipe nous disposons de modèles animaux génétiquement modifiés nous permettant d’étudier, dans des conditions proches de la pathologie humaine, les phases précoces de l’initiation tumorale. Ces souris, bien que génétiquement identiques, développeront pourtant un nombre de tumeurs intestinales très variable. En comparant les profils moléculaires de souris développant peu ou beaucoup de tumeurs intestinales, nous souhaitons mettre en évidence les facteurs épigénétiques mis en jeu pour expliquer cette différence de susceptibilité à la tumorigenèse. Pour ce faire, nous avons mis en place une stratégie visant à prélever un échantillon intestinal, par chirurgie, sur de jeunes souris qui ont ensuite été suivies jusqu’à l’âge adulte et à l’apparition des tumeurs. Cette stratégie innovante nous a permis de corréler les profils d'expression et de methylation d'ADN d’intestins développant peu ou beaucoup de tumeurs, ouvrant la possibilité de disposer de nouveaux marqueurs prédictifs quant aux chances de développer un cancer. Ces données sont complétées par une étude sur les conséquences de la perte du gène suppresseur de tumeur Apc, un gène couramment muté dans les cancers colorectaux humain. A l’aide de modèles de souris d’invalidation inductible, nous avons déterminé les conséquences de la perte d’Apc sur les profils de méthylation de l'ADN des cellules souches intestinales, et leur capacité à initier une tumeur. L’ensemble de ces différentes projets développés dans le cadre de ma thèse nous ont permis de mieux comprendre les mécanismes cellulaires liés à la prédisposition et à l’initiation tumorale et de proposer des nouvelles stratégies diagnostiques et d’évaluation du risque individuel. / Cancer initiation and progression represent the outcome of the progressive accumulation of genetic and epigenetic alterations. Global changes in the epigenome are now considered as a common hallmark of malignancies. However, most of our present knowledge represents the result of the comparison between fully established malignancies and their surrounding healthy tissue. Such comparison is not informative about the epigenetic contribution to the very early steps of cancer onset. By performing DNA methylation and gene expression profiling of the intestinal epithelium of relevant in vivo models we aim at shedding light on the correlation between the interindividual epigenetic polymorphisms within the population and the relative risk to develop malignancies, and establish the existence of a molecular signature associated with an increased susceptibility to develop intestinal cancer. Our results confirm that a considerable degree in the variability associated to cancer susceptibility cannot be ascribed to major genetic changes and that such heterogeneity seems to correlate with distinct molecular profiles associated to classes of poorly or highly susceptible isogenic animals.We also investigated in vivo the timing at which the remodeling occur at the epigenomic scale by analyzing the alterations in the DNA methylation and gene expression profiles of intestinal stem cells upon the loss of the Apc gene, the most common genetic lesion associated with human colorectal cancer initiation. We found that the loss of function of Apc in the Lgr5-positive intestinal stem cell compartment is rapidly accompanied by a reprogramming of the DNA methylation profiles resulting in altered gene expression and impaired fate determination in those cells. The results show that part of the phenotype resulting from the constitutive activation of the Wnt pathway upon Apc loss is acquired via differential epigenetic regulation of key biological processes controlling the balance between self-renewal and differentiation. By using conditional genetic ex vivo models we found part of these oncogenic effects to be reversible via the modulation of the machinery responsible for de novo methylation of the DNA.Overall, this work confirms that the epigenetic remodeling is an early event in tumorigenesis that might even precede actual cell transformation. The functional impact of our findings on cancer initiation is currently under investigation.

Mon travail a été d'étudier le rôle de l'expression de FZD9, un récepteur Wnt, dans l'hématopoïèse normale et maligne. Frizzled 9 (FZD9) est un composant clé de la voie de signalisation Wnt, une voie connue pour jouer un rôle important dans l'hématopoïèse normale et maligne. La méthylation d'un site CpG proximal de site d'initiation de la transcription du gène FZD9 est un facteur de mauvais pronostic dans les LAMs et sa déméthylation est un facteur prédictif de réponse aux thérapies épigénétiques. Sa fonction dans l'hématopoïèse n'est cependant pas connue. Au cours de ma thèse, j'ai démontré que le promoteur du gène FZD9 est dans un état non-permissif dans les cellules leucémiques. L'induction de la différenciation des cellules leucémiques restaure l'état permissif du promoteur, le recrutement du facteur E2F et de l'histone H3 acétyle permettant l'expression de FZD9. L'expression de FZD9 expression est également retrouvée au cours de la différenciation myéloïde d'une lignée IPS Dans les cellules de la lignée THP1 différenciées en monocytes, FZD9 est retrouvé dans un complexe comprenant LRP5/6 et Wnt5a. L'incubation des cellules différenciées par Wnt5a, un ligand de FZD9, déclenche la diminution de l'expression de -caténine et sa localisation nucléaire ainsi que l'expression des gènes cibles de la voie Wnt canonique (c-myc, Cyclin Dl and CD44). Nous n'avons détecté aucune augmentation des taux intracellulaires de calcium. Ces travaux suggèrent que la méthylation du promoteur de FZD9 dans les LAMs pourrait participer à la leucémogénèse en maintenant la voie b-caténine active / My goal was to study the importance of the expression of the FZD9, a Wnt receptor, in both normal and malignant hematopoiesis. Frizzled 9 (FZD9) is a key component of the Wnt signaling pathway, a pathway which has been shown to play a role in both normal and malignant hematopoiesis. Methylation of the CpG proximal to the transcription start site of the FZD9 gene is recognized as a prognostic factor in AML and its demethylation is a predictive factor for response to epigenetic therapy. Its function in hematopoiesis is however not known. The results show that the FZD9 promoter is in a non-permissive state in leukemic cells. Induction of myeloid differentiation in human myeloid leukemic cell fines restores FZD9 promoter permissiveness with recruitment of E2F and acetylated histone H3 and upregulation of FZD9 mRNA expression. FZD9 expression was progressively increased through the stages of myeloid differentiation in an IPS cell line. In differentiated monocytic THP1 cells, FZD9 was found in the LRPS/6 Wnt receptor complex. Incubation of differentiated cells with Wnt5a, a ligand of FZD9, triggered the decreased expression of - catenin and its nuclear localisation and the canonical Wnt-target genes (c-myc, Cyclin D1 and CD44). We detected no increase in calcium intracellular levels and thus activation of classical non-canonical pathway was not noted upon WntSa incubation. The results of my PhD suggest that the reported methylation of FZD9 promoter in AML and HR-MDS patients may participate in leukemogenesis by the maintenance of an activated Wnt canonical pathway.

L’huître du pacifique Crassostrea gigas est l'espèce marine la plus cultivée avec une production supérieure à 4 millions de tonnes pour l'année 2010. En France, C. gigas est cultivée depuis la fin des années 1970. Cependant, cette espèce souffre d’un syndrome de mortalité estivale depuis les années 1980, avec une amplification depuis 2008 qui touche jusqu'à 100 % des naissains. Ce syndrome de mortalité est un phénomène multifactoriel, basé sur l’interaction de nombreux facteurs: stress environnementaux, caractéristiques physiologiques et génétiques de l’huître, présence et virulence de pathogènes. L’huître du pacifique C. gigas est une espèce estuarienne qui est soumise aux pressions anthropiques comme la pollution du milieu côtier. Ces événements représentent des sources potentielles de stress en zones ostréicoles. Cependant, les connaissances sur les effets des polluants comme les pesticides sur C. gigas restent fragmentaires. Les périodes d’épandage d’herbicides coïncident parfois avec la période de reproduction des huîtres, raison pour laquelle nous considérons que ces produits chimiques pourraient affecter la génération suivante d'huîtres. Parmi les pesticides, le diuron est le plus fréquemment détecté sur les côtes françaises, avec une concentration maximale rapportée de 0,78 µgL-1. L'exposition directe aux herbicides affecte le transcriptome des huîtres qui est le premier niveau de réponse face à l'exposition du polluant. Il a été démontré que l'exposition parentale au diuron a des effets génotoxiques chez C. gigas au stade de naissain. Une variabilité phénotypique de trait d’ histoire de vie a été observée aussi pour ces naissains. Un autre effet possible des pesticides serait la modification de marques épigénétiques. Il est connu que les facteurs environnementaux telle que la pollution par des composés chimiques peuvent modifier l'épigénome et par conséquent le phénotype des individus et de leurs descendance en agissant au niveau trans-générationnel. Ces dernières observations nous permettent d’émettre l’hypothèse de l’implication de mécanismes épigénétiques suite à l’interaction avec des produits phytosanitaires. Ces mécanismes modifieraient le phénotype des huîtres au stade de naissains par l'exposition parental. Pour tester cette hypothèse nous avons étudié la méthylation globale de l'ADN (méthylome), qui est un de principal marques épigénétiques, et le transcriptome des naissains issus de géniteurs exposé au Diuron. Nous avons identifié des modifications du méthylome et du transcriptome qui ont un lien avec le phénotype de trait d'histoire de vie de ces naissains. Ces résultats démontreraient qu’une exposition indirecte ou parentale du diuron modifie la méthylation et l'expression de fonctions de gènes spécifiques, expliquant en partie la variabilité phénotypique observée. / The Pacific Oyster Crassostrea gigas is the most cultivated marine species in the world with a production superior to 4 millions of tons in 2010. In France, C. gigas is cultivated since the end of 1970s. However, this specie suffers from a syndrome of summer mortalities since the 1980s, with an amplification since 2008 affecting up to 100% of spats. This syndrome of mortality is a multifactorial phenomenon, based on the interaction of many factors: Environmental factors, genetic and physiologic features of the oysters, and the presence and virulence of pathogens. The Pacific Oyster C. gigas is an estuarine specie which is subjected to anthropogenic pressures such as pollution of the coastal environment. These events represent a potencial source of stress in oyster farm areas. However, the knowledge about the effects of pollutants such as pesticides on C. gigas remain fragmented. The herbicide application periods may coincide with the oyster breeding period, reason for which we consider that these chemicals could affect the next generation of oysters. Among pesticides, diuron is the most frequently detected on the French coast, with a maximum reported concentration of 0.78 µgL-1.The direct exposure to herbicides affects the transcriptome of oysters which is the first level of response to the exposure of pollutants. It was shown that parental exposure to diuron has genotoxic effects on C. gigas at the spat stage. A phenotypic variability of life history traits has also been observed for these spats. Another possible effect of pesticides would be the modification of epigenetic marks. It is known that environmental factors such as pollution by chemical compounds can alter the epigenome and consequently the phenotype of individuals and of their offspring acting at a transgenerational level. These last observations allow us to hypothesize the involvement of epigenetic mechanisms in response to interactions with herbicide products. These mechanisms could modify the phenotype of oysters spat state by parental exposure. To test this hypothesis we studied the genome-wide DNA methylation (methylome), which is a main epigenetic mark, and the transcriptome of the spat from diuron-exposed genitors. We identified methylome and transcriptome changes that are related to the phenotype of life history trait of these spats. These results show that an indirect or parental exposure to the diuron is able to modify the methylation and the expression of specific gene functions, partially explaining the phenotypic variability observed.

Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by progressive amyloid plaque aggregation, neurofibrillary tangles, and cortical tissue death. As the prevalence of AD is projected to climb in coming years, there is a vested interest in identifying endophenotypes by which to improve diagnostics and direct clinical interventions. The risk for complex disorders, such as AD, is influenced by multiple genetic, environmental, and lifestyle factors. Significant strides have been made in identifying genetic variants linked to AD through the genome-wide association study (GWAS). It has been estimated in more recent years, however, that GWAS-identified variants account for limited AD heritability, suggesting the role of non-sequence genetic mechanisms, such as epigenetic moderators. By influencing gene expression, epigenetic markers have been linked to age- associated decline through modulation of chromatin architecture and global genome instability, though such mechanisms are also involved with a number of normal biological processes, including neurogenesis. As the strategies of clinical genetics shift to include a heavier focus on epigenetic contributors, altered adult neurogenesis presents itself as a strong candidate for an endophenotype of AD development. This thesis proposes that, due to neuropathological dysfunction of epigenetic mechanisms in AD, new generations of neurons fail to proliferate, differentiate, and mature correctly, resulting in the larger loss of neurons and cognitive deficits characteristic to neurodegenerative disease. The plasticity of the epigenome and the role of epigenetic factors as mediators of the genome and the environment make such alterations attractive in AD research and implies the potential for therapeutic interventions. The present review submits neurogenesis as a viable target of epigenetic research in AD, highlights shared loci between neurogenesis and AD in the epigenome, and considers the promises and limitations of the neurogenic endophenotype.

Epigenetics is the study of heritable changes in phenotypes that are not the result of changes in DNA sequence. Examples of epigenetic affecters include methylation changes, chromatin modifications, transcription factors, and RNA-based changes. The molecular mechanisms behind epigenetic changes are not fully understood. Canalization is the buffering of gene expression against environmental changes over time, while habituation is semi-stable expression change over time due to selection. This work characterized the molecular changes associated with the kernel color changes of the R-sc:86-17pale allele at the maize red color1 (r1) locus to determine if the changes are epigenetic in nature. The research; 1) quantified the color differences between the progenitor and habituated sublines; 2) Determined that there are not sequence differences between the progenitor and habituated sublines at the 3` end of the Sc||nc1 gene that could account for changes in seed color; 3) and examined the cytosine methylation patterns at the 3` end of the Sc||nc1 gene of the habituated sublines and the progenitor to determine whether there are methylation differences that correspond with the kernel color changes. Quantification of the kernel colors of the R-sc:86-17pale selection sublines showed that there was a statistically significant difference in kernel color. The identical sequence of the R-sc:86 line and the R-sc:86-17pale Lightest and R-sc:86-17pale Darkest sublines at the 3` end of the Sc||nc1 gene is evidence that the kernel color change is not driven by differences in sequence within the r1 gene. The methylation data suggests that some methylation differences in the R-sc:86-17pale Lightest and R-sc:86-17pale Darkest sublines are present, and suggests that the molecular basis of the kernel color is epigenetic in nature.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the pathognomonic genomic alteration in 85% of the Ewing Family of Tumors (EWFT) a malignancy of the bone and the surrounding tissue, predominantly affecting children and adolescents. This translocation results in the formation of a chimeric oncoprotein which acts as an aberrant transcription factor that is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. The EWSR1-FLI1 translocation induces a variety of changes including dysregulation of the epigenome and altered gene expression to drive tumorigenesis, and consequently contributes to the hypersensitivity of EWFT to several classes of chemotherapeutics. We sought to exploit these intrinsic sensitivities by employing a matched pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenografts (PDX) collected at the time of relapse or autopsy, which led us to the development of two novel combination targeted therapies for EWFT. In our matched pair of EWFT cell lines, we found sensitivity to the Poly(ADP-ribose Polymerase (PARP) inhibitor olaparib was diminished following chemotherapy, despite a predicted sensitivity. In addition, we discovered increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone is insufficient to sensitize EWFT cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL (BCL2L1) in EWFT survival. These data reveal BCL-2 and BCL-XL act together to drive olaparib mediated apoptotic resistance in Ewing sarcoma and identify a novel, rational combination therapy using olaparib and the BCL-2/BCL-XL inhibitor navitoclax. In addition, using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to GSK-J4 (GlaxoSmithKline), an inhibitor of lysine 27 of histone 3 (H3K27) demethylases: ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji D3 (JMJD3). Treatment with GSK-J4 leads to a decrease in H3K27 acetylation (H3K27ac) and ultimately, the silencing of EWS-FLI1 gene targets. We sought to sensitize GSK-J4-mediated inhibition of EWS-FLI1 targets by blocking RNA polymerase II activity using the Cyclin Dependent Kinase 7 (CDK7) inhibitor THZ1. By targeting CDK7-mediated transcription we were able to sensitize EWFTs to H3K27 demethylase inhibition. We therefore propose co-targeting of H3K27 demethylases and CDK7 acts as a surrogate EWS-FLI1 inhibitor. Given the difficulties targeting EWS-FLI1, these strategies may present viable clinical therapies.

acase@tulane.edu