Delicate epigenetic modifications are essential for production of spermatids during spermatogenesis. DNA methyltransferase 3 (DNMT3), the enzymes involved in adding a methyl group to unmodified DNA, contains three members: DNMT3A, DNMT3B and DNMT3L. The latter lacks methyltransferase activity, but was closely associated with spermatogenesis in many reports. According to the presentation of mature spermatids in testis, azoospermia could be separated into obstructive and non-obstructive categories. Non-obstructive azoospermia is spermatogenesis defective, germ cells absent in seminiferous tube is the most serious type. Therefore, we would like to find out if there are differential expression of DNMT3 family transcripts in testes of azoospermia patients from infertility clinic. Using RT-PCR and qPCR, we found only 5 (29.4%) expressed DNMT3L in 17 non-obstructive patients, whereas all 20 obstructive patients expressed. Both groups were similar in expression levels of DNMT3A and DNMT3B. Nuclei of spermatogonia and spermatocyte were the main immunohisto-chemical localization of DNMT3L protein. Lost of germ cells should be the cause of undetectable DNMT3L expression in azoospermia patients. By this founding, it could serve as an indicator for ability of male germ cell culture in further applications of assisted reproduction.

Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores. / Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores. / Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Chromatin remodelling proteins support a variety of cellular functions and utilise the energy from ATP hydrolysis to either reposition or evict nucleosomes. One such protein, Lymphoid specific helicase (LSH), regulates DNA methylation in mammalian cells cooperatively with DNA Methyltransferase 3B (DNMT3B) through binding of the N-terminal domain of LSH. The correct functioning of LSH is essential for heterochromatin formation, with a knockout of LSH causing perinatal lethality or severe developmental abnormalities. There is little biochemical data and no structural data on LSH. Therefore, we aim to determine the structural characteristics and regulatory mechanism of LSH in vitro. LSH was expressed in an optimised insect cell system which increased protein yield 25-fold with greater than 95% purity. LSH is monomeric with increased thermal stability upon ATP or ADP binding. Full length LSH could not be crystallised therefore a core ATPase region of LSH missing the N-terminal domain was identified through limited proteolysis. This also provided evidence the N-terminal domain of LSH is disordered, which was proven through biophysical characterisation of LSH1-176. Expression of the LSH ATPase region was weak and the protein was unstable; suggesting the N-terminal domain of LSH is required for LSH stability. Therefore, complementary structural methods were used to study LSH. Crosslinking mass-spectrometry revealed the N and C termini are in close proximity, suggesting flexible linking regions, which was supported by limited proteolysis experiments. Negative staining Electron Microscopy defined LSH as a tri-lobal and elongated structure which could harbour the ATPase region in the two spherical lobes. 3D modelling of SAXS data obtained of LSH was in agreement with EM data. To understand molecular mechanisms of LSH, functional studies investigating LSH:DNA and LSH:DNMT3B interactions were performed. LSH had a KD for dsDNA of 0.4 μM in solution. LSH does not bind ssDNA nor does it have a greater affinity for methylated dsDNA. LSH was found to bind the dsDNA overhangs of nucleosomes but not to core nucleosomes, suggesting LSH solely interacts with DNA in chromatin and not histones. A stable complex of LSH:DNMT3B could not be achieved in vitro, however, other components for complex formation may have been missing. This study has improved our understanding of LSH structure, biophysical properties and its biochemical interaction with DNA and nucleosomes. This study has laid the foundations for the structural investigations of a LSH:nucleosome and potentially a LSH:DNMT3B complex in vitro to gain a greater understanding of how functional domains of LSH regulates its enzymatic function.

Die hydrolysestabile C-C-Bindung von Nukleosiden, deren Nukleobase über ein aromatisches oder methylen-verbrücktes Kohlenstoffatom an Ribose oder 2-Desoxyribose gebunden ist, ermöglicht die Synthese von neuartigen Strukturen und Eigenschaften, die bei N-Nukleosiden nicht stabil oder nicht gegeben wären. In dieser Arbeit wurde die die Cuprat-vermittelte Glycosylierung und die Friedel-Crafts-Alkylierung als Methoden zur Darstellung von Desoxyribose-basierenden C-Nukleosiden weiterentwickelt. Die Cuprat-vermittelte Glycosilierung ermöglichte die Synthese von C-Nukleosiden in bis zu 93% Ausbeute, wenn Grignard-basierende Normant-Cuprate verwendet wurden. Die Verwendung Organolithium-basierender Gilman-Cuprate war ebenfalls möglich. In Gegenwart von Sauerstoff wurden O-Glycoside isoliert in über 80 % Ausbeute isoliert. Mit den C-Nukleosiden wurden modifizierte Oligonukleotide, die als potentiell verbesserte Binder an M.TaqI und E.coli Dam dienen, dargestellt. Nach ihrer Charakterisierung über Schmelzwerte und Fluoreszenzeigenschaften wurde diese an die Arbeitsgruppe von Prof. Elmar Weinhold weitergereicht und dort erfolgreich als optimierte Binder an an M.TaqI und E.coli Dam getestet. Oligonukleotide, die ein oder mehrere 1,1-Binaphthyl-Chromophore als einen neuen Typus eines torsionsflexiblen Farbstoffes enthalten, wurden untersucht. Die Einführung mehrerer aufeinander folgender Binaphthyle führte zu einer thermodynamischen Stabilisierung von Duplex-Oligonukleotiden. Die geringe Neigung Binaphthyls zur Selbstlöschung bewirkte dabei einen starken Anstieg der Fluoreszenz. / The stable C-C-bond of ncleosides, whose nucleobase is attached to the ribose or 2-deoxyribose via an aromatic or methylen-bridged carbon atom, is stable to hydrolyses. This allows the synthesis of new structures and properties, which would not be available in N-nucleosides. In this work, a cuprate-mediated glycosilation and the Friedel-Crafts-alkylation as methods for the preparation of doxyribose-based C-nucleosides were developed. The cuprate-mediated glycosilation allowed the synthesis of C-nucleosides in up to 93 % yield, when Grignard-based Normant-Cuprates were used. The use of Organolithium-based Gilman-Cuprates was also possible. In the presence of oxygen O-glycosides were isolated in over 80 % yield. With the C-nucleosides modified oligonucleotides, which serve as potentially improved binders of the DNA-methyltransferases M.TaqI und E.coli, were prepared. After their charakterisation via melting point measurements and fluorescence properties, the oligonucleotides were given to the working group of Prof. Elmar Weinhold and successfully tested as improved binders of the DNA-methyltransferases M.TaqI und E.coli. Oligonucleotides, which contain one or multiple 1,1-binaphthyles as a new type of a torsionally flexible chromophore, were charakterised. The incorporation of several successive binaphthyls led to a thermodynamical stabilisation of the duplex-oligonucleotides. The low tendency of the Binaphthyl for self-quenching caused a remarkable increase of the fluorescence.

Die DNA Methyltransferasen sind verantwortlich für die spezifische Methylierung von DNA-Basen. Mehrere DNA Methyltransferasen sind bekannt, wobei die Dnmt1 das hauptsächlich vorkommende Enzym ist. Bei Säugetieren korreliert die DNA-Methylierung mit der Genaktivität und ist essentiell für die Embryonalentwicklung. Eine beeinträchtigte Funktion oder Verfügbarkeit des Enzyms kann zu pathologisch veränderten Zuständen führen. Die Regulation der Dnmt1 und die damit verbundene Bedeutung bei der Entstehung von Krankheiten ist bisher nur unvollständig untersucht. In der Frühphase der Embryonalentwicklung von Säugetieren ändert sich das Methylierungsmuster des Genoms dramatisch. In zeitlich aufeinander folgenden Phasen wird die DNA demethyliert (Verlust der Methylgruppen) und neu methyliert (De-Novo Methylierung). Die Hypothese dieser Arbeit ist, dass verschiedene Isoformen der Dnmt1 in spezifischen Entwicklungsstadien exprimiert werden und zu Veränderungen des Methylierungsmusters der DNA beitragen. Um diese Regulation zu untersuchen, wurde die Struktur der Maus Dnmt1-Gens bestimmt. Außerdem wurde in verschiedenen Gewebetypen die Transkriptionsgröße und die Transkriptionsintensität der mRNA mit Hilfe von Northern-Blots quantifiziert. Mit diesen Experimenten konnte im Hoden- und Skelettmuskelgewebe ein längeres Dnmt1-Transkript als in anderen Geweben identifiziert werden. Dieses neue Dnmt1-Transkript wurde mit Hilfe von RT-PCR und RACE-Techniken kloniert und ist in beiden Geweben identisch. Es unterscheidet sich auf DNA-Ebene in der Sequenz des 5'-Endes von der bisher bekannten Form der Dnmt1 und besitzt einen anderen Startpunkt für die Transkription. Darüber hinaus besitzt das neue Dnmt1-Transkript ein 800 Basenpaar großes erstes Exon, welches sich von dem des bekannten Dnmt1-Transkripts unterscheidet. Die spezifische zelluläre Lokalisation des neuen Transkripts wurde mit Hilfe der In-Situ-Hybridisierung analysiert. Mit dieser Technik wurde das alternative Transkript in stärker spezialisierten, haploiden spermatogenen Zellen (Spermatiden) und zu einem geringen Maß im Skelettmuskel nachgewiesen. Während der Differenzierung von Muskelzellen wurde eine verminderte Expression des bereits bekannten mRNA-Transkripts und eine verstärkte Expression des neu identifizierten mRNA-Transkripts festgestellt. Obwohl die mRNA der alternativen Isoform verschiedene, kurze offene Leserahmen enthält, welche die Translation eines spezifischen Dnmt1 Proteins verhindern könnten, wurde durch Immunofluoreszenz- und Western-Blot Analysen ein Translationsprodukt nachgewiesen. Nach den hier aufgezeigten Ergebnissen werden alternative Dnmt1 Isoformen in vivo exprimiert, welche eine aktive Rolle bei der Regulation der DNA-Methylierung spielen könnten. / DNA methyltransferases (DNA MTases) are enzymes responsible for DNA methylation (transfer of methyl groups to a base in the DNA) and are vital for the development of mammals. Several MTases have been identified in eukaryotes but the most abundant is Dnmt1. Furthermore, many pathological conditions are often attributed to an altered availability or function of this enzyme, however the understanding of the regulation of Dnmt1 and the concomitant relationship to diseases is far from being complete. In mammals the methylation of DNA correlates with gene activity, and methylation patterns change dramatically during early development when the genome of the mammalian embryo undergoes consecutive waves of demethylation (loss of methylation) and de novo methylation (methylation of DNA sites that have not been previously methylated). The hypothesis of this study was that alternative Dnmt1 isoforms are expressed at specific developmental stages and thus contribute to changes in the DNA methylation pattern. To study this regulation the structure of the Dnmt1 gene was determined. In this work, the tissue distribution and abundance of Dnmt1 mRNA was analyzed by Northern blot and a new, longer transcript was identified that is present in testis and skeletal muscle tissue. The novel isoform was cloned by a combination of RT-PCR and RACE techniques and found to be identical in both tissues. This new isoform differs from the ubiquitous cDNA in the 5' end, utilizing a new transcriptional start site and an 800 bp long alternative first exon. The cellular localization of this new transcript was determined by in situ hybridization and found to be present in the more specialized haploid spermatogenic cells, spermatids and at lower level in skeletal muscle. During muscle differentiation, the ubiquitous isoform is downregulated while the alternative isoform is upregulated. Although this mRNA codes for several short upstream ORFs which could prevent translation of the Dnmt1-specific ORF, it was found by immunofluorescence and Western blot analyses that this transcript can be translated in vivo producing a shorter Dnmt1 protein. The results shown here indicate that alternative Dnmt1 isoforms are expressed in vivo and might play an active role in the regulation of DNA methylation.

Ao longo dos últimos anos observou-se um aumento no número de idosos no mundo, com isso faz-se necessário buscar terapias que amenizem os danos relacionados e também elucidar os mecanismos envolvidos neste processo. O exercício físico tem sido sugerido como uma ferramenta importante, não farmacológica, para atenuar os déficits relacionados à idade. Ainda, estudos recentes sugerem uma relação entre o processo de envelhecimento cerebral e o desequilíbrio de mecanismos epigenéticos, contudo, estes dados ainda não são conclusivos. Sabe-se que o grau de neuroplasticidade varia com a idade e que as estruturas encefálicas podem responder diferentemente à exposição ao exercício. Estudos demonstram que o córtex pré-frontal está envolvido em funções de alta ordem como atenção, tomada de decisão e memória de trabalho. Portanto, o objetivo deste trabalho foi avaliar os efeitos de diferentes protocolos de exercício físico (sessão única e exercício diário moderado) sobre a modulação de marcadores epigenéticos em córtex pré-frontal de ratos Wistar de 3 e 21 meses de idade. Os animais foram submetidos ao protocolo de sessão única (20 minutos) ou o exercício diário moderado (20 minutos durante 14 dias), 1 hora após a última sessão foram eutanasiados. O córtex pré-frontal foi dissecado e a acetilação da H4, o conteúdo da DNA metiltransferase (DNMT1 e DNMT3b), assim como a atividade da histona metiltransferase H3K27 foram analisadas. Os resultados serão apresentados na versão completa desta dissertação. / Over the past few years the number of elderly people has increased in the world, therefore it is necessary to search therapies that ameliorate age-related deficits as well as elucidate the mechanisms involved in this process. Physical exercise has been suggested as an important non-pharmacological approach to alleviate the age-related decline. Furthermore, recent studies have suggested a relationship between the process of brain aging and imbalance of epigenetic mechanisms, however, these data are not conclusive. It is well described that prefrontal cortex is involved in higher functions like attention, decision making and working memory. Then, the aim of this study was to investigate the effects of two exercise protocols (single session and daily moderate exercise) on the modulation of epigenetic markers in the prefrontal cortex from Wistar rats of 3- and 21- months-old. Animals were submitted to single session protocol (20 minutes) or the daily moderate exercise (20 minutes for 14 days), and 1hour after the last exercise session animals were euthanized. Prefrontal cortex was dissected out and acetylation of H4, the content of DNA methyl transferase (DNMT1 and DNMT3B), as well as histone methyltransferase H3K27 activity were analyzed. Results will be presented in the full version.

Les méthyltransférases de l'ADN (DNMT) catalysent la méthylation de l'ADN, l'une des marques épigénétiques les plus étudiées. Dans les cancers, on observe une hyperméthylation spécifique de promoteurs de gènes suppresseurs de tumeurs (GST) conduisant à leur extinction génique, ce qui participe au maintien et à la progression de tumeurs. A ce jour, les mécanismes responsables de cette hyperméthylation spécifique des promoteurs de GST dans les cancers sont indéterminés. Ces travaux de thèse sont consacrés à l'inhibition des DNMT dans les cancers afin de restaurer l'expression des GST mais également à l'utilisation d'une approche innovante de chemobiologie pour l'identification de partenaires des DNMT potentiellement responsables de leur adressage vers les régions promotrices des GST. Les partenaires ainsi identifiés peuvent constituer de nouvelles cibles épigénétiques pour le ciblage indirect de la méthylation de l'ADN dans les cancers. Deux séries d'inhibiteurs de DNMT ont été étudiées. La première est la famille des chloronitro-flavanones, précédemment identifiée par criblage, pour laquelle de nouveaux dérivés de type bromonitro-flavanones ont été synthétisés afin d'améliorer la stabilité en conditions physiologiques. J'ai réalisé l'étude des effets pharmacologiques de cette famille de molécules. J'ai également entrepris la synthèse et la caractérisation pharmacologique de nouveaux inhibiteurs de type bi-substrats, analogues de l'adénosine et de la désoxycytidine, conçus par une approche rationnelle. Ces deux études ont permis respectivement d'identifier un dérivé flavanone plus stable et plus actif que le composé de référence et deux dérivés quinazoline-quinoléine très prometteurs, actifs sur les DNMT et dans les lignées cellulaires, à la fois pour la réexpression d'un gène rapporteur mais surtout dans l'induction de la déméthylation du GST CDKN2A et de sa réexpression. Pour identifier les partenaires de DNMT, nous avons employé une approche de chemobiologie (" Activity-Based Protein Profiling - ABPP ") basée sur la conception de sondes chimiques comportant un inhibiteur de DNMT. Ces sondes, utilisées sur des cellules vivantes, permettent, grâce à une étape de fonctionnalisation par chimie bioorthogonale, de purifier les protéines partenaires des DNMT. Vingt sondes ont été synthétisées et leurs activités ont été évaluées sur des modèles enzymatiques et cellulaires. Les sondes sélectionnées ont été utilisées dans des lignées cellulaires cancéreuses pour purifier les protéines partenaires qui ont ensuite été identifiées par analyse protéomique. Suite à leur validation, ces protéines pourront constituer de nouvelles cibles de la méthylation aberrante de l'ADN dans les cancers. / DNA methyltransferases (DNMTs) catalyse DNA methylation, one of the most studied epigenetic marks. In cancers, a specific hypermethylation of the promoters of the tumour suppressor genes (TSGs) is observed, which leads to their silencing. This abnormal DNA methylation pattern participates to the maintenance and the progression of the tumour. Today, the mechanisms that direct this specific hypermethylation of TSG promoters and their transcriptional repression in cancers are still unknown. The aim of my PhD is to identify DNMT inhibitors that are able to reactivated TSGs in cancer cells but also to identify the DNMT partners that address specifically these enzymes to TSG promoter regions. Such partners can constitute new anticancer "epitargets" to indirectly target DNA methylation specifically in cancer cells. Two families of DNMT inhibitors were studied. The first one starts from the chloronitro-flavanones previously identified by screening. New derivatives including bromonitro-flavanones were synthesised aiming at improving compound stability. I pharmacologically characterised these compounds and show for one of them an increased stability and activities compared to reference compound. In parallel, I synthesised and pharmacologically characterised new bi-substrate analogue inhibitors, mimicking the adenosine and the deoxycytidine. Two very promising quinazoline-quinoline derivatives were identified. They are active against DNMT and in cell lines, both for reexpression of a reporter gene but mostly in CDKN2A TSG demethylation inducing its reexpression. To identify DNMT partners we adopted a chemical biology approach (Activity-Based Protein Profiling (ABPP)) based on the use of chemical probes including in-house non- nucleoside DNMT inhibitors as bait to trap the DNMT partners. We designed and synthesised twenty chemical probes and evaluate them using enzymatic and cellular-based assays. Selected probes were used to carry out ABPP directly in living cells. After functionalization by bioorthogonal chemistry, DNMT protein partners were purified and identified by proteomic analysis. Target validation would enable to determine new targets for the aberran

Myeloid Derived Suppressor Cells (MDSC) represent a significant hurdle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous studies have reported on the myelo-depletive effects of certain chemotherapies. Using guadecitabine, a next-generation DNA methyltransferase inhibitor (DNMTi), we observed significantly reduced tumor burden in the 4T1 murine mammary carcinoma model. Guadecitabine treatment prevents excessive tumor-induced myeloid proliferation and systemic accumulation, and skews remaining MDSCs toward a beneficial antigen-presenting phenotype. Together, this alters the splenic environment to improve T cell activation and interferon-gamma (IFNg) production. Additionally, guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. Based on these findings, the immune-modulatory effects of guadecitabine can help rescue the anti-tumor immune response and could contribute to the overall effectiveness of current cancer immunotherapies. Allergies and asthma are common ailments that are on the rise around the world. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. In studies using ADAM17-deficient mast cells, percent degranulation and cytokines released by IgE-mediated activation were significantly reduced. Interestingly, ionomycin-activation was unchanged, suggesting ADAM17 may be involved in IgE-mediated mast cell activation upstream of calcium release. Additionally, ADAM17MC-/- mice showed protection from IgE-, but not histamine-, mediated passive systemic anaphylaxis (PSA). The underlying mechanism behind the reduced degranulation occurs through signaling deficiencies downstream of Lyn phosphorylation. Together, the data suggest that ADAM17 is required for proper mast cell signaling through its interaction with the Src family kinase, Lyn.

Embryos derived by somatic cell nuclear transfer (SCNT) produce few pregnancies that result in a live, healthy offspring. This has largely been attributed to the aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve the efficiency of cloning there is a great deal of research needed to determine the role of proteins involved in early embryonic reprogramming. In addition, studies are needed to determine effects on somatic and embryonic cell development as a result of altering these proteins. In this study we investigate the use of RNA interference in bovine somatic cells and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an enzyme responsible for maintenance methylation in mammalian cells. We designed our experiments to test whether or not knocking down the DNMT1 gene would lead to a decrease in global methylation. It is our hypothesis that using somatic cells with reduced methylation may be advantageous for increasing the efficiency of cloning via somatic cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral vector capable of delivering a gene that produces a short hairpin RNA targeting the mRNA of DNMT1. The construct included a sequence coding for green fluorescent protein (GFP) that will allow us to identify cells expressing the hairpin as well as a region coding for neomycin resistance so we could select for a pure population of transgenic cells to use for analysis. Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA suggests that our shRNAs are capable of an 80% knockdown. The protein blot of indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation as a result of the DNMT1 knockdown. However, double transduced cells with a high knockdown percentage of DNMT1 mRNA and protein became hypermethylated. The second experiment was conducted to determine the effect of injecting small interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic activation. This experiment was designed to give us information on the survivability and epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the sham injected oocytes. This treatment group also had decreased DNA methylation as determined by immunolabeling of methylated cytosine residues.