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1
Laget, Sophie. "Les protéines humaines MBD4, MBD5 et MBD6, et la régulation épigénétique des gènes." Paris 11, 2010. http://www.theses.fr/2010PA112148.
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Les protéines humaines se liant à l’ADN méthylé par un domaine MBD sont des éléments clefs de la chromatine. Elles peuvent se localiser à l'hétérochromatine méthylée, et, en synergie avec d'autres composants de la chromatine, réprimer l'expression des gènes. Des mutations de MeCP2, la première protéine à domaine MBD découverte, causent un trouble neurologique humain sévère. La première partie de mon travail de doctorat était d’initier la caractérisation de deux protéines humaines à domaine MBD, MBD5 et MBD6. Comme MBD5 a récemment été associée au retard mental, il était important de clarifier si elle se lie l'ADN vraiment à l’ADN méthylé. Bien qu’elles colocalisent avec l’hétérochromatine dans les cellules de culture, les protéines n’interagissent pas avec l’ADN méthylé in vitro. Elles pourraient cependant contribuer à la formation ou la fonction de l'hétérochromatine. La deuxième partie de mon travail porte sur l'étude de MBD4, une protéine impliquée dans la réparation de l'ADN, et mutée dans plusieurs types de cancer. Nous avons constaté que MBD4 interagit directement avec la méthyltransférase de l’ADN DNMT1. Nous étudions actuellement les cibles de MBD4 à l'échelle du génome humain. Ce travail pourrait éclairer le rôle fonctionnel de MBD4 dans la régulation épigénétique des gènes et le cancerHuman methyl-CpG Binding Domain (MBD) proteins are key chromatin components. They can localize to methylated heterochromatin, and, in synergy with other chromatin components, repress gene expression. Mutations in MeCP2, the first MBD protein discovered, can cause a severe human neurological disorder. The first part of my graduate work was to initiate the characterization of two human MBD proteins, MBD5 and MBD6. As MBD5 was recently reported a possible cause of mental retardation, it was important to clarify if they really bind methylated DNA. Although they colocalize with heterochromatin in cultured cells, the proteins do not interact with methylated DNA in vitro. They could however contribute to the formation or function of heterochromatin. The study of MBD4, a protein involved in DNA repair, and mutated in several types of cancer, was the second part of my work. We found that MBD4 could interact directly with DNA methyltransferase 1 (DNMT1). We are currently studying genome-wide MBD4 targets. This work could illuminate the functional role of MBD4 in epigenetic gene regulation and cancer
2
Signolet, Jason George. "Control of self-renewal and pluripotency by the Mbd3/NuRD complex." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648774.
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Brown, Shelley. "The role of MBD3 and the cell cycle in the regulation of the epigenome." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19259.
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DNA methylation patterns were thought to be stable and unchanging throughout life. However more evidence has emerged suggesting that the DNA methylation pattern is dynamic, subject to changes in response to physiological and environmental cues. Thus, it is crucial to understand when these changes occur and how the DNA methylation pattern can be manipulated. This thesis focuses on alterations in the DNA methylation pattern during the cell cycle both globally and at a specific promoter, as well as how the methylated DNA binding domain protein MBD3 is involved in regulating these changes. During the cell cycle, the levels of DNA methylation are highest during S phase, and drop after DNA replication during G0/G1. These changes in DNA methylation are not occurring at repetitive sequences, but rather at specific sequences throughout the genome, as determined using a CpG island microarray. Conversely, the DNA methylation pattern of the ribosomal RNA (rRNA) promoter follows an opposite pattern, with decreased levels of DNA methylation at G1/S and S phase, with the levels increasing as the cell progresses through the cell cycle. These changes at the rRNA promoter correlate with other epigenetic changes, including histone acetylation and transcription factor binding, indicating a coordinated epigenetic change regulated with the different stages of the cell cycle. We further determined how the DNA methylation pattern of the rRNA promoter is regulated, and found a critical role for the methylated DNA binding domain protein MBD3. MBD3 was found to bind to unmethylated rRNA promoters, together with the transcription factor Upstream Binding Factor (UBF). Manipulating the levels of MBD3 expression resulted in a change in the levels of DNA methLe patron de méthylation de l'ADN est connu pour être stable et fixes durant toute la vie. Cependant, plusieurs évidences suggèrent que le patron de méthylation de l'ADN soit dynamique, sujet aux changements en réponse à des stimuli physiologiques et environnementaux. Ainsi, il est crucial de comprendre quand ces changements se produisent et comment le patron de méthylation d'ADN peut être manipulé. Cette thèse se concentre sur les changements dans le patron de méthylation de l'ADN pendant le cycle cellulaire, à l'échelle globale et au niveau de promoteurs spécifiques, et également sur le mécanisme par lequel la protéine MBD3, qui contient un domaine de liaison à l'ADN méthylé, module ces changements. Pendant le cycle cellulaire, les niveaux de méthylation de l'ADN sont plus élevés pendant la phase de S, et chute par la suite après la réplication de l'ADN lors de la phase G0/G1. Ces changements de méthylation de l'ADN ne se produisent pas dans des séquen-ces répétitives, mais plutôt dans des séquences spécifiques dans tout le génome, tel que déterminé par microarray des îlots CpG. Réciproquement, le patron de méthylation du promoteur de l'ARN ribosomal (ARNr) suit un modèle opposé, avec un bas niveau de méthylation de l'ADN en phase G1/S et S, qui augmente progressivement pendant le cycle cellulaire. Ces changements dans le promoteur de l'ARNr corrèlent avec d'autres changements épigénétiques, y compris l'acétylation des histones et la liaison de facteurs de transcription, indiquant un changement épigénétique coordonné régulé avec les différentes étapes du cycle cellulaires. Nous avons de plus déterminé comment le patron de méthylation du promoteur de l'ARNr est régulé
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Tabaroni, Rachel. "Etude structurale du complexe de remodelage de la chromatine NuRD et sa sous-unité MBD3 liée à l'ADN." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ094.
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La régulation de la transcription est un processus dynamique faisant intervenir le recrutement de complexes protéiques impliqués dans le remodelage de la chromatine. Parmi eux, mon travail s’est focalisé sur le complexe NuRD (Nucleosome Remodeling and histone Deacetylation) et sa sous-unité de liaison à l’ADN CpG MBD3. Pour cela une approche de biologie structurale intégrative combinant la préparation biochimique, la caractérisation biophysique et l’étude structurale par cryo-EM et cristallographie aux rayons-X a été mise en place. Les caractérisations biophysiques de MBD3 ont permis de mettre en évidence son interaction avec un ADN non-modifié CpG et des cristaux diffractant jusqu’à 3.9 Å ont été obtenu. De plus la région désordonnée en aval du domaine de liaison a été identifiée et son impact dans la formation de complexe caractérisé. Des cristaux pour les différentes constructions en complexe avec l’ADN ont été obtenus et sont actuellement optimisés. Enfin l’optimisation de la purification et la préparation du complexe, ont permis la visualisation du complexe NuRD et mettent en avant pour la première fois une organisation en domaines du complexeTranscription regulation of chromatin is a very dynamic process regulated through the recruitment of chromatin-remodeling complexes. My work focuses on NuRD for Nucleosome remodeling and histones deacetylation complex a 1 MDa multi-subunit protein complex and its subunit MBD3 a CpG-binding protein and more precisely on an integrated biology approach of this molecular assembly and its interaction with DNA. It combines biochemical preparation, biophysical characterization, single particle cryo-eletron microscopy and x-ray crystallography. Biophysical analysis show that MBD domain of MBD3 interacts with unmodified CpG DNA, a crystal diffracting up to 3.9 Å were obtained. Moreover a C-terminal intrinsically disordered region of MBD3 were identified and despite is inherent disorder seems to increase the binding affinity of MBD3 for DNA. Crystals were obtained for both constructs in complex with DNA and are currently optimized.Cryo-EM study of NuRD complex allows us to develop and optimized purification and grids preparation for the visualization of the complex. The present results reveal a domain organization of the complex never identify before
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Cramer, Jason. "EVOLUTION AND DIVERGENCE OF THE STRUCTURAL AND PHYSICAL PROPERTIES OF DNA BINDING BY METHYL-CYTOSINE BINDING DOMAIN FAMILY MEMBERS 2 AND 3." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3517.
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The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of MBD2 and MBD3. This study provides information regarding previously unidentified MBD3 binding properties and potential biological function. In chapter three, we show that sponges demonstrate a MBD2-mediated capacity for binding methylated DNA sites, recruit NuRD components in vitro, and knockdown of MBD2 in the freshwater desmosponge, Ephydatia muelleri, promotes an abnormal growth phenotype.
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Ee, Ly-Sha. "Regulation of Pluripotency and Differentiation by Chromatin Remodeling Factors." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/921.
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Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions.
In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of >2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms.
The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes.
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Ee, Ly-Sha. "Regulation of Pluripotency and Differentiation by Chromatin Remodeling Factors." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/921.
Full textAbstract:
Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions.
In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of >2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms.
The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes.
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Lyst, Matthew James. "Biochemical analysis of MBD1." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/3931.
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Methylation of cytosines within CpG dinucleotides is a feature of vertebrate DNA. The precise role of DNA methylation is unknown to date, although it has been implicated in several processes relating to transcriptional regulation. One approach to study DNA methylation is the characterization of proteins that bind specifically to methylated DNA. One such family of proteins is the methyl-CpG binding domain (MBD) containing family and MBD1 is a member of this family. MBD1 is implicated in transcriptional repression and various mechanisms by which it might bring about gene silencing have been proposed. These are mainly based on studies reporting interactions between MBD1 and various proteins that regulate chromatin structure. Also MBD1 function can be modified by PIAS proteins, which stimulate its conjugation to SUMO (small ubiquitinlike modifier).The original aim of this work was to address two questions about MBD1: (1) Does MBD1 form part of a stable complex with other factors, and if so, what are the identities of the other components? Purification of MBD1 revealed the presence of no stably bound interacting proteins. However, some evidence indicates MBD1 may interact with itself and form dimers, a finding which impacts on many aspects of the function of MBD1. Also a proteomics screen for transient interaction partners identified candidate binding partners for MBD1 and the related protein MeCP2, which may throw light on the function of these proteins. (2) Are there any activities which regulate MBD1 function by the removal of SUMO from this protein? No activities capable of removing SUMO from native MBD1 were found but it was demonstrated that this modification leads to the destabilization of MBD1 in vitro. The relevance of this finding in vivo is yet to be determined.
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Morey, Ramonell Lluís. "Chromatin alterations imposed by the oncogenic transcription factor PML-RAR." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7138.
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En mamíferos, así como en plantas, mutaciones en AND helicasas/ATPasas del la família SNF2, no solo afectan a la estructura de la cromatina, sino que también afectan al patrón global de la metilación del ADN. Sugiriendo una relación funcional entre la estructura de la cromatina y la epigenética. El complejo NuRD, el cual posee una ATPasa de la familía SNF2, está relacionado con la represión de la transcripción y en el remodelamiento de la cromatina. Nuestro laboratorio demostró que la proteína leucémica PML-RARα reprime la transcripción de sus genes diana por el reclutamiento de DNMTs y el complejo PRC2. En esta tesis, demostramos una relación directa del complejo NuRD en la represión génica y en los cambios epigenéticos en la leucemia promielocítica aguda (APL). Mostramos que PML-RARα se une y recluta NuRD a sus genes diana, incluyendo el gen supresor de tumores RAR2, facilitando que el complejo de Polycomb se reclute y metile la lisina 27 de la histona H3. Tratamiento con Acido Retinóico (RA), el qual se utiliza en pacientes, reduce la ocupación de NuRD en células leucémicas. Eliminando NuRD no solo provoca que las histonas no se deacetilen y que la cromatina no se compacte, sino que también provoca que tanto la metilación del ADN y de las histonas no se produzca, así como la represión génica del gen RAR2, favoreciendo la diferenciación celular. Nuestros resultados caracterizan un nuevo papel del complejo NuRD en el establecimiento de los patrones epigenéticos en APL, demostrando una relación esencial entre la estructura de la cromatina y epigenética durante el desarrollo de la leucemia, pudiéndose aplicar a la terapia de esta enfermedad.In mammals, as in plants, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigentic marks. The SNF2-like containing NuRD complex is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PMLRARα represses target genes through recruitment of DNMTs and Polycomb complex. In this thesis, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leucemia (APL). We show that PML-RARα binds and recruits NuRD to target genes, including to the tumor-suppressor gene RAR2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment reduced the promoter occupancy of the NuRD complex. Knock-down of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction, but also impaired DNA and histone methylation as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.
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MacDougall, Eilidh Fiona. "Functional analysis of the DNA repair protein MBD4." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/12502.
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The methylation of cytosine plays a fundamental role in mediating transcriptional repression. However, 5-methylcytosine can undergo spontaneous hydrolytic delamination to form thymine. It the resulting T:G mismatch is replicated prior to being repaired, a C:G to T:A transition mutation will be present in one of the two daughter DNA molecules. Methyl-CpG-binding domain protein 4 (MBD4) is a DNA glycosylase that can excise thymine from mismatches with guanine, and that acts preferentially on T:G mismatches within CpG dinucleotides in vitro. In order to test the hypothesis that MBD4 repairs the product of 5-methylcytosine delamination in vivo, MBD4-deficient mice were crossed onto the Big Blue genetic background. This background enables the frequency and spectrum of in vivo mutations in a bacteriophage lambda cII transgene to be determined. As predicted, Mbd4-/- mice have a significantly increased frequency of C:G to T:A mutation sat CpG dinucleotides. T:G mismatch-specific thymine DNA glycosine (TDG) can also attempt to excise thymine from T:G mismatches within CpG dinucleotides in vitro. In an attempt to determine the relative contributions of MBD4 and TDG to the repair of 5-methylcytosine delamination-induced T:G mismatches in vivo, the mutation frequencies and spectra in cell lines lacking MBD4 and/or TDG were measured. An additional line of research focused on potential mechanisms by which the DNA repair activity of MBD4 may be regulated. A novel protein has previously been shown to interact with MBD4 in a yeast two-hybrid screen that used MBD4 as the bait protein. This interaction was further characterised by mapping of the interaction domains using the yeast two-hybrid assay, and by immunocytochemistry. Finally, it was also shown that MBD4 may be post-translationally modified by sumoylation.
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Auriol, Émilie. "Spécificité des protéines à domaine de liaison sur l’ADN méthyle (MBD) pour un locus cible : fixation du represseur MBD2 sur la région constitutivement méthylée de l’ilot de CpG des gènes BRCA1 et NBR2." Lyon 1, 2005. http://www.theses.fr/2005LYO10134.
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Chez les mammifères, la méthylation de l’ADN induit une répression transcriptionnelle, grâce à la fixation de protéines à domaine liant les CpG méthylés (MBD), qui recrutent des complexes enzymatiques capables de compacter la chromatine. L’existence d’une spécificité des répresseurs MBD1, MBD2 et MeCP2 pour leur cible reste contestée. Par immunoprécipitation de chromatine et interférence à l’ARN, nous avons pu démontrer qu’une région méthylée de l’îlot de CpG incluant le promoteur bidirectionnel des gènes BRCA1 et NBR2 était la cible spécifique de MBD2. De plus, MBD2 réprime sélectivement la transcription du gène NBR2. Les profils de méthylation sont souvent altérés dans les tumeurs humaines, affectant l’expression génique. Aussi, si l’observation d’une telle spécificité s’étendait à d’autres gènes cibles, des stratégies visant à réduire l’expression de ces répresseurs pourraient constituer une approche thérapeutique anti-tumorale, alternative aux thérapies déméthylantes généralistesThe methyl binding domain proteins (MBD) are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the breast cancer predisposition gene, BRCA1, and the Near BRCA1 2 (NBR2) gene. Quantitative chromatin immunoprecipitation assays and RNA interference strategies indicated that MBD2 is specifically associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. Furthermore, MBD2 represses the expression of the NBR2 gene. Our data indicate that MBD2 has specific targets and that its presence at these targets is indispensable for gene repression. Methylation patterns are altered in tumours, leading to misexpression of genes. Also, specific binding of MBD proteins and reexpression of targeted genes by RNAi approaches could be of great interest for cancer therapy
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Mullegama, Sureni. "Uncovering the molecular pathways of MBD5 in neurodevelopmental disorders." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/459.
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Neurodevelopmental disorders (NDs) are a growing public health concern. These complex disorders cause failure of normal brain development, which leads to intellectual disability (ID) or autism in 3% of children. Accurate diagnosis of NDs is difficult due to complex overlapping phenotypes. Moreover, associations between phenotypically similar NDs and their overlapping molecular mechanisms remain unidentified. The chromosome 2q23.1 region is a newly discovered disease region. We have recently identified a novel ND, 2q23.1 deletion syndrome. The phenotype includes severe ID, significantly delayed speech, behavioral problems, seizures and short stature. This syndrome shares characteristics in common with other genetic syndromes, including Smith-Magenis (SMS, RAI1), Pitt-Hopkins (PTH, TCF4), Angelman (AS, UBE3A) and Rett (RTT, MECP2) syndromes, including ID, speech impairment, and seizures, in addition to other autism spectrum disorder (ASD)-associated phenotypes (associated with mutation of MBD1). The methyl-CpG binding domain protein 5 (MBD5) is thought to be the causative gene for the core phenotype seen in del2q23. We propose that MBD5 is a dosage dependent gene, wherein deletion or duplication results in two distinct syndromes. We hypothesize that deletions, mutations, and duplications in MBD5 and its associated overlapping gene networks are responsible for causing the phenotype seen in 2q23.1 disorders. Furthermore, we hypothesize that syndromic neurodevelopmental genes are involved in common biological networks that, when dysregulated, result in the overlapping phenotypes present in many of these neurodevelopmental disorders. We first show that the causative gene for 2q23.1 deletion syndrome is MBD5. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosome 2q23.1, acquiring 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints, aligned microdeletions to determine the critical region, assessed effects on mRNA expression, and examined medical records, photos, and clinical evaluations. We identified MBD5 as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency, intellectual disability, epilepsy, and autistic features. Sixteen alterations disrupted MBD5 alone, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. We surveyed MBD5 coding polymorphisms among 747 ASD subjects compared to 2,043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG binding domain missense variant, G79E (p=0.012). Thus, we establish that haploinsufficiency of MBD5 is the primary causal factor in 2q23.1 microdeletion syndrome and that mutations in MBD5 are associated with autism. Secondly, we show that MBD5 is a dosage dependent region, wherein deletion or duplication results in altered gene dosage. We previously established the 2q23.1 microdeletion syndrome and report herein 23 individuals with 2q23.1 duplications, thus establishing a complementary duplication syndrome. The observed phenotype includes intellectual disability, motor delay, language impairments, infantile hypotonia and gross motor delay, behavioral problems, autistic features, dysmorphic facial features (pinnae anomalies, arched eyebrows, prominent nose, small chin, thin upper lip), and minor digital anomalies (fifth finger clinodactyly and large broad first toe). The microduplication size varies among all cases and ranges from 680 kb to 53.7 Mb, encompassing a region that includes MBD5. Phenotypic analyses suggest that 2q23.1 duplication results in a slightly less severe phenotype than the reciprocal deletion. The features associated with a deletion, mutation, or duplication of MBD5 and the gene expression changes observed support MBD5 as a dosage sensitive gene critical for normal development. Dup(2)(q23.1) causes a phenotype similar to del(2)(q23.1) and other NDs, like SMS and autism, suggesting shared molecular pathways. Finally, chromatin-modifying genes play an important role in the genetic etiology of many NDs, including intellectual disability, epilepsy, and autism. Many monogenic NDs are caused by chromatin modifying genes, including 2q23.1 deletion and duplication, SMS, RTT, AS, fragile X syndrome (FXS), and PTH. Many of these disorders have overlapping features that include language, sleep, and behavioral anomalies. Investigation of relative gene expression by quantitative PCR and microarray of cell lines from individuals with disorders due to altered expression of MBD5, RAI1, MECP2, UBE3A, TCF4, and MBD1 revealed molecular signatures that allowed for the generation of a novel neurodevelopmental molecular network supporting the overlapping features across these syndromes. Further, knockdown of MBD5 and RAI1 in SH-SY5Y and HEK293T cell lines expanded the repertoire of genes involved in these pathways and showed that other chromatin modifying genes, as well as developmental genes are dysregulated. Pathway analyses showed that MBD5 and RAI1 function in chromatin remodeling, circadian rhythm, neuronal development, and cell growth/survival pathways. From these studies, precise gene dosage of chromatin modifying genes, such as RAI1 and MBD5 are clearly a requirement for normal neurodevelopment and function. Taken together, these studies have given us insight into the role of MBD5 as a dosage sensitive gene in two NDs. Furthermore, we gained insight of how dosage effects of MBD5 and RAI1 affect molecular pathways that are linked to neuronal and behavioral development. We have unveiled pathways and genes, which are important to normal human development, neurodevelopment and behavior. These findings support further investigations into the relationships among causative neurodevelopmental genes, which will lead to common points of regulation that may be targeted toward therapeutic intervention.
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Devailly, Guillaume. "Les protéines MBD2 et ZBTB4 répriment la transcription de nombreux gènes méthylés. MBD2 est redistribuée sur l’ADN méthylé dans des modèles de transformation oncogénique." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10316.
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La méthylation de l'ADN est une marque épigénétique répressive impliquée dans de nombreux processus physiologiques et pathologiques. Des hyperméthylations de promoteurs sont ainsi responsables de répressions transcriptionnelles de gènes suppresseurs de tumeurs dans les cancers. La méthylation de l'ADN serait capable d'induire une répression transcriptionnelle par la combinaison de deux mécanismes principaux : l'éloignement de facteurs de transcription activateurs, et le recrutement de protéines répressives liant spécifiquement l'ADN méthylé. MBD2 est une protéine de liaison à l'ADN méthylé capable de recruter les complexes répresseurs NuRD et SIN3A. ZBTB4 est capable de se lier à l'ADN méthylé in vitro et induit une répression de la transcription de plasmides méthylés lorsqu'elle est surexprimée. Son rôle de répresseur transcriptionnel dépendant de la méthylation de l'ADN reste toutefois peu documenté. Nous avons identifiés par RNAseq les modifications du transcriptome induites par une déplétion de MBD2 ou de ZBTB4. Les gènes surexprimés après déplétion de MBD2 ou ZBTB4 sont méthylés sur leur promoteur, et sont aussi surexprimés après traitement avec des agents déméthylants. Des résultats d'immuno-précipitations de chromatine réalisées contre les deux protéines endogènes montrent que la quasi-totalité des sites de fixation de MBD2 et qu'une partie des sites de fixations de ZBTB4 correspondent à des régions méthylés. Ces résultats confirment à l'échelle du génome que MBD2 endogène est bien un interprète majeur de la méthylation de l'ADN, et que ZBTB4 réprime bien la transcription de gènes méthylés. Nous avons aussi observé une redistribution importante de MBD2 sur le génome dans des modèles de progression tumorale. Nos résultats montrent que les gènes réprimés pendant la transformation oncogénique le sont en partie par MBD2. L'expression de certains de ces gènes peut être induite dans les lignées transformées par déplétion de MBD2 par siRNADNA methylation is an epigenetic mark that plays a role in many physiological and pathological processes. Indeed, silencing of tumor suppressor genes in cancer is frequently caused by promoter hypermethylations. Transcriptional repression induced by DNA methylation is likely caused by the combination of two mechanisms: the repulsion of activator transcription factors, and the recruitment of repressor proteins able to specifically recognize methylated DNA. MBD2 is a methyl DNA binding protein that cans recruits NuRD or SIN3A repressor complexes. ZBTB4 is able to bind methylated DNA in vitro, and can repress the transcription of methylated plasmids when overexpressed. Its methylationdependent transcriptional repressor function remains poorly documented. By RNAseq, we have identified transcriptomic modifications induced by the depletion of either MBD2 or ZBTB4. Genes up regulated after MBD2 or ZBTB4 depletion were methylated on their promoter, and were also up regulated after treatment with demethylating agents. Chromatin immunoprecipitations experiments against endogenous proteins showed that almost all MBD2 binding sites, and that a part of ZBTB4 binding sites, correspond to methylated DNA regions. These results confirmed at genome wide scale that endogenous MBD2 is a major reader of DNA methylation and that ZBTB4 does repress the transcription of methylated genes. We observed an important redistribution of MBD2 on the genome in models of tumor progression. Our results showed that MBD2 plays role in gene repressions occurring during oncogenic transformation. Some of those repressed genes can be re-expressed in transformed cell lines after depletion of MBD2 by siRNA
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Ibrahim, Abdulkhaleg. "Regulation of DNA methylation by DNA glycosylases MBD4 and TDG." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ019/document.
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Chez les mammifères, la méthylation est une marque épigénétique ciblant la cytosine principalement dans un contexte CpG pour produire une 5mC. 5mC est très sensible à une déamination spontanée ou enzymatique, conduisant à la formation d'un mésappariement G/T. La 5mCpeut également être oxydée pour former successivement la 5hmC, la 5fC et la 5caC. Ces modifications de la 5mC participent aux processus actifs de déméthylation de l’ADN. Chez les mammifères, la thymine, dans le mésappariement G/T, est clivée par TDG et MBD4. TDG est également en mesure d'exciser 5fC et 5caC. Cette thèse avait pour but de clarifier la fonction de TDG et MBD4 dans la dynamique de la 5mC. Nous avons montré que MBD4 est associée aux protéines de réparation des mésappariements. Les tests enzymatiques, in vitro, montrent que le complexe MBD4/MMR a une activité bifonctionnelle (glycosylase/lyase) spécifique pour G/T, qui est régulée par la méthylation. Pour TDG, nous avons ciblé cette enzyme dans les cellules MEF et caractérisé la distribution des cytosines modifiées. Les résultats montrent des profils de méthylation/oxydation d'ADN qui sont régulés par TDG et surviennent principalement au niveau des répétitions de CA et dans les rétroéléments spécifiques de la lignée sourisIn mammals, methylation is an epigenetic mark targeting cytosine mainly in a CpG context, producing 5mC. 5mC is highly sensitive to a spontaneous or enzymatic deamination leading to G/Tmismatch. 5mC can also be oxidized to 5- 5hmC, 5fC and 5caC. These modifications of 5mC participate in the active demethylation processes. In mammals, the thymine in G/T mismatch is cleaved by TDG and MBD4 glycosylases. TDG is able also to excise the 5fC and 5caC.This thesis was to clarify the function of TDG and MBD4 in the dynamics of 5mC. We showed that MBD4 is associated with PMS2, MLH1, MSH2 and MSH6 proteins, four proteins involved in DNA mismatch repair (MMR). The in vitro enzymatic tests show that MBD4/MMR complex has a bifunctional glycosylase/lyase activity specific for G/T and is regulated by methylation.For TDG, we targeted this enzyme in MEF cells and characterized the distribution of modified cytosines. The results show that DNA methylation/oxidation patterns are regulated by TDG and occur mainly at CA repeats and at the mouse-lineage specific retro-elements
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Millar, Catherine Bridget. "Functional analysis of the methyl-CpG-binding DNA glycosylase MBD4." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/15385.
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The methyl-CpG-binding domain protein MBD4 can bind to methylated DNA and has glycosylase activity against thymine or uracil mismatched with guanine, preferentially within a CpG site (Hendrich et al., 1999). This particular combination of activities indicates that MBD4 may be involved in the recognition and repair of deamination-induced mismatches at CpG sites. A prediction of this model is that the number of C:G-T:A mutations would be elevated in the absence of MBD4. This hypothesis has been tested by the use of an in vivo mouse mutational assay to compare wild-type and MBD4-deficient animals. Analysis of the mutational status of these mice shows that MBD4-deficiency markedly increases the incidence of C:G-T:A mutations at CpG sites in vivo, thus demonstrating a role for MBD4 in the initiation of repair at deamination-induced G-T mismatches. Since a DNA repair defect can predispose to tumour formation, the consequences of MBD4 deficiency in relation to tumour formation were also examined. The data indicate that MBD4 acts as a tumour supressor in the intestine. A separate avenue of investigation has been to examine whether MBD4 acts alone or with partner proteins. Two-hybrid screening in yeast identified the mismatch repair protein MLH1, the kinase ZIP, and a novel protein as potential partners of MBD4. The possible biological roles of the interaction of MBD4 with these proteins have been probed using biochemical assays. In summary, the work presented in this thesis demonstrates that MBD4 acts in vivo to initiate repair at deamination-induced mismatches. In addition, the interactions of MBD4 with ZIP kinase, as well as its role in tumour suppression in the intestine indicate that this protein may have other, previously undescribed functions.
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Zhu, Yanhua. "MBD genes and Hedgehog signalling in cancer." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/27742.
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In this study, two sets of candidate genes in colon and lung cancer tumourigenesis were studied. The first set comprised members of a family of genes whose proteins are important in the recognition of the methylation/epigenetic status of other genes. The second set were members of a pathway that normally regulate tissue development but whose abnormal, epigenetic loss of activity could lead to tissue dysregulation and tumourigenesis. MBD3 and MBD2 are two members of the MBD family of proteins with a methyl-CpG-binding domain (MBD) involved in transcriptional silencing of methylated genes. Both genes are located in chromosomal regions that suffer loss of heterozygosity in colon and lung cancers. By SSCP analysis and methylation sensitive restriction followed by PCR, 2 mutations were found in 28 cell lines and in no cases was there evidence of gene silencing by hypermethylation of putative promoter regions. RT-PCR and northern hybridisation showed expression of MBD3 in all cancer cell lines examined. The results indicate that neither MBD2 nor MBD3 are major targets of genetic and epigenetic alteration in colon and lung cancers. The Hedgehog (Hh) pathway is a highly conserved signalling cascade involved in many developmental processes. In this study, two genes of this pathway, SMO and GLI3 were investigated for expression and epigenetic alterations in colon and lung cancers. In three cell lines expression of SMO was absent, the putative SMO promoter was fully methylated and GLI3 was not expressed. Two other cell lines had a methylated wild-type SMO allele and expressed mutant SMO, and also did not express GLI3. The results indicate that SMO is silenced by CpG island hypermethylation in colon and lung cancer cell lines, that GLI3 is also silenced in colon and lung cancer cell lines by an as yet unrevealed mechanism and that GLI3 is possibly regulated by SMO in a manner outside the normal sequence of steps currently thought to comprise the Hh pathway.
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Salamé, Patrick-Georges. "The epigenetic mechanism involved in MBD2-mediated induction of interleukin-33." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95057.
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Il-33, the most recently discovered member of the il-1 family of cytokines, is mainly expressed by fibroblasts, epithelial cells, and endothelial cells and signals via the ST2 receptor to promote Th2 type immune responses. This newly discovered cytokine has a well established role in airways inflammation, most notably asthma, and is involved in a wide range of diseases such a s atherosclerosis and atopic dermatitis, but its role in cancer remains unknown. Previous studies in our lab have demonstrated that expression of ectopic MBD2 transforms mouse fibroblasts NIH3T3 cells into highly invasive metastatic cancerous cells. Il-33 is the top gene induced by upregulation of the putative DNA demethylase MBD2 suggesting an undiscovered role of this new cytokine in tumorigenesis. The aim of this thesis is to assess the mechanisms underlying MBD2-mediated induction of il-33. We identify here using high-density tiling arrays with a combination of mDIP and ChIP-on-chip a regulatory region of il-33 which is partially demethylated by increasing the levels of methylated DNA binding protein domain 2 (MBD2) in the cell. Luciferase reporter assays confirm that this region bearing promoter activity is silenced upon in vitro methylation as well as show MBD2-dependent activation of a reporter gene. We further demonstrate using bisulfite pyrosequencing that similar methylation patterns are observed in murine tissues and cell types expressing il-33. Taken all together, our data suggest that il-33 is silenced by DNA methylation and activated by MBD2 triggering cell transformation and invasion. Thus, the new mechanism of il-33 regulation discovered in our studies might have important therapeutic implications in cancer growth and metastasis.Il-33, le plus récemment découvert membre de la famille il-1 des cytokines, est principalement exprimé par les fibroblastes, les cellules épithéliales et les cellules endothéliales et signale via le récepteur ST2 pour promouvoir des réponses immunitaires de type Th2. Cette cytokine nouvellement découverte a un rôle bien établi dans l'inflammation des voies respiratoires notamment l'asthme et est impliquée dans un large éventail de maladies comme l'athérosclérose et la dermatite atopique, mais son rôle dans le cancer reste inconnu. Des études antérieures dans notre laboratoire ont démontré que l'expression ectopique de MBD2 transforme des cellules NIH3T3 fibroblastes de souries en cellules cancéreuses hautement envahissantes et métastatiques. Il-33 est à la tête des gènes induits par la régulation positive de la putative ADN déméthylase MBD2 suggérant un rôle inconnu de cette nouvelle cytokine dans la tumorigenèse. L'objectif de cette thèse est d'évaluer les mécanismes sous-jacents l'induction d'il-33 médiée par MBD2. Nous avons identifié en utilisant des puces à ADN de carrelage haute densité avec une combinaison de mDIP et ChIP-on-chip une région de régulation d'il-33 qui est partiellement déméthylée en augmentant les niveaux de methylated DNA binding protein domain 2 (MBD2) dans la cellule. Les dosages rapporteurs de la luciférase confirment que l'activité de cette région du promoteur est réduite au silence avec la méthylation in vitro ainsi que l'activation d'un gène rapporteur par MBD2. Nous montrons également à l'aide du pyroséquencage bisulfite que des modèles de méthylation similaires sont observés dans les tissus murins et des types de cellules exprimant il-33. Prises dans leur ensemble, nos données suggèrent qu'il-33 est réduit au silence par la méthylation de l'ADN et activé par la déméthylation par MBD2 déclenchant la transformation cellulaire et l'invasion. Ainsi, le nouveau mécanisme de
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Massie, Charles Edward. "The functional and transcriptional consequences of attenuating MBD2 in cancer cells." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613667.
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Desai, Megha. "Structural and Functional Characterization of the MBD2-NuRD Co-Repressor Complex." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3617.
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The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex.
In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2IDR). Biophysical analyses show that MBD2IDR is an intrinsically disordered region (IDR). Despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical area of contact requiring two contiguous amino acid residues, Arg286 and Leu287. Mutation of these critical residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene Prostasin in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.
In Chapter 3, we have discussed a novel mechanism for MBD2-mediated silencing of the fetal γ-globin gene. Through microarray expression analyses in adult erythroid cells of MBD2-/- mice, we identified ZBTB32 and miR-210 as downstream targets of MBD2. Over-expression of ZBTB32 and miR-210 in adult erythroid cells causes increased expression of the silenced fetal γ-globin gene. Thus, our results indicate that MBD2 may regulate γ-globin gene expression indirectly though ZBTB32 and miR-210 in adult erythroid cells.
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Deaton, Aimée M. "Role of CpG island methylation and MBD2 in immune cell gene regulation." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4758.
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The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.
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Jones, Gareth-Rhys. "Role of methyl-CpG-binding domain protein-2 (MBD2) in colonic inflammation." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/15974.
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The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.
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Meng, Huan. "Functional characterization of the DNA glycosylase, methyl-CpG binding domain protein 4 (MBD4)." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11818.
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DNA methylation is a major form of epigenetic modification and involves the addition of a methyl group covalently to the 5-position of the cytosine pyrimidine ring, mostly within the context of CpG dinucleotides in vertebrate somatic cells. Methylation of CpG dinucleotides at promoter regions is generally associated with transcriptional repression. In this context, the methyl-CpG binding proteins (MeCPs) that are capable of recognition of methylated CpG dinucleotides are proposed to play a central role in DNA methylation associated transcriptional repression. Methyl-CpG binding domain protein 4 (MBD4) is an MeCP that possesses a glycosylase domain at its C-terminal, which can excise and repair both G:T and G:U mutations derived from DNA deamination at CpG dinucleotides, in addition to its Nterminal MBD binding domain. MBD4 has been associated with a number of pathways including DNA repair, apoptosis, transcriptional repression, and possibly DNA demethylation processes. However, the precise contribution of MBD4 to these processes remains unclear. To explore the functional repertoire of MBD4 I decided to undertake multiple protein interaction studies to identify potential partner proteins. I performed yeast 2-hybrid screens with an 11.5 day mouse embryonic cDNA library and multiple mass spectrometry of immunoprecipitates of tagged versions of MBD4 that were over-expressed in human cell lines. I detected ~380 potential interacting candidates with these assays. A significant number of candidates were detected in both assay systems. Chosen candidates were further validated by reciprocal co-IP of expressed partners and by immunofluorescence (IF) microscopy to determine their potential co-localisation in mouse and human cell lines. Subsequently, I identified the intervening domain of MBD4 as a novel protein interaction region for tested candidates. My analysis suggests that MBD4 can have a role in regulation of post-replication methyl-error repair/methylation machinery through its direct interaction with DNMT1 (previously shown), UHRF1 (novel) and USP7 (novel), as well as possible cross-talk to histone modification and chromatin remodelling pathways, through partners such as PRMT5 and ACF1. Interestingly the transcription regulatory components KAP1 and CFP1 not only interact with but also dramatically influence the stability of exogenously expressed MBD4 in human cells. In general positive validation by IP and IF demonstrates the robustness of the initial screens, and implies that MBD4 may impact upon several transcriptional and epigenetic networks along with a number of nuclear pathways that include transcriptional repression, DNA repair and RNA processing. To test for transcriptional aberration in the absence of Mbd4 function I profiled two independent mouse cell lines that lack MBD4 activity using Illumina MouseWG-6 v2.0 Expression BeadChip arrays. A number of genes were identified that are significantly up- or down- regulated in both Mbd4-/- MEFs. This included mis-expression of insulin-like growth factor-binding proteins and two paternally imprinted genes Dio3 and H19. The cohort of genes that were mis-expressed in the Mbd4-/- MEFs overlap with genes that responsed to tamoxifen exposure in an ER-positive ZR-75-1 xenograft model. In response to this observation I identified a potential interaction between MBD4 and estrogen receptor α (ERα) by co-IP and IF co-localisation. This suggests that MBD4 might potentiate transcription of estrogen regulated genes via a direct interaction with ERα, supporting a possible link between replication repair remodelling and steroid/thyroid hormone receptor transcriptional regulation. Additionally I performed a pathway analysis by which several developmental genes including Sox9, Klf2 and Klf4, were prioritised as possible MBD4 targets. On this basis I propose a role for MBD4 in acquired diseases such as cancers and autoimmune diseases via transcriptional regulation. I also performed a comparison of MBD4 DNA binding activity with MBD4 homologues from the Medaka fish (Oryzias latipes) and the amphibian, Xenopus laevis. I could show that DNA binding specificity to a series of methylated and mismatched probes is conserved regardless of the poor sequence conservation of the MBD domain of MBD4 between the species. I conclude that MBD4 is integrated in multiple pathways in the nucleus that includes DNA repair, chromatin remodelling, transcriptional regulation and genome stability.
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Mian, Omar. "The Role of the Methyl DNA Binding Domain Protein 2 (MBD2) in Breast Cancer." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/39.
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Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..
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Kennedy, Nathan. "Reservation Prices and Willingness to Accept Price Offers for Nonindustrial Forest Landowners in Western Virginia." Thesis, Virginia Tech, 2001. http://hdl.handle.net/10919/33766.
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The purpose of this thesis is to examine what
motivates nonindustrial private forest landowners
to accept bids of various levels for harvesting.
Through the use of a survey we specifically
consider what preferences and landowner
characteristics effect these decisions.
Landowners were randomly selected from counties
in Southwest Virginia. The participants were
presented a payment table in which they were asked
to indicate the level of certainty with which they
would accept bids of various levels for their
timber. The information obtained for the survey
was used in a LOGIT model to examine which
variables were most important both in determining
the certainty respondents attached to different
bid levels, and the likelihood of accepting a bid
of any size. Our most important results show that
factors such as bequest motives, tract size,
absentee status, and environmental preferences
influence the bid acceptance decision for
landowners in the sample.Master of Science
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Mallikarjuna, Rao Tarun. "Modelling and simulation of Research Concept Vehicle using MBD-FEM approach." Thesis, KTH, Maskinkonstruktion (Inst.), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-183008.
Full textAbstract:
This work highlights the design process to build a MBD (Multi-Body Dynamics) model with flexible parts for a RCV (Research Concept Vehicle). Full vehicle dynamic simulations of the RCV model with flexible parts were performed for different load cases and the results were compared with that of a MBD model with rigid body components. In addition, FE modelling of the RCV body parts, selection of attachment nodes, generation and verification of Modal Neutral Files (MNFs) are discussed. RCV is a concept vehicle developed at KTH Royal Institute of technology as a research platform to implement, validate and demonstrate results of various research projects. The vehicle consists of body, suspension and tire subsystems which were designed and developed as individual projects. The body subsystem comprises of rollcage, subframe and a composite baseplate. In this project, a MBD model of the RCV was developed in ADAMS/CAR to measure the forces acting at the interface of these body components and also to consider the suspension forces acting on the individual front and rear subframe parts. Finite element (FE) models were incorporated to consider the flexibility of the body components. The RCV is a vehicle constantly evolving with addition of new components to implement and test various research results. To study the application of this method, two Models of the RCV with design modifications were developed and studied. A model of the RCV without rollcage and a model with a rigid link connecting the body components were built and the results of dynamic simulations were compared with that of the existing RCV design. When flexibility of the baseplate was considered in the models, an overall change in dynamics of the body components was observed. Further, observing the results from models with design modifications, it was evident that this method can be used to study the effect of these modifications on the dynamic behaviour of the vehicle.Det här arbetet belyser konstruktionsprocessen för att bygga en MBD-modell (Multi-Body Dynamics) med flexibla komponenter av konceptfordonet RCV (Research Concept Vehicle). Fullständiga fordonsdynamiska simuleringar med flexibla komponenter utfördes för olika lastfall och resultaten jämfördes med en MBD-modell med stela komponenter. Dessutom diskuteras FE modellering av RCVs olika delsystem, val av kopplingsnoder, generering och verifiering av ”Modal Neutral Files” (MNFs). RCV är ett konceptfordon som utvecklats vid Kungliga Tekniska Högskolan, KTH, som en forskningsplattform för att implementera, validera och demonstrera resultaten av olika forskningsprojekt. Fordonet består av delsystemen; chassi, hjulupphängning, och däck, vilka har utvecklats tidigare i separata projekt. Chassit består i sin tur av delsystemen; ”rollcage”, ”subframe” och ”baseplate”. I detta projekt har en MBD-modell av RCV utvecklats i ADAMS/CAR för att simulera olika körfall och beräkna de krafter som verkar mellan dessa delsystem och att också studera skillnaden i belastning av främre resp. bakre ”subframe”. FE modeller importeradesäven till modellen för att studera effekten av elasticiteten hos komponenterna på fordonets beteende.RVC är ett fordon som konstant utvecklas med tillägg av nya komponenter för att implementera och testa olika forskningsresultat. För att studera tillämpningen av denna metod skapades två modeller av RCV med olika konstruktiva förändringar vilkas inverkan på fordonet studerades. En modell av RCV utan ”rollcage” och en modell med styv länk som förbinder olika delar av chassit skapades och resultaten av dynamiska simuleringar jämfördes med simuleringsresultat för den befintliga RCV-designen. När flexibiliteten hos basplattan beaktades i modellerna observerades förändringar i dynamiken hos chassit vad gäller vertikala förskjutningar och vinkelförskjutningar. Utifrån dessa simuleringar kan vi dra slutsatsen att den utvecklade metoden är användbar för att studera effekter av konstruktionsförändringar på det dynamiska beteendet hos fordonet.
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Rodrigues, Manuel. "Génétique des mélanomes oculaires." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS107.
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Les mélanomes oculaires sont des tumeurs rares représentant environ 5% des mélanomes. Les mélanomes oculaires peuvent provenir de deux tissus : l’uvée (~500 cas/an en France) et la conjonctive (~30 cas/an). Les mélanomes uvéaux présentent un très faible taux de mutations somatiques. Ces tumeurs sont également porteuses d’altérations du nombre de copies caractéristiques (gains du 8q, 1q, 6p, pertes du 3, du 1p, du 6q ou du 8p). L’évolution du génome de ces tumeurs durant la progression métastatique est à ce jour mal décrit. Afin d’explorer l’évolution métastatique du mélanome uvéal, nous avons séquencé les exomes de 14 tumeurs primaires et 79 métastases provenant de 24 patients. Il existait une grande proximité génétique entre tumeurs primaires et métastases avec une médiane de 11,5 mutations dans les tumeurs primaires, et 14 dans les métastases. Bien que les mutations SF3B1 et EIF1AX soient des facteurs pronostiques majeurs dans les mélanomes uvéaux, leurs fréquences dans les métastases étaient similaires à celle observée dans les séries historiques de tumeurs primaires. Les métastases présentaient quelques altérations de nombre de copies supplémentaires par rapport aux tumeurs primaires correspondantes. Parmi les altérations du nombre de copies les plus souvent acquises lors du processus métastatique, les gains du 8q étaient présents dans 92% des métastases. Lors de ce travail, nous avons découvert un mélanome uvéal présentant un phénotype hypermuté CpG>TpG chez une patiente ayant présenté une réponse exceptionnelle à une immunothérapie anti-PD1. Ce phénotype hypermutateur a été expliqué par une mutation germinale délétère de MBD4 (Methyl-CpG Binding Domain 4) avec une inactivation bi-allélique dans la tumeur. Deux autres tumeurs hypermutées CpG>TpG porteuses d’une mutation de MBD4 germinale, un mélanome uvéal et un glioblastome, ont été identifiées dans les bases de données publiques. La biologie des mélanomes conjonctivaux et leurs profils génomiques sont mal connus. Nous avons séquencé les génomes de 6 tumeurs, puis procédé à un séquençage ciblé de 47 autres tumeurs. Nous avons montré que ces tumeurs présentent un profil hypermuté C>T induit par l’exposition aux ultra-violets. Ces tumeurs présentaient un profil de mutations proche des mélanomes cutanés avec une fréquence moindre de mutations BRAF (33%), et des mutations plus spécifiques des mélanomes muqueux telles que des mutations activatrices de KIT et SF3B1 dans les mélanomes conjonctivaux non exposés au soleil. Nous avons également identifié des mutations de CTNNB1 dans les tumeurs développées sur des nevi conjonctivaux. L’ensemble de ces travaux illustrent comment la description moléculaire des tumeurs rares permet d’envisager de nouvelles stratégies de médecine de précisionOcular melanomas are rare tumors representing about 5% of all melanomas. Ocular melanomas may arise from two tissues: the uvea (~ 500 cases / year in France) and the conjunctiva (~ 30 cases / year). Uveal melanomas have a very low rate of somatic mutations. These tumors also carry specific distinctive copy number alterations (gains of 8q, 1q, 6p, losses of 3, 1p, 6q or 8p). The evolution of the genome of these tumors during metastatic progression has been poorly described.To explore the metastatic evolution of uveal melanoma, we whole-exome sequenced 14 primary tumors and 79 metastases from 24 patients. Primary tumors and metastases presented close genetic profiles with a median of 11.5 mutations in primary tumors, and 14 in metastases. Although SF3B1 and EIF1AX mutations are major prognostic factors in uveal melanomas, their frequencies in metastases were similar to those observed in historical primary tumors. The metastases showed some additional copy number alterations compared to the corresponding primary tumors. Among the alterations acquired during the metastatic process, 8q gains were present in 92% of metastases.Thanks to this work, we found a uveal melanoma with a CpG> TpG hypermutated phenotype in a patient who had an exceptional response to anti-PD1 immunotherapy. This hypermutated phenotype was explained by a deleterious germline mutation of MBD4 (Methyl-CpG Binding Domain 4) with bi-allelic inactivation in the tumor. Two other hypermuted CpG> TpG tumors with germline MBD4 mutation, a uveal melanoma and a glioblastoma, were identified in public databases.The biology of conjunctival melanomas and their genomic profiles have been scarcely described. We sequenced the genomes of 6 tumors and then target-sequenced 47 other tumors. We showed that these tumors had a C> T hypermuted profile induced by ultraviolet exposure. These tumors presented a pattern of mutations close to cutaneous melanomas with a lower frequency of BRAF mutations (33%), and mutations that were more specific of mucosal melanomas such as activating mutations of KIT and SF3B1 in conjunctival melanomas not exposed to the sun. We also identified CTNNB1 mutations in tumors developed on conjunctival nevi.All of these works illustrate how the molecular description of rare tumors opens new avenues for precision medicine
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Lillie, Kirsten L. "The structure of the methyl-binding domain of MBD2, and studies of the MBD:DNA interactions." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/11062.
Full textAbstract:
DNA methylation is a powerful epigenetic control mechanism, essential for the regulation of transcriptional processes. In mammals, methylation occurs mainly at CpG dinucleotides and the modified sequence is recognised by members of the methylated DNA-binding domain (MBD) family. Methylated DNA binding domain 2 (MBD2) is a 44 kDa protein with the ability to distinguish between methylated and unmethylated DNA. It is associated with histone deacetylases and nucleosome remodelling complexes in vivo, and functions as a transcriptional repressor. MBD2 expression levels correlate with certain types of cancer and MBD2 may contribute to tumour development, by inappropriate silencing of tumour suppressor genes. MBD2 is a link between DNA methylation, chromatin structure and certain disease mechanisms. Understanding how methylated DNA is recognised by MBD2, will help establish its specific role as a methylation interpreter. This thesis describes the structure determination of the 72 residue MBD from mouse MBD2, using solution NMR spectroscopy. The MBD has a characteristic wedge-shaped fold, with one face made up of 13-sheets, and the other of an a-helix plus a hairpin loop. Loop Li, between two long beta-strands, is much more rigid in this structure compared to the equivalent region in other members of the MBD family. It appears to be a pre-formed DNA binding loop and overlays well with the structure of the MBD (from MBD1):DNA complex, solved by another group. NMR was subsequently used to follow the titration of the MBD from MBD2, with a 12 base-pair DNA oligomer, symmetrically methylated at a central CpG pair. Large chemical shift changes in the '5N-HSQC spectrum were observed for Li loop residues, and for an arginine residue at the base of the a-helix. This corresponds to the DNA binding interface in the MBD1 MBD:DNA complex structure. Surface plasmon resonance was used to compare the affinities of three different MBDs for DNA oligomers in various methylation states. The MBD2 MBD showed weaker binding to symmetrically methylated DNA, when compared to MeCP2 and MBD4 MBDs. In general, there was no difference in affinity for any of the methylated oligomers, and no construct bound unmethylated DNA. There is some evidence that oligomer length affects binding, although this was not studied in detail.
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Thigpen, Ashley Lauren Clark C. Randall. "Chromatographic and mass spectral studies on mass equivalent substituted phenethlamines related to MDEA, MDMMA and MBDB." Auburn, Ala., 2006. http://repo.lib.auburn.edu/2006%20Fall/Theses/THIGPEN_ASHLEY_11.pdf.
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Perriaud, Laury. "Étude systémique des cibles génomiques de la methyl-CpG binding domain protein 2 (MBD2), un répresseur transcriptionnel dépendant de la méthylation de l'ADN : évolution de la distribution de MBD2 dans un modèle syngénique de progression tumorale mammaire." Phd thesis, Université Claude Bernard - Lyon I, 2010. http://tel.archives-ouvertes.fr/tel-00833153.
Full textAbstract:
Les protéines à " Methyl-CpG-binding domain " (MBD) jouent un rôle important dans l'interprétationde la méthylation de l'ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted'expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l'échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L'impact sur l'expression génique de l'inhibition de MBD2 par interférence àl'ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n'induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l'ADN, de liaisons de MBD2 et de l'ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l'analyse de l'acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l'ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l'ADN méthylé.
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Billard, Lise-Marie. "Expression des gènes codant les protéines liant l'ADN méthylé (MBD) dans les cancers du sein : implication des MBD dans la répression transcriptionnelle de gènes suppresseurs de tumeurs BRCA1 et CDX1." Lyon 1, 2003. http://www.theses.fr/2003LYO1T083.
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Ejermo, Emil. "MBD-sjuk, DAMP-unge och ADHD-barn : Koncentrationssvårigheter i grundskolan ur ett historiskt perspektiv." Thesis, Linnéuniversitetet, Institutionen för kulturvetenskaper (KV), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-81337.
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MBD, DAMP, ADD and ADHD are all concepts that through history have been used as labels to explain people with concentration difficulties. The purpose of this essay is to illustrate how the school's view on pupils with concentration difficulties has changed over the years. In addition, it shows how the school's view of pupils with concentration difficulties has affected both the teaching, the role of the teachers and the students. In the 1950s, the concept of MBD was used. In the 1990s, the term DAMP was used and today children with concentration problems are diagnosed with ADD or ADHD. In this essay I follow how perceptions of these difficulties has changed in line with the concept differences.
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Chatagnon, Amandine. "Spécificité de liaison et de répression de la " Methyl-CpG-Binding Domain protein 2 " (MBD2) : identification de gènes cibles impliqués dans les cancers." Phd thesis, Université Claude Bernard - Lyon I, 2009. http://tel.archives-ouvertes.fr/tel-00603777.
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De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l'expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l'expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l'analyse globale des profils de distribution de MBD2, de la méthylation de l'ADN, ainsi que de l'ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d'un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.
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Bonnet, Céline. "Micro-réarrangements chromosomiques et déficience intellectuelle : identification de nouveaux gènes et caractérisation des conséquences moléculaires de ces micro-réarrangements sur les gènes cibles." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0308.
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De nombreux gènes impliqués dans la déficience intellectuelle (DI) restent encore à découvrir. Une des voies permettant l'identification de ces nouveaux gènes est la caractérisation de micro-réarrangements chromosomiques chez des patients atteints de DI. L'hybridation génomique comparative sur microréseau permet de détecter des déséquilibres génomiques de très petite taille touchant un ou quelques gènes. Les conséquences moléculaires de ces microdélétions ou microduplications sont différentes en fonction de la position du gène par rapport à leurs bornes. Nous avons ainsi montré l'implication du gène MBD5 dans le syndrome microdélétionnel 2q23.1 et la DI grâce à la caractérisation de trois délétions partielles, une duplication partielle et la première mutation non sens décrite dans ce gène. Nous avons également décrit chez des patients avec DI sévère un nouveau syndrome associé aux délétions de la région 4q21 et deux gènes candidats : PRKG2 et RASGEF1B. Ce syndrome est associé à un phénotype clinique reconnaissable, marqué surtout par un retard de croissance majeur et un retard psychomoteur sévère prédominant sur le langage. Par ailleurs nous avons étudié chez des garçons avec DI, deux duplications situées en Xq24q25 affectant le gène GRIA3. La première touche partiellement le gène, la seconde est situé en amont du gène et est responsable d'un effet de position. Pour finir nous avons étudié une famille consanguine dans laquelle ségrége une duplication 8p22 touchant partiellement le gène TUSC3 impliqué dans la DI de transmission autosomique récessiveA lot of intellectual disability (ID) genes have to be discovered. One of the approaches to identify new ID genes is to characterize chromosomal aberrations in affected patients. Array-CGH (Comparative Genomic Hybridization) made it possible to detect small CNV (Copy Number Variations) affecting only one or a few genes. Molecular outcomes of these microdeletions and microduplications are different depending on the position of the gene relative to the breakpoints. We have thus shown the involvement of MBD5 gene in the 2q23.1 microdeletion syndrome and in ID with the characterization of three partial deletions, a partial duplication and the first nonsense mutation described in this gene. We have also described in patients with severe ID a new syndrome associated with 4q21 deletions involving two candidate genes: PRKG2 and RASGEF1B. This syndrome is associated with a recognizable clinical phenotype with marked growth restriction, severe psychomotor delay and absent or severely delayed speech. In addition, we have studied two Xq24q25 duplications affecting GRIA3 gene in boys with ID. The first one affects partially the gene, the second one is located upstream of the gene and is responsible for a position effect. Finally we have studied a consanguineous family with a 8p22 duplication affecting partially TUSC3 gene which is involved in autosomal recessive ID
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Athrey, Ankith Suresh. "Design and Analysis of Electric Over-actuated Vehicle Suspension." Thesis, KTH, Skolan för industriell teknik och management (ITM), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-281708.
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The main aim of this master thesis is to improve the performance of the Research Concept Vehicle (RCV). The RCV is an electric over-actuated vehicle developed at Integrated Transport Research Lab (ITRL) at KTH Royal Institute of Technology. The vehicle steer, camber, drive, and brake on each wheel of the vehicle. The RCV also has various operation modes such as 2WD, 4WD, 2WS and 4WS. The RCV is used as a research platform to implement, validate, and demonstrate results of various research projects. The RCV was developed in the year 2012. There is now a requirement to improve the performance of the vehicle to create a more dynamically capable platform to do more dynamic tests with. The main aim of this thesis is to explore the possibility of upgrading the suspension system with integrated wheel hub motor, electric steering actuator and electric camber actuator. It also involves packaging of the new battery pack system and reinforcing the chassis to reduce the flex during operation. Steps followed involves analysis of the current electric steering and electric camber actuator systems using MBD method to test the performance. With this as the base, requirements are decided as to what must be done to improve the performance by creating another MBD model to obtain the new performance figures. Also, the new battery pack is to be positioned on the base plate of the vehicle and this is achieved by placing the new battery pack onto the existing CAD model. The chassis is to be reinforced with the help of cross members, also designed on CAD software. The damper unit needs to be repositioned to accommodate the battery pack. Based on the changes in the vehicle, new hardpoints are decided for the new steering system, camber system and suspension system. Based on the new performance figures obtained from MBD, the requirements of the new electric steering and camber actuator systems are presented. The strength in the new frame is tested using FEM method. The new position of the damper unit is tested for performance using an MBD software. With the end of this thesis, the requirements to develop the new and improved RCV was obtained, thereby allowing for more dynamic testing to be done with the electric vehicle.Huvudsyftet med detta examensarbete är att förbättra prestanda för Research Concept Vehicle (RCV). RCV är ett elektriskt överaktuerat fordon utvecklat vid Integrated Transport Research Lab (ITRL) vid KTH Royal Institute of Technology. Fordonet styr, reglerar cambervinkeln, kör och bromsar med varje hjul i fordonet. RCV har också olika driftlägen som 2WD, 4WD, 2WS och 4WS. RCV används som en forskningsplattform för att implementera, validera och demonstrera resultat från olika forskningsprojekt. RCV utvecklades år 2012. Nu är det nu ett krav att förbättra fordonets prestanda för att skapa en mer dynamiskt kapabel plattform att göra mer dynamiska tester med. Huvudsyftet med denna avhandling är att undersöka möjligheten att uppgradera upphängningssystemet med integrerad hjulnavmotor, elektrisk styrmanöverdon och elektrisk cambermanöverdon. Det handlar också om förpackning av det nya batteripaketet och förstärkning av chassit för att minska flex under drift. Stegen som följs innefattar analys av de nuvarande elektriska styrsystemen och de elektriska camber-ställdonssystemen med MBD-metoden för att testa prestandan. Med detta som bas bestäms krav på vad som måste göras för att förbättra prestandan genom att skapa en annan MBD-modell för att erhålla de nya prestandasiffrorna. Det nya batteripaketet ska också placeras på fordonets bottenplatta med hjälp av CAD-programvara. Chassit ska förstärkas med hjälp av tvärbalkar, även utformade på CAD-programvara. Spjällenheten måste placeras om för att rymma batteripaketet. Baserat på förändringarna i fordonet bestäms nya hårda punkter för det nya styrsystemet, camber-systemet och upphängningssystemet. Baserat på de nya prestandasiffrorna som erhållits från MBD presenteras kraven för de nya elektriska styr- och camber-ställdonssystemen. Styrkan i den nya ramen testas med FEM-metoden. Spjällenhetens nya position testas för prestanda med hjälp av en MBD-programvara. I slutet av denna avhandling erhölls kraven för att utveckla den nya och förbättrade RCV, vilket möjliggjorde en mer dynamisk testning med elfordonet.
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Andersson, Henric. "Aircraft Systems Modeling : Model Based Systems Engineering in Avionics Design and Aircraft Simulation." Licentiate thesis, Linköping University, Linköping University, Machine Design, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-17573.
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Aircraft developers like other development and manufacturing companies, are experiencing increasing complexity in their products and growing competition in the global market. One way to confront the challenges is to make the development process more efficient and to shorten time to market for new products/variants by using design and development methods based on models. Model Based Systems Engineering (MBSE) is introduced to, in a structured way, support engineers with aids and rules in order to engineer systems in a new way.
In this thesis, model based strategies for aircraft and avionics development are studied. A background to avionics architectures and in particular Integrated Modular Avionics is described. The integrating discipline Systems Engineering, MBSE and applicable standards are also described. A survey on available and emerging modeling techniques and tools, such as Hosted Simulation, is presented and Modeling Domains are defined in order to analyze the engineering environment with all its vital parts to support an MBSE approach.
Time and money may be saved by using modeling techniques that enable understanding of the engineering problem, state-of-the-art analysis and team communication, with preserved or increased quality and sense of control. Dynamic simulation is an activity increasingly used in aerospace, for several reasons; to prove the product concept, to validate stated requirements, and to verify the final implementation. Simulation is also used for end-user training, with specialized training simulators, but with the same underlying models. As models grow in complexity, and the set of simulation platforms is expanded, new needs for specification, model building and configuration support arise, which requires a modeling framework to be efficient.
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Andrews, Stephen. "MBD2 transforms normal cells into highly invasive cancer cells by causing DNA demethylation and the activation of pro-cancerous genes." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92225.
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The epigenome, comprised of the chromatin and the DNA methylome, sets up and maintains gene expression patterns. Disruption of the epigenome and its components is a hallmark of cancer, particularly global DNA hypomethylation. The existence of a bona fide DNA demethylase has long been disputed. This thesis focuses on studying methylated DNA-binding protein-2's (MBD2) role in DNA demethylation and cancer.MBD2 was concurrently classified as a DNA demethylase and a transcriptional repressor. The effects of MBD2 overexpression and depletion in normal and cancerous cells on the methylome strongly supports MBD2's role in participating in the DNA demethylation reaction, as do demethylase assays with purified MBD2 and with nuclear extracts prepared from cells that are either over-producing or under-producing MBD2.
Global DNA hypomethylation is a hallmark of cancer. We therefore tested whether increased MBD2 expression could transform a normal cell into a cancerous cell. Over-production of MBD2 in normal cells, including primary human fibroblasts, results in highly invasive and migratory cancer cells with hypomethylated genomes. MBD2 depletion in several cancer cell lines blocks cellular transformation and results in silencing and methylation of MBD2 target genes.
MBD2's ability to transform cells led us to study whether MBD2 mediates transformation by well-characterized oncogenes such as the proto-oncogene RAS. MBD2 depletion from cells transformed by RAS resulted in an increase in global DNA methylation and a reduction of transformation.
Microarray gene expression analysis revealed novel targets of MBD2 and genes that had not yet been classified as oncogenic, such as the dual function cytokine Il-33. We show here that Il-33 signaling is capable of turning a normal cell to become invasive and that Il-33 can act as a chemo-attractant for normal cells to migrate. Furthermore, abrogation of the cytokine effect of Il-33 in cancer cell by using a recombinant decoy receptor as a pharmacological inhibitor results in an abrogation of the cell's transformed phenotype.
Taken together, the data presented in this thesis show that MBD2 is involved in the DNA demethylation reaction and that MBD2, through DNA demethylation and activation of pro-metastatic genes, can transform normal cells into highly metastatic cancer cells. This thesis supports investigating pharmacological inhibitors of DNA demethylation as anti-metastatic drugs.
L'épigénome, composé de la chromatine et ADN methylome, met en place et maintient les profils d'expression génique. Des perturbations de l'épigénome et de ses composantes est une caractéristique de cancer, particulièrement hypométhylation de l'ADN. L'existence d'un ADN demethylase a longtemps été contestée. Cette thèse vise à étudier le rôle de MBD2 dans la déméthylation de l'ADN et le cancer.
MBD2 a été classé séparément mais en même temps comme étant un demethylase ADN et un répresseur transcriptionnel. Les effets de la surexpression d'MBD2 et l'appauvrissement dans les cellules normales et cancéreuses sur le methylome appuie fermement le rôle MBD2 en tant que participation à l'activité de déméthylation de l'ADN comme le font des tests de demethylase sur MBD2 purifiée et sur des extraits nucléaires préparés à partir de cellules qui sont soit en situation de surproduction ou de sous-production MBD2.
L'hypométhylation global de l'ADN est une des caractéristiques du cancer. Nous avons donc testé si une élévation d'MBD2 pourrait transformer une cellule normale en cellule cancéreuse. La surproduction de MBD2 dans les cellules normales, y compris les fibroblastes humains primaires, donne comme résultats des cellules cancéreuses très envahissantes et migratrices dont les génomes sont hypo-méthylées. L'appauvrissement d'MBD2 dans plusieurs lignées cellulaires cancéreuses bloque la transformation cellulaire et à comme effet atténuer et de la méthylation de gènes cibles d'MBD2.
La capacité remarquable MBD2 à transformer des cellules nous a amené à étudier si MBD2 médiatise la transformation de certains oncogènes bien caractérisés tels que le proto-oncogène ras. Appauvrissement d'MBD2 dans des cellules transformées par RAS a entraîné une augmentation globale de la méthylation de l'ADN et une réduction globale de la transformation.
L'analyse Microarray de l'expression génique ont révélé de nouvelles cibles de MBD2 et des gènes qui n'avaient pas encore été classée comme oncogène, tels que la double fonction de cytokines IL-33. Nous montrons ici que la signalisation l'IL-33 est capable de transformer une cellule normale en cellule envahissante et que l'IL-33 peut agir comme un chimio-attractant en permettant à des cellules normales de migrer. En outre, le phénotype transformé induit par l'IL-33 peut être abrogé à l'aide d'un recombinant decoy receptor comme un inhibiteur pharmacologique.
Les données présentées dans cette thèse démontre que MBD2 est directement impliqué dans la réaction de déméthylation de l'ADN et que MBD2, par la déméthylation de l'ADN et l'activation de gènes pro-métastatiques, peut transformer des cellules normales en cellules cancéreuses hautement métastatique. Cette thèse soutient l'enquête inhibitrice pharmacologique de déméthylation de l'ADN en tant que médicaments anti-métastatique.
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Fukuma, Shingo. "Prescription Patterns and Mineral Metabolism Abnormalities in the Cinacalcet Era: Results from the MBD-5D Study." Kyoto University, 2013. http://hdl.handle.net/2433/180604.
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Gnanapragasam, Merlin Nithya. "The Role of Methyl CpG Binding Domain Protein 2 (MBD2) in the Regulation of Embryonic and Fetal β-type Globin Genes." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2315.
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The reexpression of the fetal γ-globin gene in adult erythrocytes is of therapeutic interest due to its ameliorating effects in β-hemoglobinopathies. We recently showed that Methyl CpG Binding Domain Protein2 (MBD2) contributes to the silencing of the chicken embryonic ρ-globin and human fetal γ-globin genes. We further biochemically characterized an erythroid MeCP1 complex that is recruited by MBD2 to mediate the silencing of these genes. These observations suggest that the disruption of the MeCP1 complex could augment the expression of the fetal/embryonic globin genes. In the studies presented in chapter 2, we have pursued a structural and biophysical analysis of the interaction between two of the six components of the MeCP1 complex: MBD2 and p66α. These studies show that the coiled coil regions from MBD2 and p66α form a highly stable heterodimeric complex. Further, overexpressing the p66α coiled coil domain in adult erythroid cells can augment the expression of the chicken ρ-globin and human γ-globin genes, by disrupting the assembly of a functional MeCP1 complex. This indicates that the exogenously expressed p66α coiled coil peptide competes with the endogenous p66α for the interaction with the coiled coil domain of MBD2. These studies show that the coiled coil interaction between MBD2 and p66α could serve as a potential targets for the therapeutic induction of fetal hemoglobin. The laboratory showed that knockout of MBD2 in transgenic mice carrying the human β-globin gene cluster, results in an elevated expression of γ-globin in adult erythrocytes. However, MBD2 does not directly bind to the γ-globin gene to mediate its silencing. In the work presented in chapter 3, we have tested the hypothesis that MBD2 may suppress γ-globin gene transcription in adult erythrocytes indirectly, by binding to and repressing transcription of intermediary gene/s which may be involved in γ-globin gene regulation. Employing microarray technology, we have identified Gab1 and ZBTB32 as candidate genes that may be involved in the MBD2 mediated silencing of γ-globin.
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Anbo, Anders. "Identification of extreme load cases for a surface drill rig by means of MBS simulations." Thesis, Uppsala University, Division of Scientific Computing, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130228.
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This master thesis is Atlas Copco Craelius’ first step in incorporating numericalmethods in load case analysis during the development process. Atlas Copco needs toconstantly evolve and refine their methods in the design process to remain as thenumber one manufacturer of mining and construction equipment. Poor knowledge ofloads results either in structural failures or in oversized structures, both very costlyfor Atlas Copco.The main goal of this thesis is to examine the potential in MBS software by using it toidentify extreme load cases in one of Atlas Copco Craelius’ surface drill rigs, Mustang5. The MBS-software ADAMS View is used to build a model of the Mustang 5 drill rigand evaluate simulation results. The rig model is subject to motions which representreal case scenarios. The feed positioning possibilities are examined thoroughly since it was expected that the positioning has impact on the load levels. 25 different feedpositioning are simulated.The main conclusion is that the load levels are highly dependent on the feedpositioning. For example, the load levels in the boom raising cylinder can increaseseven times, if the rig is operated with the most unfavorable positioning compared tothe recommended. It could also be concluded that not only one positioning isextreme in terms of loads; it depends on which part of the boom system is beingobserved. Thus, several positioning cases have to be taken into consideration in orderto optimize the design of parts in the boom system.
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Walavalkar, Ninad. "Structural basis of DNA binding complexes." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3162.
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The nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex, which couples histone deacetylation and chromatin remodeling ATPase activities, and has a broad cellular and tissue distribution. Although the working model of how this complex forms and functions is not well known, we have demonstrated that the coiled-coil interaction between two proteins (MBD2 and p66α) is critical for DNA methylation dependent gene silencing in vivo. Chapter one: ‘Unique features of the anti-parallel, heterodimeric coiled-coil interaction between methyl-cytosine binding domain 2 (MBD2) homologues and p66α dictate high affinity binding’ describes this unique coiled coil interaction. Coiled-coils were studied using a variety of biophysical techniques including analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and circular dichroism (CD). Results were compared across homologues and mutation studies were carried out to test our hypotheses. The studies reported in this chapter add to our understanding of coiled-coil interaction and thereby facilitate development of small peptide based drugs which target such interactions in nature.A number of proteins have been identified in humans that specifically bind to methylated CpG via a methyl binding domain (MBD). The human genome encodes at least five MBD proteins: MeCP2 and MBD1 through MBD4, which are homologous in their methyl binding domains but not many similarities are seen outside the MBD. Out of the five MBDs, MBD4 has a c-terminal glycosylase domain through which it recognizes mCpG.TpG mismatch and is important for base excision repair system. Chapter two: ‘Dynamic behavior of MBD4 in methylated DNA recognition’ focuses on MBD4 and its preference for DNA methylation mark. Techniques of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy are used to study binding affinity for variations of methylated DNA mark. Chemical exchange studies are used to demonstrate how MBD4 scans for methylation mark and these studies have added a new dimension to our understanding of how MBD proteins ‘read’ DNA methylation marks. Chapter three: ‘Solving the solution structure of MBD domain of MBD4 on methylated DNA by NMR’ describes a process of structure determination using NMR spectroscopy. The focus of this chapter is not on developing a new technique but rather on using current resources to solve a protein structure, which can be used to further understand our biological system. Here, I have discussed the workflow used to determine a final three-dimensional structure starting from sample preparation, data collection, data analysis to structure calculation.
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Haiberger, Ilka [Verfasser], and Bernd [Gutachter] Lenz. "Genexpression von DNMT-1, -3a und -3b, MBD2, CREB, HERP und Alpha-Synuclein während des frühen Alkoholentzugs / Ilka Haiberger ; Gutachter: Bernd Lenz." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2017. http://d-nb.info/1148105107/34.
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Mathot, Pauline. "Mécanismes épigénétiques et réponse des cellules cancéreuses au microenvironnement : implication de la méthylation de l’ADN et de l’un de ses interprètes, MBD2." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1163/document.
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Les cancers du sein ont la particularité de développer un microenvironnement tumoral important où les fibroblastes associés au cancer (CAF) jouent un rôle crucial dans la tumorigénèse via la sécrétion de différents facteurs de croissance, cytokines, protéases et composants de la matrice extracellulaire. Ces différents facteurs secrétés par les CAFs sont impliqués dans de nombreuses voies de signalisation suggérant que la reprogrammation des cellules cancéreuses par les CAFs peut affecter de nombreux gènes.Le séquençage des ARN messagers (RNAseq) de lignées cellulaires de cancer du sein cultivées en présence de milieux conditionnés de CAF, nous a permis d'identifier 372 gènes surexprimés in vitro par les facteurs sécrétés par les CAF et in vivo selon la teneur en cellules stromales des tumeurs mammaires. De façon inattendue, nous avons pu constater que les facteurs sécrétés par les CAF ainsi que le contenu en cellules stromales des tumeurs n'induisent pas de changements significatifs de méthylation de l'ADN mais activent de manière spécifique des gènes caractérisés par une signature méthylation. Différentes approches expérimentales, telles que l'inhibition de la méthylation de l'ADN, l'inhibition de l'expression de la protéine MBD2 (protéines de liaison à l'ADN méthylé) et des expériences d'immuno-précipitation de la chromatine (ChIP) ont permis de montrer l'implication de ces marques de méthylation et des protéines de liaison à l'ADN méthylé dans la réponse des cellules cancéreuses aux facteurs sécrétés par les CAFs. Ces résultats ont permis l'identification d'événements moléculaires impliqués dans la réponse des cellules tumorales aux signaux sécrétés par les cellules stromales dans les tumeurs mammaires mettant en lumière l'importance des marques épigénétiques dans la reprogrammation des cellules cancéreuses induites par les cellules stromalesBreast cancers develop in complex tissue environments where cancer associated fibroblasts (CAF) play a crucial role in tumorigenesis by secreting various growth factors, cytokines, proteases and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation, and epigenetic modulation suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation signature: hypermethylation at transcription start site (TSS) and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of MBD2, and ChIP assays) demonstrated the implication of DNA methylation and methyl DNA binding protein in the response of cancers cells to CAF-secreted factors. These data put in light the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicate that the interpreters of the DNA methylation signal play a major role in the response of the cancer cells to the microenvironment
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Frydén, Cecilia, and Nadia Omri. "Effektivisering av konstruktörens arbete i produktutvecklingsprocessen med hjälp av 3D-modeller och detaljritningar." Thesis, Högskolan i Skövde, Institutionen för ingenjörsvetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-14037.
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I kursen Examensarbete i integrerad produktutveckling har ett projekt utförts av designingenjörsstudenterna Cecilia Frydén och Nadia Omri våren 2017 på Högskolan i Skövde. Projektet genomfördes i samarbete med konsultföretaget ÅF Industry AB i Skövde och några av deras externa tillverkare. I ÅF:s produkt-utvecklingsprocess ägnar konstruktörerna många timmar åt att framställa tillverkningsunderlag, 3D-modeller och detaljritningar. Tillverkningsunderlagen misstänks innehålla överflödig information som inte används av tillverkarna, vilket innebär att konstruktören ägnar tid åt arbetsmoment i onödan. Av den anledningen vill ÅF att det undersöks vilken information i tillverknings-underlaget som är överflödig och att alternativ på hur det utformas tas fram. Aktionsforskning är den övergripande metodiken som används. Projektet har varit fokuserat på att kartlägga arbetsflödet, identifiera mottagarna av tillverkningsunderlaget och deras behov samt definiera tekniska möjligheter för att generera lösningsförslag på alternativa arbetsmetoder. Lösningsförslagen har utvärderats med både konstruktörer och med mottagarna hos tillverkarna. Utifrån utvärderingarna har ett lösningsförslag valts som består av en 3D-modell med Model Based Definition (MBD) där Product Manufacturing Information (PMI) reducerats och förmedlas tillsammans med en 3D-PDF.This project was made for the Bachelor Degree Project in Product Design Engineering course at the University of Skövde by Cecilia Frydén and Nadia Omri in cooperation with the engineering company ÅF during the spring of 2017. ÅF’s designers spends several hours creating 3D-models and detail drawings as manufacturing information for the manufacturing phase of the product development. Some of this time is suspected to be spent unnecessary since some of the information generated by the designers might not be used during the manufacturing process. The aim of the project is to generate a solution to increase the efficiency of the designer’s workflow while generating Product Manufacturing Information (PMI) without compromising the manufacturer’s understanding of the information. Research upon the product manufacturing information generation and handling was done in order to identify its main issues. Observation and interviews were done in order to identify its users and clarify the main purpose of the manufacturing information. The result provides reduced PMI in the 3D-model and uses a 3D-PDF to communicate the PMI to the manufacturers. This was decided from evaluations with the manufacturers and designers and is believed to improve the communication between them but also to make the designer’s workflow more efficient.
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Conger, Michael Anthony. "Validation of CFD-MBD FSI for high-gidelity simulations of full-scale WAM-V sea-trials with suspended payload." Thesis, University of Iowa, 2015. https://ir.uiowa.edu/etd/1960.
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High-fidelity CFD-MBD FSI (Computational Fluid Dynamics - Multi Body Dynamics Fluid-Structure Interaction) code development and validation by full-scale experiments is presented, for a novel hull form, WAM-V (Wave Adaptive Modular Vessel). FSI validation experiments include cylinder drop with suspended mass and 33 ft WAM-V sea-trials. Calm water and single-wave sea-trails were with the original suspension, while the rough-water testing was with a second generation suspension. CFDShip-Iowa is used as CFD solver, and is coupled to Matlab Simulink MBD models for cylinder drop and second generation WAM-V suspension. For 1DOF cylinder drop, CFD verification and validation (V&V) studies are carried out including grid and time-step convergence. CFD-MBD results for 2DOF cylinder drop show that 2-way coupling is required to capture coupled physics. Overall, 2-way results are validated with an overall average error value of E=5.6%DR for 2DOF cylinder drop. For WAM-V in calm water, CFD-MBD 2-way results for relative pod angle are validated with E=14.2%DR. For single-wave, CFD-MBD results show that 2-way coupling significantly improves the prediction of the peak amplitude in pontoon motions, while the trough amplitudes in suspension motions are under-predicted. The current CFD-MBD 2-way results for single-wave are validated with E=17%DR. For rough-water, simulations are carried out in regular head waves representative of the irregular seas. CFD-MBD 2-way results are validation with E=23%D for statistical values and the Fourier analysis results, which is reasonable given the differences between simulation waves and experiments.
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Wang, Yisong. "LOcating Non-Unique matched Tags(LONUT) -improving the detection of the enriched regions for ChIP-seq and MBD-seq data." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306170566.
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Dibra, Harpreet Kaur. "Determination of an interaction between the DNA repair proteins MLH1 and sMBD4 and aspirin regulation of DNA repair gene and protein expression in colorectal cancer." Thesis, University of Wolverhampton, 2010. http://hdl.handle.net/2436/109190.
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The base excision repair protein, MBD4 (also known as MED1) is known to be transcriptionally coupled to a mismatch repair protein MLH1. To date the significance of this coupling has not been elucidated and the significance of MBD4 within the mismatch repair system and apoptotic pathway is still being understood. Recently a novel alternatively spliced form of MBD4 has been identified and termed sMBD4. To date the significance of sMBD4 is unknown. MBD4 and sMBD4 share a common glycosylase domain and this is the domain through which MBD4 is reported to interact with MLH1. It was the aim of this study to determine if sMBD4 was also a binding partner of MLH1 to help elucidate a potential role of sMBD4 and to further characterise the binding domain between MLH1 and MBD4. Recombinant proteins were utilised in binding assays however, a specific protein – protein interaction could not be determined. Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of DNA mismatch repair (MMR) proteins in DNA MMR proficient cells. As MBD4 has previously been suggested to be coupled to MLH1 expression by a post‐translational mechanism the cytotoxicy of aspirin in relation to MBD4 expression was examined. This study reports that aspirin does not up‐regulate MBD4 gene transcription in vitro in the DNA mismatch repair proficient/p53 mutant colorectal cancer cell line SW480. However, MBD4 gene transcription was up‐regulated upon treatment with the aspirin precursor, salicylic acid. The suggested involvement of the DNA repair proteins in the mechanism of action of aspirin promoted the investigation into the expression of DNA damage signalling pathways genes upon aspirin exposure. This study utilised a commercially available PCR array to analyse the expression of 84 DNA damage signalling genes in the SW480 colorectal cancer cell line upon aspirin treatment. It is reported that treatment of the SW480 cell line with aspirin caused changes in mRNA expression of several key genes involved in DNA damage signalling including a significant down‐regulation in expression of the genes encoding ATR, BRCA1 and MAPK12 and increases in the expression of XRCC3 and GADD45α genes. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo‐protective effect of aspirin in vivo.Further to this, protein expression was analysed to determine if correlation could be established with the changes in mRNA expression observed. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45α, an increase in XRCC3 protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This study indicates that alterations in gene expression seen in microarray studies need to be verified at the protein level. Furthermore, this study reports the novel discovery of XRCC3 gene and protein expression being susceptible to exposure to the non‐steroidal anti‐inflammatory drug, aspirin.
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Tervaniemi, Ulrika. "DAMP -hur skolsituationen kan underlättas för elever med DAMP-svårigheter." Thesis, Linköping University, Department of Educational Science (IUV), 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-947.
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Syftet med detta arbete är att genom litteraturstudier och en empirisk undersökning få en bättre insikt i vad DAMP är och vad det kan innebära för elever med denna diagnos. Jag vill även ta reda på vad skolans ansvar är och hur lärare i skolan kan hjälpa och underlätta för elever med dessa svårigheter.
Arbetet består av två delar, varav den första är en litteraturgenomgång där olika forskares syn och åsikter om DAMP är presenterade och sammanställda. Den andra delen består av en undersökande del där intervjuer med fyra olika lärare, om deras kunskaper om hur lärare kan underlätta för DAMP-elever, presenteras.
Genom litteraturen och den empiriska undersökningen har det framkommit att DAMP är ett osynligt handikapp som kan bero på många olika faktorer, av vilka forskarna inte är riktigt överens om. För ett stort antal barn innebär DAMP en mängd olika svårigheter som de behöver hjälp och stöd för att kunna hantera. Det finns inte heller några specifika åtgärder som man kan tillämpa på samtliga barn med dessa svårigheter. Man måste hela tiden se till den enskilda individens behov och förutsättningar och utefter det komma fram till vad som passar bäst för den enskilda eleven. Genom kunskap och med hjärta och hjärna i samspel kan man hjälpa barn med särskilda svårigheter. Det gäller bara att vara lyhörd och öppen för deras behov och att anpassa sig efter vad de behöver.
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Pettersson, Viktoria, and Malin Magnusson. "Efficiency and Automation in the Interface between Airframe Development and Production : A study to identify and reduce time-consuming activities with focus on the methodology of In-Process Part Definition." Thesis, Linköpings universitet, Maskinkonstruktion, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-159933.
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This thesis started as an initiative from one of the co-authors that previously worked at SAAB AB during summer 2018. During the summer she worked with the design process of In-process part Definition (IPPD) and an interest emerged for making it more efficient. The design process of IPPD (DPOI) is where a design article, designed in CATIA, become manufacturable and adapted for assembly. The DPOI can be seen as the interface between the department of Airframe development and Production at SAAB AB. The first step was to investigate the current DPOI and conduct a pre-study to find time-consuming activities. The pre-study consisted of five interviews, an observational study and a time study were the aims was to collect employees' own opinions, approve a pre-defined workflow divided into twelve elements and find problem areas. Element 1.0-11.0 is tasks within the DPOI and element 12.0 is the first step in the review process called Checker. Element 4.0 and 8.0 were divided further into parallel activities where the operators in the time study performs either, e.g., E4.0 (macro) or E4.1 (manually). To find time-consuming activities a time study was performed. The authors of this thesis acted observers and clocked each element while three operators denoted A-C designed 24 IPPDs. The results from the time study showed that elements 1.0, 3.0, 4.1 and 7.0 were time-consuming and E4.1 had potential to become automated. The selection of 2-3 problems was carried out through two Weighted Sum Models (WSM) where criteria was defined and solutions was listed. Each solution was weighted to each criterion and got a total grade. The selected problems, based on the total grade, were: Documents and Combined macro. Documents and manuals for scenario 5, 6 and the entire design process of IPPD was developed to make new employees learning process more efficient. A draft macro for scenario 5 and new complete macros for scenario 1 and 6 was developed and used in the comparative study. The comparative study was conducted like the previous time study but instead the new developed macros was used to make E4.0 more efficient and eliminate E4.1. In the comparative study only E4.0 was clocked for all 24 IPPDs in the time study. The result showed that E4.0 has become average 60% more efficient for all IPPDs and the total time with the new developed macros for E4.0 vs E4.1 has become 14,3% more efficient. Problems and time-consuming activities has been found and improved. The performed comparative study shows that the DPOI can be minimized further in terms of time; there are possibilities to make more elements from the DPOI automated.
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Söderberg, Åsa. "DAMP : ett osynligt handikapp." Thesis, Linköping University, Department of Educational Science (IUV), 1999. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-601.
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Syftet med detta examensarbete är att ta reda på vad DAMP (Dysfunktion i fråga om Avledbarhet, Motorik och Perception) är och hur vi kan hjälpa barn med denna problematik i skolan. För att finna svar på mina frågor i ämnet har jag läst litteratur samt gjort egna empiriska studier. DAMP-problematiken är oerhört utbredd i Sverige idag. Man räknar med att det finns minst ett barn i varje klass som har DAMP i våra skolor. Min viktigaste slutsats i detta arbete är att barn som har DAMP är individer liksom alla människor i samhället. Därför kan man aldrig upprätta ett handlingsprogram som generellt skall användas till elever med DAMP, utan man alltid måste utgå ifrån indivdens särskilda behov.
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Limbach, Anne-Sophie [Verfasser], and Sarah [Akademischer Betreuer] Seiler-Mußler. "Analyse des neuen CKD-MBD-Parameter nox-PTH als renaler und kardiovaskulärer Prädiktor bei chronischer Nierenerkrankung / Anne-Sophie Limbach ; Betreuer: Sarah Seiler-Mußler." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2019. http://d-nb.info/119175569X/34.
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