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Transcription and pre-mRNA processing, e.g., splicing, occur at the same place and time in the context of chromatin. A growing amount of evidence supports the hypothesis that these processes are interconnected. Prp45/SKIP is one of the factors which are believed to mediate the interconnection. The human ortholog, SKIP, is known for affecting mRNA formation on the levels of transcription initiation and elongation. Moreover, it interacts with chromatin modifiers and it is a splicing factor, too. The function of the Saccharomyces cerevisiae ortholog, Prp45, has been so far connected only to pre-mRNA splicing. In this work, we characterized the role of Prp45 in splicing and elaborated the results connecting Prp45 to transcription and chromatin modifications. RNA-seq results showed that pre-mRNA is accumulated in prp45(1-169) cells. This accumulation is not caused by the reduced activity of pathways responsible for RNA degradation. The extent of the splicing inefficiency in prp45(1-169) cells did not depend on either the canonicity of the 5' splice site and branch site or the distance between the branch site and the 3' splice site. Using chromatin immunoprecipitation, we found that prp45(1-169) mutation causes delay in U2 snRNP recruitment to assembling spliceosome. This delay transfers to the later...

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Chronic kidney disease (CKD) is a major public health concern, characterized by an irreversible reduction in renal function. Currently, creatinine-based GFR estimation is predominantly used clinically to characterize CKD. However, this method is known to be an insensitive test for early losses of kidney function. Since patient prognosis relies heavily on slowing further decline of kidney function, uncovering novel biomarkers for kidney function, in conjunction with eGFR, will help improve patient outcome. Epigenetic-based biomarkers have been identified in numerous cancers, as DNA methylation changes alter cellular function. Thus, the objective of this study is to determine novel DNA methylation patterns reflecting altered kidney function. Five healthy participants that have undergone a nephrectomy have donated urine samples before and after their surgery, and global DNA methylation changes were analyzed through the 450K HumanMethylation microarray. Site- and region-level analyses were conducted to determine significant differentially methylated probes post-nephrectomy. The differential associations observed post-nephrectomy are statistically significant in both the site-level and regional analyses. Nineteen significant candidate probes have been systematically selected for validation, based on involvement in kidney function and consistent direction of methylation. Pyrosequencing assays have also been successfully designed and tested with control DNA, however replication of the microarray findings in participant DNA was unsuccessful. The inability to validate these candidate probes may be attributed to many influencing factors, and with this in mind, uncovering novel methylation patterns is still a promising biomarker for evaluating kidney function. / Thesis / Master of Science (MSc)

The multiple physiological effects of the indoleamine hormone melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Our group has shown an upregulation of melatonin receptors following treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and Trichostatin A, in cultured cells and/or in the rat brain. VPA increases histone H3 acetylation at the MT1 gene promoter region in rat C6 glioma cells, indicating that this epigenetic mechanism underlies its upregulation of MT1 expression. Since HDAC inhibitors can also alter DNA methylation, the possible involvement of this second major epigenetic mechanism in the regulation of MT1 expression, was examined. C6 cells were treated with the DNA demethylating agent, azacytidine (AZA, 1 - 25 µM), for 24 or 48 hours. Treatment of C6 cells with AZA caused a significant upregulation of MT1 mRNA expression, as compared with controls (DMSO 0.05%). Moreover, treatment with AZA (10 or 20 µM) for 24 or 48 hours, suppressed or abolished DNMT1 protein expression, and inhibited DNMT1 mRNA expression, which indicates inhibition of the DNMT1 enzyme activity. A combination of VPA and AZA caused a trend toward additive upregulation of the MT1 receptor. These results show that DNA demethylation plays a role in the regulation of the MT1 receptor, consistent with the well-known effects of this epigenetic mechanism on gene transcription. Epigenetic regulation of melatonin receptor expression could provide a novel strategy for modulating the therapeutic effects of this hormone and its clinically relevant agonists, such as agomelatine, and could also provide avenues for enhancing the antioxidant, neuroprotective, oncostatic and other benefits of this hormone and its agonists. / Thesis / Master of Science (MSc) / The hormone, melatonin, is involved in maintenance of the sleep cycle, and has many neuroprotective effects, initiated by its binding to specific proteins called receptors. Epigenetic or reversible chemical modifications which alter DNA, without changing its sequence, can alter the levels of these receptors. This process can be modulated by drugs, which can increase levels of the melatonin receptor. In this study, the drug 5-Azacytidine (AZA) was used to cause specific chemical changes to DNA, termed demethylation. This thesis shows for the first time, that AZA causes an increase in melatonin receptors. AZA’s ability to cause demethylation was confirmed by observing decreased levels of the protein responsible for DNA methylation, DNA methyltransferase. Melatonin receptors in the brain exhibit changes in disorders such as Alzheimer’s and Parkinson’s disease. Understanding the mechanisms underlying the regulation of these receptors could provide avenues for enhancing the neuroprotective benefits of melatonin and related drugs.

Two different mechanisms participate in the loss of SFRPs expression in cancer cells: allelic loss and epigenetic silencing [1]. Methylation status in grade II and grade III glioma differs. From previous studies which were performed in Germany, 2010 by S. Gotze, M. Wolter, G. Reifenberger, O. Muller and S. Sievers [2], it is known that in grade II glioma SFRP2 gene supposed to be methylated (12.5% - 2 samples from 16 were methylated) , while in grade III glioma commonly is unmethylated (0% - from 14 samples no samples were methylated). SFRP2 gene mRNA expression decreases during transformation of grade II to grade III glioma, what’s why our researches were based on idea that SFRP2 expression level decreases because of methylation on this gene. Secondly, another hypothesis we supported that SFRP2 gene could be silenced due to loss of heterozygosity (LOH). IDH1 (isocitrate dehydrogenase1) mutation occurs in tumor tissues while is absent in normal tissue. Third hypothesis which we done, that IDH1 mutation is a common event in malignant glioma tissues, and can be used for glioma diagnostic as biomarker. Our object for this master thesis was to find SFRP2 (Secreted frizzled related protein-2) gene promoter methylation level in grade II and grade III gliomas. To compare the results with the results publicated in previous studies. The main goal of this thesis is to perform methylation study of the promoter region of SFRP2 gene on 86 samples (42 WHO... [to full text] / Centrinės nervų sistemos (CNS) gliomos yra navikai, kilę iš paraminio nervinio audinio, esančio smegenyse. Yra keletas skirtingų rūšių paraminio nervinio audinio ląstelių: astrocitų, oligodendrocitų ir ependimocitų. Pirminiai centrinės nervų sistemos navikai sudaro apie 2 % visų vėžių atveju. Tuo tarpu gliomos sudaro 64-72% visų pirminių smegenų vėžių. Pirminiai CNS navikai yra lyderiaujanti priežastis, sukelianti vaikų mirtis ir ketvirtoje vietoje pagal svarbą įtakojanti suaugusiųjų (virš 54 metų) mirtis. Kasmet Jungtinėse Amerikos valstijose yra registruojama apie 14 000 naujų gliomų atveju, t.y. 5 nauji atvejai/ 100 000 gyventojų. Prancūzijoje šis skaičius yra 3000 naujų gliomų per metus, t.y. 8 nauji atvejai 100 000 gyventojų. Pagal statistinius duomenis, Lietuvoje 2001 metais buvo užregistruota 247 nauji pacientai sergantys gliomomis ( 7.1 nauji atvejai / 100 000 gyventojų), o 2007 metais 211 naujų susirgimų gliomomis ( 6.2 / 100 000 gyventojų). Pagal Pasaulinės Sveikatos Organizacijos (PSO) priimtus kriterijus (pyktibiškumo laipsnį) gliomos yra skirstomos į keturias stadijas : I- policitinė astrocitoma ; II- astrocitoma, oligo-astrocytoma, oligodendroglioma ; III- anaplastinė astrocitoma, anaplastinė oligoastrocitoma, anaplastinė oligodendrolglioma ; IV- glioblastoma. Daugumos atliktų studijų metu nustatyta, jog gyvenimo prognozė labai priklauso nuo piktybiškumo laipsnio. I stadijos gliomos nėra pavojingos, po sėkmingo gydymo sergantieji išgyvena... [toliau žr. visą tekstą]

Chez Saccharomyces cerevisiae, la methylation de la lysine 4 de l'histone H3 (H3K4) est catalysée par le complexe à activité methyltransférase Set1, conservé au cours de l'évolution. Durant la méiose, l'absence de Set1 conduit à un retard de démarrage de la phase S, et à un défaut dans la formation des coupures double-brin de l'ADN (CDBs). Nous avons cherché à mieux caractériser ces deux conséquences phénotypiques liées à l'absence de Set1. Nous montrons que le retard de réplication est lié à la perte de méthylation de H3K4 mais qu'il ne résulte pas d'un défaut d'activité des kinases responsables de l'activation des origines de réplication ou de l'activation des voies canoniques de surveillance moléculaire liées aux dommages de l'ADN. L'importante diminution de fréquence de CDB sur la majorité des points chauds chez le mutant set1∆ a été corrélée à l'absence de la marque de H3K4 triméthylée. Nous avons confirmé le role de la méthylation de H3K4 sur la base de la diminution générale de la fréquence des CDBs observée en absence des différentes sous-unités du complexe associé à Set1 (COMPASS) ou chez un mutant exprimant une histone H3 non-méthylable (H3K4R). Pour tester la relation de causalité entre méthylation et CDBs, différentes sous-unités du COMPASS, telles que Set1 et Spp1, ont été fusionnées avec le domaine de fixation à l'ADN de Gal4 pour les cibler vers des régions non méthylées et dépourvues de CDB. Gal4BD-Spp1 stimule fortement la fréquence des CDBs à certains loci, y compris en contexte mutant H3K4R. Ainsi, le ciblage de Spp1 peut etre suffisant pour recruter et/ou activer la machinerie de CDB. / In Saccharomyces cerevisiae, the methylation of the lysine 4 of histone H3 (H3K4) is catalysed by the evolutionary conserved Set1 methyltransferase complex. During meiosis, the absence of Set1 leads to a delay of S-phase onset and to a defect in the formation of double-strand breaks (DSBs). Our work was intended to give some clues about these two phenotypic consequences of Set1 loss. We show that the replication delay is linked to the absence of H3K4 trimethylation but does not result from a defect of the kinases responsible for the activation of replication origins or the activation of the canonical DNA-damage checkpoints. The severe decrease of DSB levels at the majority of recombination hotspots in set1∆ has been correlated with the specific marking of DSB sites by H3K4 trimethylation at some loci. We have confirmed the role of H3K4 methylation by observing a general decrease in DSB frequency similar to that of set1∆ in mutants lacking various subunits of the Set1- associated complex (COMPASS) or expressing a nonmethylatable histone H3 (H3K4R). To test for a causal relationship between H3K4 methylation and DSB formation, we have fused different proteins of the COMPASS, such as Spp1 or Set1, with the DNA binding domain of Gal4, in order to target them to H3K4-unmethylated and DSB-cold regions. Remarkably, Gal4BD-Spp1 strongly stimulates DSB formation in naturally cold DSB regions, even in the H3K4R mutant context. Thus, the specific tethering of Spp1 to a chromosome site is sufficient to recruit and/or activate the DSB machinery.

Les bactéries sont capables de vivre dans une grande variété d'environnements différents et sont confrontées à des conditions en constante évolution. Par conséquent, elles doivent rapidement adapter leur métabolisme en utilisant différentes régulations aux niveaux transcriptionnel et traductionnel. Ces régulations sont largement étudiées et bien caractérisées. Cependant, les implications du ribosome sur la modulation de la traduction au cours de la réponse aux stress commencent à être explorées. Dans ce contexte de régulation ribosomique, l'hétérogénéité de la machinerie pourrait jouer un rôle important. En effet, le ribosome n'est pas une particule invariable et ses composants (ARNr, protéines ribosomiques) et leurs modifications peuvent varier. Les modifications des ARNr sont situées dans les sites fonctionnels du ribosome et sont particulièrement conservées, ce qui sous-entend leur potentielle importance. Cependant, leur rôle physiologique n'est pas toujours bien défini. Nous nous sommes intéressés aux méthylations de l'ARNr 16S et avons étudié leur rôle dans la traduction, dans des conditions favorables et stressantes. Nous avons démontré que l'absence de certaines méthylations augmente la traduction dans des conditions stressantes et non stressantes. Ainsi, les ribosomes modifiés peuvent jouer un rôle bénéfique lors de la réponse au stress. Une autre façon d'agir sur la traduction dans des conditions stressantes consiste à cibler les ARNm. C'est notamment le cas des toxines endoribonucléases qui sont spécifiquement produites lors de conditions stressantes. Ainsi, nous avons caractérisé le système toxine-antitoxine HicAB de Sinorhizobium meliloti. Nous prévoyons d’utiliser la toxine HicA afin d’étudier la réponse à son activité endoribonucléase chez les mutants ne possédant pas certaines modifications ribosomiques. / Bacteria are able to live in a large variety of environments and they face constantly changing conditions. Therefore they have to adapt quickly to their metabolism using different regulations at the transcriptional and translational levels. Those types of regulation are extensively studied and well characterized. However, the implications of the ribosome in modulation of translation during stress response remains poorly understood. In this context of ribosomal regulation, the heterogeneity of the machinery could play a relevant role. Indeed, the ribosome is not an invariable particle and its components (rRNAs, r-proteins) and their modifications may vary. Modifications of ribosomal RNAs are clustered in the functional sites of the ribosome and are particularly conserved, underlying their potential importance. However their physiological role is still unclear. We focused on methylations of the 16S rRNA and investigated their role in translation under favourable and stressful conditions. We successfully demonstrated that lack of some methylations increases translation under stressful and non stressful conditions. So, lack of methylation may give an advantage to ribosomes during stress response. Another way to act on translation under stressful conditions resides in targeting mRNAs. This is particularly the case for endoribonuclease toxins that are specifically produced during detrimental conditions. Thus, we characterized S. meliloti toxin-antitoxin system HicAB. We plan to use it in order to study the response to HicA toxin of mutants lacking some ribosomal modifications.

Leung Sau Ching. / Thesis submitted in: October 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-165). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / Abstract (English Version) --- p.iii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Multiple Myeloma (MM) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.3 / Chapter 1.1.2 --- Clinical and Pathologic Features of MM --- p.3 / Chapter 1.1.3 --- Diagnosis and Staging --- p.4 / Chapter 1.1.4 --- Prognosis --- p.6 / Chapter 1.1.5 --- Treatment --- p.7 / Chapter 1.2 --- Molecular Abnormalities of MM --- p.8 / Chapter 1.2.1 --- Genetic Alterations: Chromosomal Aberrations --- p.8 / Chapter 1.2.2 --- Genetic Alterations: Ras Mutations --- p.11 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.12 / Chapter 2.1 --- Epigenetic Alterations: DNA Methylation --- p.12 / Chapter 2.1.1 --- Characteristics of CpG Island --- p.14 / Chapter 2.1.2 --- Mechanism of Methylation-Related Gene Silencing --- p.14 / Chapter 2.1.3 --- DNA Methylation Is Important for Normal Cellular Functions --- p.17 / Chapter 2.1.4 --- DNA Methylation Changes in Cancer Cells --- p.17 / Chapter 2.1.5 --- Global DNA Hypomethylation --- p.18 / Chapter 2.1.6 --- Regional DNA Hypermethylation --- p.20 / Chapter 2.1.6.1 --- De Novo Methylation --- p.21 / Chapter 2.1.6.2 --- DNA Hypermethylation Acts as a Third Pathway to Loss of Function in Carcinogenesis --- p.21 / Chapter 2.1.6.3 --- DNA Hypermethylation Contributes to Tumorigenesis --- p.25 / Chapter 2.1.6.4 --- Methodologies in the Study of DNA Hypermethylation --- p.26 / Chapter 2.1.6.5 --- Single Gene Hypermethylation --- p.28 / Chapter 2.1.6.6 --- Multiple Gene Hypermethylation --- p.30 / Chapter 2.1.6.7 --- Potential Clinical Applications of DNA Hypermethylation --- p.36 / Chapter 2.1.6.7.1 --- Tumor Cells Detection by 5'CpG Island Hypermethylation --- p.37 / Chapter 2.1.6.7.2 --- Prognostic and Predictive Significances of DNA Hypermethylation --- p.39 / Chapter 2.1.6.7.3 --- Therapeutic Intervention of CpG island Hypermethylation --- p.40 / Chapter 2.2 --- DNA Hypermethylation in MM and MGUS --- p.43 / Chapter 2.3 --- Six-Genes Panel for the Hypermethylation Study --- p.45 / Chapter 2.3.1 --- Apoptotic Pathway: DAP-kinase --- p.45 / Chapter 2.3.2 --- Retinoid Signaling Pathway: RARβ --- p.50 / Chapter 2.3.3 --- Angiogenic Pathway: THBS-1 --- p.52 / Chapter 2.3.4 --- Cell cycle Regulatory Pathway: pl6 and p15 --- p.57 / Chapter 2.3.5 --- Ras Signaling Pathway: RASSF1A --- p.62 / Chapter CHAPTER 3 --- BACKGROUND OF STUDY --- p.67 / Chapter 3.1 --- Rationale --- p.67 / Chapter 3.2 --- Hypothesis --- p.69 / Chapter 3.3 --- The Objectives of Study --- p.70 / Chapter CHAPTER 4 --- MATERIALS AND METHODS --- p.71 / Chapter 4.1 --- Culture of Human Multiple Myeloma (MM)-derived Cell Lines --- p.71 / Chapter 4.2 --- Demethylation Treatment --- p.72 / Chapter 4.3 --- Patient and Control Samples --- p.72 / Chapter 4.4 --- DNA Extraction --- p.73 / Chapter 4.5 --- MS-PCR --- p.73 / Chapter 4.6 --- Plasma Cell Isolation --- p.77 / Chapter 4.7 --- RNA Extraction and RT-PCR --- p.78 / Chapter 4.8 --- Statistics --- p.82 / Chapter CHAPTER 5 --- RESULTS --- p.84 / Chapter 5.1 --- Patient Characteristics --- p.84 / Chapter 5.2 --- Single Gene Hypermethylation --- p.87 / Chapter 5.2.1 --- Normal PB Did Not Show Methylation --- p.87 / Chapter 5.2.2 --- DNA Hypermethylation in Human MM-derived Cell Lines --- p.87 / Chapter 5.2.3 --- DNA Hypermethylation in Primary MM --- p.89 / Chapter 5.3 --- Demethylation Treatment --- p.93 / Chapter 5.4 --- Concurrent Hypermethylation --- p.96 / Chapter 5.5 --- Statistical Analyses of Primary MM --- p.101 / Chapter 5.5.1 --- Statistical Analyses Between Single Gene Hypermethylation and Clinical Parameters (Categorical) --- p.101 / Chapter 5.5.2 --- Statistical Analyses Between Single Gene Hypermethylation and Clinical Parameters (Non-Categorical) --- p.101 / Chapter 5.5.3 --- Survival Analyses of Single Gene Hypermethylation --- p.105 / Chapter 5.5.4 --- Correlation Analyses of Concurrent Hypermethylation --- p.107 / Chapter 5.5.5 --- Correlation Analyses Between Concurrent Hypermethylation and Clinical Parameters --- p.107 / Chapter CHAPTER 6 --- DISCUSSION --- p.110 / Chapter 6.1 --- Involvement of Cellular Pathways by Hypermethylation --- p.111 / Chapter 6.1.1 --- Apoptotic Pathway: DAP-kinase and RARβ --- p.111 / Chapter 6.1.2 --- "Cell Cycle Regulatory Pathway: p16, p15 and RASSF1A" --- p.113 / Chapter 6.1.3 --- Angiogenic Pathway: THBS-1 --- p.117 / Chapter 6.2 --- Hypermethylation-Associated Gene Silencing --- p.119 / Chapter 6.3 --- Hypermethylation in Cell Lines and Primary MM --- p.120 / Chapter 6.4 --- Concurrent Hypermethylation --- p.122 / Chapter 6.4.1 --- DNA Hypermethylation is Common in MM --- p.122 / Chapter 6.4.2 --- Extent of Hypermethylation --- p.123 / Chapter 6.4.3 --- Involvement of Cellular Pathways by DNA Hypermethylation --- p.124 / Chapter 6.4.4 --- Concurrent p16 and DAP-kinase Hypermethylation --- p.126 / Chapter 6.5 --- Clinical Applications of DNA Hypermethylation --- p.129 / Chapter 6.5.1 --- Methylation As Tumor Markers for MM --- p.129 / Chapter 6.5.2 --- Prognostic Implications of DNA Hypermethylation in MM --- p.130 / Chapter 6.5.3 --- Correlations Between DNA Hypermethylation and Clinical Parameters --- p.131 / Chapter 6.6 --- MS-PCR --- p.136 / Chapter CHAPTER 7 --- CONCLUSION --- p.137 / Chapter CHAPTER 8 --- FURTHER STUDIES --- p.140 / References --- p.142