Дисертації: "Repeat instabilty"

1

De, Pontual Laure. "Identification de nouveaux facteurs chimiques capables de moduler l'instabilité des répétitions CTG dans la dystrophie myotonique de type 1." Electronic Thesis or Diss., Sorbonne université, 2024. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2024SORUS198.pdf.

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La dystrophie myotonique de type 1 (DM1) est la dystrophie la plus fréquente chez l'adulte avec une prévalence estimée à 1 : 8000 individus. C'est une maladie multi-systémique caractérisée par des atteintes musculaires, cardiaques, cognitives et digestives responsables d'une réduction de l'espérance et de la qualité de vie des patients. Elle est causée par une expansion anormale de répétitions CTG en 3'UTR du gène DMPK. Dans la population générale, le nombre de répétitions est inférieur à 35 CTG tandis qu'il dépasse 50 CTG et peut atteindre jusqu'à plusieurs milliers de répétitions chez les patients. Comme dans d'autres maladies causées par une expansions de triplets répétés, l'expansion CTG est instable chez les patients DM1. La taille des répétitions CTG augmente entre les générations (instabilité intergénérationnelle) et au sein des tissus au cours de la vie des patients (instabilité somatique). Or, le nombre de répétitions héritées ainsi que le niveau d'instabilité somatique corrèlent avec l'âge d'apparition des symptômes ainsi qu'avec leur sévérité. S'attaquer à la mutation elle-même pour stabiliser ou réduire le nombre de répétitions CTG est la voie thérapeutique la plus prometteuse puisque cela permettrait d'agir sur l'ensemble des mécanismes physiopathologiques qui découlent de la présence de la mutation.Dans un premier temps, mes travaux de thèse ont porté sur l'identification de molécules chimiques de repositionnement capables de moduler l'instabilité des répétitions. Le criblage des 1280 molécules de la chimiothèque Prestwick m'a permis d'identifier 39 molécules candidates qui modifient l'expression d'un gène rapporteur suggérant qu'elles pourraient moduler l'instabilité des répétitions. Après étude directe de leur effet sur l'instabilité, j'ai pu exclure quatre de ces molécules qui ne modulent pas l'expression des répétitions. J'ai montré qu'une cinquième molécule, la clomipramine, était capable de moduler l'instabilité des répétitions dans le modèle cellulaire de criblage mais pas dans des modèles cellulaires DM1 murins et humains.Parallèlement, j'ai démontré que RGFP966, un inhibiteur sélectif d'HDAC3, induisait des contractions des répétitions CTG dans des fibroblastes murins DM1 avec environ 650 répétitions CTG. Cet effet semble dépendre de la dose de RGFP966 ou de la taille de la répétition CTG car il n'a pas été reproduit dans des fibroblastes humains DM1 avec 350 CTG. Une approche RNA-seq dans les cellules murines traitées avec RGFP966 a permis d'identifier plusieurs gènes candidats impliqués dans la réplication de l'ADN, comme modificateurs possibles de l'instabilité. J'ai également montré une diminution de la transcription bidirectionnelle de DMPK associée à une probable hyperméthylation en aval des répétitions dans les cellules murines DM1. En conclusion, mes données suggèrent que RGFP966 module l'instabilité des répétitions CTG dans la DM1 par de multiples mécanismes, incluant potentiellement une modification de la structure chromatinienne au locus DM1 et des altérations de la réplication de l'ADN.L'ensemble de mon projet de thèse a permis de progresser dans la compréhension des mécanismes de l'instabilité et dans l'identification de molécules chimiques modulant la dynamique de l'instabilité. Mes travaux de thèse ont également permis de relever les limites de chacun des modèles utilisés et la complexité d'identifier de petites molécules modifiant la dynamique des triplets CTG en utilisant des modèles cellulaires rapporteurs. Par ailleurs, j'ai participé au développement pour la DM1 du séquençage à longues lectures (avec et sans amplification), un nouvel outil rapide et très informatif pour analyser la mosaïque somatique
Myotonic dystrophy type 1 (DM1) is the most common dystrophy in adults, with an estimated prevalence of 1:8000 individuals. It is a multisystemic disease characterized by muscle, cardiac, cognitive, and digestive impairments, which contribute to a reduction in both life expectancy and quality of life for patients. DM1 is caused by an abnormal expansion of CTG repeats in the 3'UTR of the DMPK gene. In the general population, the number of repeats is under 35 CTG, whereas in patients, it exceeds 50 CTG and can reach several thousand repeats. As in other diseases caused by repeat expansions, the CTG expansion in DM1 is unstable. The repeat size increases across generations (intergenerational instability) and within tissues during a patient's lifetime (somatic instability). The number of inherited repeats and the level of somatic instability correlate with the age of onset and severity of symptoms. Thus, targeting the mutation itself to stabilize or reduce CTG repeat length is the most promising therapeutic strategy, as it would address all the pathophysiological mechanisms resulting from the mutation.Initially, my thesis work focused on identifying repositioned chemical molecules capable of modulating repeat instability. Screening the 1280 molecules from the Prestwick Chemical Library allowed me to identify 39 candidate molecules that alter the expression of a reporter gene, suggesting they could modulate repeat instability. After directly studying their effect on instability, I excluded four of these molecules that do not modulate repeat expression. I demonstrated that a fifth molecule, clomipramine, can modulate repeat instability in the screening cell model but not in murine and human DM1 fibroblasts.Concurrently, I showed that RGFP966, a selective HDAC3 inhibitor, induced contractions of CTG repeats in murine DM1 fibroblasts with approximately 650 repeats. This effect appears to depend on the dose of RGFP966 or the size of the CTG repeat, as it was not replicated in human DM1 fibroblasts with 350 CTG repeats. An RNA-seq approach in murine cells treated with RGFP966 identified several candidate genes involved in DNA replication as possible modifiers of instability. I also showed a decrease in bidirectional DMPK transcription associated with a probable hypermethylation downstream of the repeats in murine DM1 cells. In conclusion, my data suggest that RGFP966 modulates CTG repeat instability in DM1 through multiple mechanisms, potentially including chromatin structure modification at the DM1 locus and alterations in DNA replication.Overall, my thesis project contributed to the understanding of repeat instability mechanisms and the identification of chemical compounds that modulate instability dynamics. My work also highlighted the limitations of each model used and the complexity of identifying small molecules that alter CTG triplet dynamics in reporter cell models. Additionally, I participated in developing long-read sequencing (with and without amplification) for DM1, providing a rapid and highly informative new tool for the analysis of somatic mosaicism

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2

Gadgil, Rujuta Yashodhan. "Instability at Trinucleotide Repeat DNAs." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472231204.

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3

Ubink-Bontekoe, Carola Jacoba Maria. "CGG repeat instability and FXR proteins." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2001. http://hdl.handle.net/1765/12091.

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4

Beaver, Jill M. "Trinucleotide Repeat Instability is Modulated by DNA Base Lesions and DNA Base Excision Repair." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/3056.

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Trinucleotide repeat (TNR) expansions are the cause of over 40 human neurodegenerative diseases, and are linked to DNA damage and base excision repair (BER). We explored the role of DNA damage and BER in modulating TNR instability through analysis of DNA structures, BER protein activities, and reconstitution of repair using human BER proteins and synthesized DNA containing various types of damage. We show that DNA damage and BER can modulate TNR expansions by promoting removal of a TNR hairpin through coordinated activities of BER proteins and cofactors. We found that during repair in a TNR hairpin, coordination between the 5’-flap endonuclease activity of flap endonuclease 1 (FEN1), 3’-5’ exonuclease activity of AP endonuclease 1 (APE1), and activity of DNA ligase I (LIG I) can resolve the double-flap structure produced during BER in the hairpin loop. The resolution of the double-flap structure resulted in hairpin removal and prevention or attenuation of TNR expansions and provides the first evidence that coordination among BER proteins can remove a TNR hairpin. We further explored the role of BER cofactors in modulating TNR instability and found that the repair cofactor proliferating cell nuclear antigen (PCNA) facilitates genomic stability by promoting removal of a TNR hairpin. Hairpin removal was accomplished by altering dynamic TNR structures to allow more efficient FEN1 cleavage and DNA polymerase β (pol β) synthesis and stimulating the activity of LIG I. This study provides the first evidence that a DNA repair cofactor plays an important role in modulating TNR instability. Finally, we explored the effects of sugar modifications in abasic sites on activities of BER proteins and BER efficiency during repair in a TNR tract. We found that an oxidized sugar inhibits the activities of BER enzymes, interrupting their coordination and preventing efficient repair. Inefficient repair results in accumulation of repair intermediates with DNA breaks, contributing to genomic instability. Our results indicate that DNA base lesions and BER play a crucial role in modulating TNR instability. The research presented herein provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

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5

Ueki, Junko. "Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability." Kyoto University, 2018. http://hdl.handle.net/2433/230991.

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6

Schmidt, Kristina H. "CTG trinucleotide repeat instability in Escherichia coli." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/14353.

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In order to identify cellular factors that affect trinucleotide repeat stability, changes in the length of a (CTG)₄₃ repeat were studied over 140 generations in wild-type Escherichia coli and in strains that are deficient in post-replicative mismatch repair, secondary structure repair and homologous recombination. It is shown that (CTG)₄₃ inserted into pUC18 expands and contracts in wild-type E. coli in an orientation-dependent manner that is unaffected by transcription. In cells deficient in post-replicative mismatch repair (CTG)₄₃ repeat instability is greater than in wild-type cells but orientation-independent. The observation of single trinucleotide insertions and deletions in these mutator mutants indicates that replication slippages of 3 bp occur in vivo leading to repeat expansion and contraction if left unrepaired. Compared to wild-type cells large deletions are reduced in these mutator mutants, but only if the CTG sequence serves as the lagging strand. Based on the opposing effects of mismatch repair a model is proposed in which orientation-dependent CTG repeat instability in mismatch repair proficient cells is caused by the repair of 3-bp slippages. This leads to the creation of larger deletions during repair synthesis due to the formation of unusual secondary structures by the CTG sequence on the lagging strand. Mutations in the recA and sbcCD genes do not affect the stability of plasmid-borne CTG repeats. Similarly the viability of recA-deficient strains carrying chromosomal insertions of (CTG)₂₅ and (CTG)₄₃ suggests that, unlike long palindromes, these trinucleotide repeats are not substrates for the structure-directed nuclease complex SbcCD or, alternatively, they do not form secondary structures frequently enough to cause lethality in recA-deficient hosts. In contrast, a mutation in the recG gene, also involved in homologous recombination, severely destabilises the (CTG)₄₃ repeat in a strongly orientation-dependent manner that exceeds all other tested mutants. Possible explanations for this observation are discussed.

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7

Zahra, Rabaab. "CAG.CTG trinucleotide repeat instability in the E.coli chromosome." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/11667.

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In order to identify the molecular basis of genetic instability, a polymerization-independent strategy is developed to generate expanded repeat arrays. The repeat tracts are integrated in the 5’end of lacZ gene in the Escherichia coli chromosome. Using this model system, instability is studied in wild type E. coli and in strains deficient in cellular pathways such as DNA repair, replication and recombination. The work demonstrates that instability (expansion and contraction) in wild type cells is length and orientation dependent. Longer tracts are more unstable than shorter ones and the orientation where CAG repeats are on the leading strand template is more unstable than the opposite where CTG repeats are on the leading strand template. This orientation-dependence of CAG·CTG trinucleotide repeat instability is determined by the proofreading subunit of DNA polymerase II (DnaQ) in the presence of the hairpin nuclease SbcCD. The analysis of the sizes of deletions observed in wild type and mutant cells is consistent with the formation of secondary structures in vivo. The mismatch repair pathway does not affect the instability of CTG repeats in the E. coli chromosome but influences the CAG orientation. It is suggested that MutS stabilizes CAG repeats by initiating a “repair” process and protecting hairpins from SbcCD, which can cleave hairpins in the presence of MutL and MutH. Finally, the roles of two helicases, Rep and UvrD are analyzed. A mutation in rep helicase strongly destabilizes CTG repeats with no effect on the CAG orientation UvrD mutants show instability in both orientations. The increase in instability in the uvrD mutant depends on RecF in the CTG orientation.

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8

Chan, Kara Y. "MECHANISMS OF TRINUCLEOTIDE REPEAT INSTABILITY DURING DNA SYNTHESIS." UKnowledge, 2019. https://uknowledge.uky.edu/toxicology_etds/29.

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Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

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9

Pickett, Hilda A. "Molecular characterisation of instability in human telomere repeat arrays." Thesis, University of Leicester, 2002. http://hdl.handle.net/2381/30343.

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Telomere instability was investigated at the proximal ends of human telomeres in normal and abnormal cells, with the aim to identify the frequency and types of mutations underlying telomere repeat turnover. Analysis of the interspersion patterns of telomere and variant repeat types at the proximal ends of the 12q and Xp/Yp telomeres in human pedigrees gave a germline mutation frequency of 0.6% per telomere per gamete over the proximal 1kb of the telomere repeat array. No somatic telomere mutations were identified in normal fibroblast cells, but the upper limit for the mutation frequency was estimated as 7.468xlO-3 per cell. Each of 7 germline mutation events involved increases or decreases in small numbers of repeats. These events can be explained by intra-allelic mutational mechanisms, such as replication slippage and unequal sister-chromatid exchange. Localised telomere instability was associated with the CTAGGG variant repeat type in 6/7 germline mutations. Approximately 15% of sporadic colon cancers and the majority of tumours from patients with hereditary non polyposis colon cancer (HNPCC) are caused by defects in genes involved in the mismatch repair pathway. Such defects result in an accummulation of mutations, particularly at microsatellite loci. A high mutation frequency was observed in the 12q and Xp/Yp telomere repeat arrays in colon cancers and was particularly associated with tumours showing microsatellite instability. The observed tumour mutations also involved increases or decreases in blocks of like-repeat types and can be explained by intra-allelic mutations. In one tumour, a complex telomere mutation was provisionally identified and may represent an inter-allelic mutation event. No telomeric mutations were observed in the first kilobase of the 12q and Xp/Yp telomeres in cell-lines derived from patients suffering from the premature ageing disorder Werner syndrome. The lack of telomere mutations in Werner syndrome cells is discussed, with respect to overall telomere stability.

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10

Chan, Nelson Lap Shun. "IDENTIFICATION OF ACTIVITIES INVOLVED IN CAG/CTG REPEAT INSTABILITY." UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/832.

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CAG/CTG repeat instability is associated with at least 14 neurological disorders, including Huntington’s disease and Myotonic dystrophy type 1. In vitro and in vivo studies have showed that CAG/CTG repeats form a stable hairpin that is believed to be the intermediate for repeat expansion and contraction. Addition of extra DNA is essential for repeat expansion, so DNA synthesis is one of the keys for repeat expansion. In vivo studies reveal that 3’ CTG slippage with subsequent hairpin formation (henceforth called the 3’ CTG slippage hairpin) occurs during DNA synthesis. It is proposed that hairpin tolerance machinery is activated because prolonged stalling of DNA polymerase triggers severe DNA damage. As a means toward studying the hairpin-mediated expansion, we created a special hairpin substrate, mimicking the 3’ CTG slippage hairpin, to determine which polymerase promotes hairpin bypass. Our studies reveal polymerase β (pol β) is involved in the initial hairpin synthesis while polymerase δ (pol δ) is responsible for the resumption of DNA synthesis beyond the hairpin (extension step). Surprisingly, we also found that the pol δ can remove the short CTG hairpin by excision of the hairpin with its 3’ to 5’ exonuclease activity. Besides repairing the hairpin directly, resolving the hairpin is an alternative pathway to maintain CAG/CTG repeat stability. With limited understanding of which human helicase is responsible for resolving CAG/CTG hairpins, we conducted a screening approach to identify the human helicase involved. Werner Syndrome Protein (WRN) induces the hairpin repair activity when (CTG)35 hairpin is formed on the template strand. Primer extension assay reveals that WRN stimulates pol δ synthesis on (CAG)35/(CTG)35 template and such induction was still found in the presence of accessory factors. Helicase assay confirms that WRN unwinds CTG hairpin structures. Our studies provide a better understanding of how polymerases and helicases play a role in CAG/CTG repeat instability. Considering CAG/CTG repeat instability associated disorders are still incurable, our studies can provide several potential therapeutic targets for treating and/or preventing CAG/CTG repeat associated disorders.

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11

Warner, Stuart A. "Roles of recombination in trinucleotide repeat instability in E.coli." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/13211.

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12

Mihaescu, Camelia. "Investigation of trinucleotide repeat instability in the Escherichia coli chromosome." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/12655.

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The expansion of trinucleotide repeat tracts is the cause of nearly twenty genetic disorders. Almost all these diseases are characterised by anticipation, which means an earlier age of onset and an increased severity of the symptoms from one generation to the next. The mechanisms of trinucleotide repeat expansion are not understood. In the course of this project, I have investigated the instability of a trinucleotide repeat array of 43 copies integrated at the attB site of chromosomes of various Escherichia coli mutants. The trinucleotide repeat tract (CTG)₄₃was integrated into the E. coli chromosome in both possible orientations using an intermediate vector and exploiting site-specific recombination between the attB site of the chromosome and the attP site of the vector. Using this method I have constructed 60 mutation strains of E. coli which contain the trinucleotide repeat tract and are deficient in genes involved in replication, recombination, secondary structure repair or mismatch repair. Techniques for the analysis of the instability of the trinucleotide repeat arrays were developed and used to quantify repeat instability. These included: digestion of chromosomal DNA with a rare-cutting restriction endonuclease and PAGE of the labelled fragments; PCR of the trinucleotide repeat tract, followed by restriction enzyme digestion and PAGE; fluorescent PCR and f-TRAMP (fluorescent trinucleotide amplification which uses just one primer in repeated cycles of linear primer extension): products were separated by capillary electrophoresis and analysed using Gene Scan software. Intensive analyses of different E. coli mutant showed that the trinucleotide repeat arrays integrated into the chromosome are stable. Except in one case, no instability was observed in any mutant deficient in replication, recombination, mismatch repair or secondary structure repair. The only strain, which showed instability, was a mutD mutant (impaired in the proof-reading activity of DNA Polymerase III). Possible explanations for this observation are discussed.

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13

Ren, Yaou. "Trinucleotide Repeat Instability Modulated by DNA Repair Enzymes and Cofactors." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3762.

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Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.

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14

Barber, Ruth Caroline. "Radiation-induced instability at mouse expanded simple tandem repeat (ESTR) loci." Thesis, University of Leicester, 2002. http://hdl.handle.net/2381/34456.

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Expanded Simple Tandem Repeat (ESTR) loci provide a useful system to assess the effect of exposure to ionising radiation on the germline of male mice; however, little is known about the mutation mechanism(s) at these loci. Information about mutation processes at these loci may provide important clues concerning the damaging effects of irradiation at the DNA level. A number of approaches have been used to investigate possible mutation mechanisms. No correlation was observed between the levels of meiotic recombination and ESTR mutation rate in the germline of exposed male mice, ruling out the possibility that radiation induced mutation at ESTR loci resulted from an increase in meiotic crossing-over. The analysis of the murine scid mutation on ESTR mutation rate demonstrated that the process of non-homologous end-joining (NHEJ) is important in the stability of ESTR loci in the germline of non-exposed mice, but was unable to ascertain whether the activation of NHEJ could provide a plausible explanation for radiation-induced increases in ESTR mutation rate. A transgenerational study of the descendants of directly exposed male mice provided evidence for a long-term effect of ionising radiation on ESTR stability in the mouse germline. ESTR instability was observed in the germline of the offspring and grandoffspring of the initially irradiated males, with no evidence for a decrease in mutation rate. This analysis also provided additional information about the inheritance of ESTR instability in the mouse germline, demonstrating the possibility that the transmission of instability was epigenetic in nature, and showing that the effect could be observed after exposure to both high- or low-LET sources of irradiation. The data also showed transgenerational effects in three different mouse strains, and that there were no differences in the inheritance of ESTR instability between the male and female germlines. The work presented here provides the basis for a number of new and exciting directions to further analyse radiation-induced mutation at ESTR loci.

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15

Narayanan, Vidhya. "Inverted repeats as a source of eukaryotic genome instability." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24774.

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Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2009.
Committee Chair: Lobachev, Kirill; Committee Co-Chair: Chernoff, Yury; Committee Member: Crouse, Gray; Committee Member: Goodisman, Michael; Committee Member: Streelman, Todd.

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16

Xu, Meng. "Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1576.

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Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

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17

Kim, Hyun-Min. "Genome instability induced by triplex forming mirror repeats in S.cerevisiae." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/33874.

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The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

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18

Rindler, Paul Michael. "Eukaryotic replication, cis-acting elements, and instability of trinucleotide repeats." Oklahoma City : [s.n.], 2009.

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19

Bourn, Rebecka Lynn. "Effects of the mismatch repair system on instability of trinucleotide repeats." Oklahoma City : [s.n.], 2009.

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20

Zhang, Yu. "Mechanisms of chromosomal instability induced by unstable DNA repeats in yeast S.cerevisiae." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52185.

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DNA repetitive sequences capable of adopting non-B DNA structures are a potent source of instability in eukaryotic genomes. They are strong inducers of chromosomal fragility and genome rearrangements that cause various hereditary diseases and cancers. In addition, a subset of repeats also has an ability to expand, which leads to more than 20 human genetic diseases that are collectively known as repeat expansion diseases. However, the mechanisms underlying the potential of these structure-prone motifs to break and expand are largely unknown. In this study, a systematic genome-wide screen was employed in yeast Saccharomyces cerevisiae to investigate the contributing factors of the instability of two representative non-B DNA-forming repeats: the triplex-adopting GAA/TTC tracts and the inverted repeats that can form hairpin and cruciform structures. The GAA/TTC screen revealed that DNA replication and transcription initiation are the two major pathways governing the GAA/TTC stability in yeast, as corresponding mutants strongly induce both fragility and large-scale expansions of the repeats. The inverted repeats screen and follow-up experiments revealed that both replication-dependent and -independent pathways are involved in maintaining the stability of palindromic sequences. We propose that similar mechanisms could operate in the human cells to mediate the deleterious metabolism of GAA and inverted repeats.

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21

Seriola, Petit Anna. "Pluripotent stem cells as research models: the examples of trinucleotide repeat instability and X-chromosome inactivation." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/325148.

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Els models de malalties són una eina bàsica per la comprensió de les malalties humanes. Actualment, la majoria de la informació de la que disposem de malalties humanes es basa en models animals. Tot i això, els models animals difereixen molecular i fenotípicament dels humans, i no sempre reprodueixen fidelment la malaltia humana. En les últimes dècades, les cèl·lules mare humanes s’han establert com una opció molt interessant en el camp de la modelització cel·lular. En aquest treball hem volgut caracteritzar les cèl·lules mare embrionàries com a models per a l’estudi de la inestabilitat de la repetició de trinucleotids a la distròfia miotonica tipus 1 (DM1) i la malaltia de Huntington (HD). Així mateix, hem volgut estudiar la inactivació del cromosoma X amb la intenció de fer servir linees cel·lulars com a models per l’estudi del desenvolupament embrionàri humà. A la primera part d’aquest treball, hem observant una inestabilitat de repeticions de trinucleotids significativa al locus de la malaltia DM1 de les cèl·lules mare estudiades. La diferenciació d’aquestes cèl·lules va estabilitzar el número de repeticions. L’estabilització de les repeticions va ser concomitant amb la regulació a la baixa de l’expressió de gens involucrats en els mecanismes de reparació cel·lular. Posteriorment a la publicació del nostre article, altres grups varen reproduir els nostres resultats, però en aquest cas utilitzant cèl·lules mare induïdes. Els estudis recolzen la reproductibilitat dels nostres resultats, suggerint que poden ser extrapol·lats a altres linees de cèl·lules mare arreu del mon. Referent a la mutació de HD, varem trobar que era estable en totes les condicions estudiades, en cèl·lules indiferenciades, diferenciades a progenitors d’os, teratomes i progenitors neurals. Aquests resultats estan en concordancia amb els resultats obtinguts per altres grups que descriuen un baix nombre de repeticions al locus de HD. Per altra banda, varis grups han descrit la presencia de inestabilitat de les repeticions en cèl·lules diferenciades a la linea neural. La discrepància entre els nostres resultats i aquests últims podria ser deguda a la obtenció de cèl·lules neurals menys madures en el moment del nostre estudi. A la segona part d’aquesta tesis hem estudiat la inactivació del cromosoma X en 23 línies femenines de cèl·lules mare pluripotents. Vàrem observar una ràpida progressió de les cèl·lules de dependència de XIST en la inactivació del cromosoma X cap a un estat d’adaptació al cultiu que es caracteritza per un estadi de inactivació independent de l’expressió de XIST i amb una erosió de la metilació. També describim un patró d’inactivació esbiaixat en la majoria de les línies estudiades, contrari al patró aleatori observat en cèl·lules femenines adultes. A més a més, aquest patró és independent de XIST, de l’origen del cromosoma X i d’aberracions cromosòmiques. Aquests resultats suggereixen que el patró esbiaixat observant esta dirigit provablement per l’activació o repressió d’al·lels específics que es troben en el cromosoma X i que li confereixen a la cèl·lula un avantatge respecte a les altres cèl·lules. En conclusió, les cèl·lules mare pluripotents semblen ser un bon model in vitro per a l’estudi d’ambdues malalties, DM1 i HD, ja que presenten el mateix patró d’inestabilitat de la repetició del trinucleotid que s’observa in vivo. Cal remarcar també la depencia Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the MMR machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicated.
Disease modelling is an essential tool for the understanding of human disease. Currently, much of the information we have on human diseases is based on animal models. However, animal models differ molecularly and phenotypically from humans, and are not always suitable to reproduce with fidelity human diseases. In the past decades, human pluripotent stem cells (hPSC) have emerged as an interesting option in the field of cellular modelling, this development recently having taken up much momentum. In this work, we aimed at characterizing hPSC as models for the study of Myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD) trinucleotide repeat (TNR) instability and to investigate the status of the X-chromosome inactivation with an eye on using these cells as models for early human development. In the first part of our work, we observed a significant TNR instability for the DM1 locus in hESC, and that differentiation resulted in a stabilization of the repeat. This stabilization was concommitant with a downregulation of the mismatch repair (MMR). Our results were later replicated in hiPSC by other researchers, showing their reproducibility and suggesting they may be extrapolated to other hPSC lines worldwide. Regarding the HD repeat, we found it was very stable in all conditions studied, both in undifferentiated hESC and cells differentiated into osteogenic progenitor-like cells, teratoma cells and neural progenitors. This is in line with other studies showing that hESC show very limited TNR in the HD locus. On the other hand, some groups have now reported some instability of this locus in cells differentiated into the neuronal lineage. The instability seen in neuronal lineage in later studies, not in our study, is probably explained by the use of hPSC derived neurons more similar to the cells showing in vivo instability than the ones we were able to generate at the time of the study. In the second part of the thesis we studied the X-chromosome inactivation in 23 female hPSC lines. We found that hPSC rapidly progress from a XIST-dependent XCI state to a culture-adapted, XIST-independent XCI state with loss of repressive histone modifications and erosion of methylation. We also report a remarkably high incidence of non-random XCI patterns, and that this skewing of the methylation patterns is independent from the transition to the XIST-independent XCI state, the origin of the X chromosome or chromosomal aberrations. These results suggest that XCI skewing is possibly driven by the activation or repression of a specific allele on the X chromosome, conferring a growth or survival advantage to the cells. Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the MMR machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicated.

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22

Jones, Hope. "Genetic Characterization and Analysis of Cis and Trans-elements That Facilitate Genome Stability in Saccharomyces cerevisiae." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/193584.

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Chromosomal fragile sites are specific loci associated with a high frequency of breakage and recombination. A cell's ability to repair and/or replicate through a lesion is prerequisite to the maintenance of genomic stability. An improved understanding of fragile site biology and its contribution to replication defects and genomic instability is critical for prevention, intervention, and diagnosis of genetic diseases such as cancer. This work seeks to identify and characterize both trans and cis fragile sites associated elements involved in instability onset and progression. An array of Saccharomyces cerevisiae isogenic DNA repair deficient mutants were utilized to identify genes contributing to the stability or instability of a natural fragile site ~ 403 kb from the left telomere on chromosome VII. Findings suggest that the RAD52 epistasis group, the MRX complex, non-homologous end-joining (NHEJ) pathways, MUS81 and SGS1 helicases, translesion polymerases, and a majority of the post replication repair (PRR) proteins are all required for faithful replication of the 403 fragile site and likely other fragile sites as well. In contrast I found that MMS2, previously thought to be specific to the PRR pathway, is required to prevent the fusion of repetitive elements within the 403 site. mgs1 (homolog of the human Werner helicase interacting protein, WHIP) and pol3-13 (a subunit of the DNA polymerase delta) mutants also exhibited reduced instability in checkpoint deficient cells. These findings suggest previously uncharacterized function of Mgs1, Pol3 and Mms2 in regulation of genome regions at risk of replication damage. We further find the presence of inverted repeats (IR) are sufficient to induce instability. Two IR's proximal to the 403 site consistently fuse to generate acentric and dicentric chromosomes involving the 403 fragile site and a newly identified site on chromosome VII as well. The frequency of fusion events is aggravated by chromatin traffic stressors such as tRNA transcription induced fork stalling and replisome termination regions.

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23

Jackson, Adam. "Effect of helicases on the instability of CTG・CAG trinucleotide repeat arrays in the escherichia coli chromosome." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4782.

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A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

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24

Dere, Ruhee J. "The molecular mechanisms involved in the genetic instability of the CCTG. CAGG repeats associated with myotonic dystrophy type 2." Texas A&M University, 2005. http://hdl.handle.net/1969.1/3783.

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Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTGÂCAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTGÂCAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTGÂCAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOSÂ¯ strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.

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25

Fortune, Maria Teresa. "Developmental timing and the role of cis and trans acting modifiers on CTG repeat instability in murine models." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341709.

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26

Tian, Lei. "BIOCHEMICAL CHARACTERIZATION OF HUMAN MISMATCH RECOGNITION PROTEINS MUTSα AND MUTSβ". UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/43.

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The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs and MutSβ recognizing 2-16-nt ID heteroduplexes. The goal of this dissertation research was to understand the mechanism underlying differential mismatch recognition by human MutSα and MutSβ and to characterize the unique functions of human MutSα and MutSβ in MMR. In this study, recombinant human MutSα and MutSβ were purified. Binding of the proteins to a T-G mispair and a 2-nt ID mispair was analyzed by gel-mobility assay; ATP/ADP binding was characterized using a UV cross-linking assay; ATPase activity was measured using an ATPase assay; MutSα amd MutSβ’s mismatch repair activity was evaluated using a reconstituted in vitro MMR assay. Our studies revealed that the preferential processing of base-base and ID heteroduplexes by MutSα and MutSβ respectively, is determined by the significant differences in the ATPase and ADP binding activities of MutSα and MutSβ, and the high ratio of MutSα:MutSβ in human cells. Our studies also demonstrated that MutSβ interacts similarly with a (CAG)n hairpin and a mismatch, and that excess MutSβ does not inhibit (CAG)n hairpin repair in vitro. These studies provide insight into the determinants of the differential DNA repair specificity of MutSα and MutSβ, the mechanism of mismatch repair initiation, and the mechanism of (CAG)n hairpin processing and repair, which plays a role in the etiology and progression of several human neurological diseases.

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27

Kaochar, Salma. "Fusion of Inverted Repeats Leads to Formation of Dicentric Chromosomes that Cause Genome Instability in Budding Yeast." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/204271.

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Large-scale changes are common in genomes, and are often associated with pathological disorders. In the work presented in this dissertation, I provide insights into how inverted repeat sequences in budding yeast fuse during replication. Fusion leads to the formation of dicentric chromosomes, a translocation, and other chromosomal rearrangements.Using extensive genetics and some molecular analyses, I demonstrate that dicentric chromosomes are key intermediates in genome instability of a specific chromosome in budding yeast. I provide three pieces of evidence that is consistent with this conclusion. First, I detect a recombination fusion junction that is diagnostic of a dicentric chromosome (using a PCR technique). Second, I show a strong correlation between the amount of the dicentric fragment and the frequency of instability of the entire chromosome. Third, I demonstrate that a mutant known to stabilize dicentric chromosomes suppress instability. Based on these observations, I conclude that dicentric chromosomes are intermediates in causing genome instability in this system.Next, we demonstrate that fusion of inverted repeats is general. Both endogenous and synthetic nearby inverted repeats can fuse. Using genetics, I also show that many DNA repair and checkpoint pathways suppress fusion of nearby inverted repeats and genome instability. Based on our analysis, we propose a novel mechanism for fusion of inverted repeats that we term `faulty template switching.'Lastly, I discuss two genes that are necessary for fusion of nearby inverted repeats. I identified a mutant of the Exonuclease 1 (Exo1) and a mutant of anaphase inhibitor securin (Pds1) that suppress nearby inverted repeat fusion and genome instability. Studies of Exo1 and Pds1 provide us with insights into the molecular mechanisms of fusion.Our finding that nearby inverted repeats can fuse to form dicentric chromosomes that lead to genome instability may have great implications. The generality of this fusion reaction raises the possibility that dicentric chromosomes formed by inverted repeats can lead to genome instability in mammalian cells, and thereby contribute to a cancer phenotype.

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28

Paek, Andrew Luther. "Formation of Dicentric and Acentric Chromosomes, by a Template Switch Mechanism, in Budding Yeast." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/194260.

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Chromosomal rearrangements occur in all organisms and are important both in the evolution of species and in pathology. In this dissertation I show that in Saccharomyces cerevisiae, or budding yeast, one type of chromosomal rearrangement occurs when inverted repeats fuse, likely during DNA replication by a novel mechanism termed "faulty template switching". This fusion can lead to the formation of either a dicentric or acentric chromosome, depending on the direction of the replication fork. Dicentric chromosomes are inherently unstable due to their abnormal number of centromeres, and thus undergo additional chromosomal rearrangements and chromosome loss.

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29

Greene, Malorie. "Étude des conséquences génomiques et fonctionnelles de l'instabilité des microsatellites dans le cancer colorectal." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066592/document.

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L’instabilité des séquences répétées microsatellites du génome (courtes répétitions en tandem d’un à cinq nucléotides) est une conséquence de l’inactivation du système MMR (MisMatch Repair), en charge de la réparation des erreurs produites au cours de la réplication de l’ADN. Cette instabilité est associée à un processus de transformation cellulaire original, observé chez l’homme dans des pathologies tumorales fréquentes, nommées MSI (pour Microsatellite Instability). Les localisations primaires les plus fréquentes de ces tumeurs sont le côlon, l’endomètre et l’estomac. Elles peuvent avoir une origine héréditaire (prédisposition familiale ; syndrome de Lynch et apparentés), mais sont dans la majorité des cas de survenue sporadique. La transformation des cellules MMR-déficientes s’observe dans le contexte de l’accumulation de nombreuses mutations somatiques dans l’ADN tumoral. Certaines ont un caractère oncogénique en favorisant la troncature et la perte de fonction de gènes suppresseurs de tumeur ou apparentés, impliqués dans des voies de signalisations diverses et qui contiennent des microsatellites codants (mutations indels d’une à deux paires de base, décalant le cadre de lecture, fréquemment rapportées dans ces tumeurs). Les travaux présentés dans le cadre de mon doctorat visent à mieux comprendre le rôle de l’instabilité microsatellitaire dans la tumorigenèse MSI. Ils s’inscrivent dans le contexte du décryptage et de l’analyse des données de séquençage d’exome de 47 cancers colorectaux primitifs MSI. Dans le contexte d’un niveau élevé d’instabilité génomique caractérisant ces tumeurs, la mise au point par mon laboratoire d’accueil de modèles probabilistes a permis de dresser une liste restreinte de gènes, remarquables par le fait qu’ils sont affectés par des mutations somatiques dont les fréquences sont exceptionnellement élevées ou basses dans l’ADN tumoral. Sous l’hypothèse que de tels évènements somatiques affectent des gènes clés de la tumorigenèse MSI colique, j’ai focalisé mes recherches sur les gènes dont les altérations sont peu fréquentes. Brièvement, j’ai pu démontrer le caractère délétère d’un petit nombre d’altérations microsatellitaires codantes dont la survenue semble soumise à une pression de sélection négative (N=13). Mes résultats indiquent que ces mutations semblent fragiliser le phénotype tumoral des cellules dans lesquelles elles surviennent, la perte de fonction des gènes qu’elles affectent conduisant à diverses conséquences délétères en fonction du gène candidat (e.g. sensibilisation à la mort cellulaire, perte des capacités proliférative et migratoire, ralentissement de la croissance tumorale). Ces résultats rapportent pour la première fois et à grande échelle, la sélection négative de mutations dans des tumeurs à forte instabilité génomique MSI. Ils ouvrent de nouvelles voies pour la compréhension de ce mode particulier de transformation cellulaire, et sont potentiellement d’intérêt pour la mise au point de thérapies personnalisées pour les patients
Since the discovery of a link between mismatch repair (MMR) deficiency and cancer, microsatellite instability (MSI) is thought as a process underlying cell transformation and tumour progression and invasion. MSI tumours are a subset of frequent human neoplasms, both inherited and sporadic, associated with several primary locations (colon, stomach, endometrium…). In MMR-deficient cells, MSI generates hundreds of frameshift mutations in genes (MSI Target Genes, MSI-TGs) containing coding microsatellite sequences (e.g. -1/+1 bp, insertions/deletions, i.e. indels). Some of these mutations affect genes with a role in human carcinogenesis and are thus expected to promote the MSI-driven tumorigenic process. During my PhD, I aimed to decipher the role of MSI in colon tumorigenesis. I exploited exome-sequencing data available in my lab that were generated from the analysis of a series of 47 human MSI primary colorectal cancer (CRC). Through biostatistics analysis and mathematical models that we designed to interpret mutation rates in the context of the high background for instability characterizing MSI in CRC, we identified a few microsatellites containing genes coding mutations that were negatively selected in MSI colon tumours (N=13). Under the hypothesis that these events may have a negative impact in colon tumorigenesis, I demonstrated that the silencing of these MSI target genes (siRNA/shRNA) was deleterious for MSI cancer cells using in vitro and in vivo models (impairment of proliferation and/or migration and/or response to chemotherapy and/or tumour growth) (Jonchère*, Marisa*, Greene* et al., submitted)

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30

Ezzatizadeh, Vahid. "Friedreich ataxia : investigating the relationships between mismatch repair gene expression, FXN gene expression and GAA repeat instability in human and mouse cells and tissues." Thesis, Brunel University, 2012. http://bura.brunel.ac.uk/handle/2438/7626.

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Friedreich ataxia (FRDA) is the most common inherited ataxia disorder, caused by a GAA repeat expansion mutation within the first intron of the FXN gene. The subsequent deficiency of frataxin protein leads to neurological disability, increased risk of diabetes mellitus, cardiomyopathy and premature death. The exact FRDA disease mechanism is not yet clear, despite some understanding of epigenetic, transcriptional and DNA repair system effects that lead to frataxin reduction. Previous studies have shown that mismatch repair (MMR) genes can affect other trinucleotide repeat disorders by destabilisation of the repeats. Furthermore, it has been proposed that frataxin deficiency might lead to cell malignancy by an as yet undefined mode of action. Therefore, the principle aim of this thesis was to use human and genetically altered mouse cells and tissues to understand the effects of MMR proteins on GAA repeat instability and FXN transcription, and also to identify potential changes in MMR transcription that might cause malignancy in FXN-defective human cells. Firstly, by using FXN and MMR genetically altered mice, MMR proteins were shown to be involved in both intergenerational and somatic GAA repeat instability, although their effects in the two systems were different. Thus, Msh2 or Msh3 were both found to protect against intergenerational transmission of GAA contractions, while loss of Msh2 or Msh3 reduced somatic GAA repeat expansions and increased levels of FXN transcription in brain and cerebellum tissues. Loss of Msh6 induced both intergenerational GAA repeat expansions and contractions, while the frequency of somatic GAA repeat expansions was reduced. Curiously, the level of FXN transcription was also reduced in Msh6-deficient brain and cerebellum tissues. On the other hand, Pms2 was found to protect against both intergenerational and somatic GAA repeat expansions, with loss of Pms2 causing increased GAA repeat expansions and decreased levels of FXN transcription in brain and cerebellum tissues. Finally, loss of Mlh1 led to a reduced frequency of both intergenerational and somatic GAA repeat expansions, but the level of FXN transcription was also reduced in brain and cerebellum tissues. Furthermore, upregulation of MMR mRNA expression was detected in human FRDA fibroblast cells, but downregulation was seen in FRDA cerebellum tissues, suggesting tissue-dependent control of FXN and MMR expression. In summary, these studies indicate that the MMR system can affect GAA repeat expansion instability and FXN transcription through different mechanisms of action. Furthermore, frataxin deficiency can also affect the levels of MMR mRNA expression in a tissue-dependent manner. These findings will assist future investigations aimed at identifying novel FRDA therapies.

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31

Abdelrazik, Donia. "The determinants of audit fees and report lag : a comparative study of Egypt and the UK." Thesis, University of Plymouth, 2017. http://hdl.handle.net/10026.1/9510.

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The determinants of audit fees and report lag: A comparative study of Egypt and the UK Despite the occurrence of recent economic and political events such as the global financial crisis and Arab spring in the Middle East, researchers have not addressed the effects of such events on the auditing profession. That is has given a motive to this study to explore this point of research. This study has three main objectives. The first objective is to investigate the determinants of audit fees and audit report timeliness in the Egyptian and UK contexts. The second objective is to point out how the economic and political events could affect these determinants. The third objective is to make a comparison between the response of auditors towards economic and political instability in both countries. These objectives are set to solve the research problem of this study which is to investigate how the price behaviour of audit fees and report timeliness can vary in two different contexts: Egypt and the UK, and to highlight how auditors respond to such economic (Global Financial Crisis) and political events (Egyptian Revolution). A special attention has been addressed to tourism industry while investigating audit pricing and timeliness decisions throughout this study for two reasons. First, tourism industry play a critical role for the economy of many developing and developed countries. Second, tourism industry is highly affected by any economic and political events. For these reasons, tourism industry is surrounded by high risk during the economic or political instability, and thus this might result in special procedures and decisions taken by the auditors regarding tourism industry clients during instable periods. To take into account the most recent economic and political events, the study sample covers the period of six financial years from 2008 to 2013. This sample period has been chosen to capture the global financial crisis that has taken place during 2008-2009 and also to investigate the effects of the Egyptian revolution that has taken place on January 25, 2011, and subsequent political events. The study sample includes 212 Egyptian companies listed in the Egyptian stock market and the top 350 companies (FTSE 350) listed in the London Stock Exchange. For guaranteeing the preciseness of the findings, advanced panel data Prais-Winsten statistical analysis technique has been used throughout this study. Results of this study reveal consistency between Egypt and UK in most signs of coefficients of audit fees determinants. However, a lot of differences exist between the audit report lag determinants in the Egyptian and UK context that suggest that a one-size-fits-all approach cannot be generalized in audit report lag determinants for various countries. Despite that tourism is a very risky industry that is easily affected by economic and political instability, results reported in Egypt and UK reveal that audit fees charged and audit delay reported for tourism did not differ from other industries in both contexts. Results also reveal that Big N auditing firms in the UK have competitive advantages of not charging an audit fee premium and offering a more timely audit report than non-big N. These advantages increase the demand of Big N in the UK and increase their dominance. On the other side, in Egypt, Big N auditing firms do not offer such advantage of timely audit reports than non-big N, besides, they charge their clients with audit fee premium. That enabled medium sized and small auditing firms to penetrate the Egyptian auditing market and increase their market share, and thus, Big N dominance is not high in the Egyptian audit market as that in the UK. Different auditor responses to global financial crisis (GFC) have been documented in both countries. As auditors in Egypt decreased their audit fees and offered more timely audit report to face the economic recession and the anxiety of investors accompanied with the GFC. However, neither the pricing of auditing services nor the audit report lag have been affected during the GFC in the UK audit market. According to the results of this study, during the Egyptian Revolution, auditors tended to charge audit fees premium without increasing/decreasing audit delay. This implies that the increase in audit fees during revolution was a risk premium due to the instability in economic and political conditions and was not accompanied by any increase in audit effort and delay.

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32

Ithurbide, Solenne. "Variabilité génétique chez la bactérie radiorésistante Deinococcus radiodurans : la recombinaison entre séquences répétées et la transformation naturelle." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112193/document.

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La bactérie Deinococcus radiodurans est connue pour sa capacité à résister à un grand nombre de traitements génotoxiques parmi lesquels on peut citer l’exposition aux rayons ionisants, aux ultra-violets, à la mitomycine C, à la dessication et au stress oxydant. Elle est capable lors d’une exposition à des doses extrêmes de rayons γ générant des centaines de cassures de l’ADN de reconstituer un génome intact en seulement 2 à 3 heures via un mécanisme original, l’ESDSA, impliquant une synthèse massive d’ADN pendant la phase de réparation des cassures de l’ADN. En plus de mécanismes efficaces de réparation de l’ADN, elle possède un kit de survie comprenant une compaction importante du nucléoïde, des mécanismes de protection des protéines contre l’oxydation, une réponse originale aux lésions de l’ADN et des protéines spécifiques induites après irradiation. Tous ces facteurs contribuent au maintien de l’intégrité du génome et à la survie de la cellule lors de l’exposition à différents agents génotoxiques. Souvent considéré comme un organisme ayant une stabilité génomique exceptionnelle, cette bactérie possède dans son génome un grand nombre de séquences répétées et des éléments mobiles et est par ailleurs naturellement compétente. Ce sont autant de facteurs pouvant participer à la variabilité génétique de cette espèce. Je me suis donc intéressée lors de ma thèse à deux processus pouvant participer à l’instabilité génétique chez D. radiodurans : la recombinaison entre séquences répétées et la transformation naturelle.L’introduction dans le génome de D. radiodurans de séquences répétées directes de 438 pb séparées par des régions d’ADN d’une longueur allant de 1479 pb à 10 500 pb m’a permis de mettre en évidence le rôle majeur joué par l’appariement simple brin (Single Strand Annealing ou SSA) impliquant la protéine DdrB, spécifique des Deinococcaceae, joue un rôle majeur dans la recombinaison « spontanée » entre les séquences répétées en absence de la recombinase RecA. L’absence de DdrB dans des souches déficientes pour la recombinaison augmente davantage la perte de viabilité observée dans ces souches ce qui suggère que le SSA participe à la prise en charge de fourches de réplication bloquées, source majeure d’instabilité génétique en absence de stress extérieur, si ces fourches ne peuvent être prise en charge par des voies impliquant des protéines de recombinaison. Je me suis également intéressée à la transformation naturelle et aux protéines impliquées dans ce processus chez D. radiodurans. J’ai pu démontrer que la protéine DprA impliquée dans la protection de l’ADN simple brin et le chargement de RecA sur l’ADN simple brin internalisé lors de la transformation de nombreuses espèces comme Streptococcus pneumoniae, Bacillus subtilis ou Helicobacter pylori, est également impliquée dans la transformation chez D. radiodurans. J’ai pu montrer également qu’en plus de jouer un rôle majeur dans la transformation par de l’ADN plasmidique, DdrB est impliquée dans la transformation par de l’ADN génomique si la protéine DprA est absente
The bacterium Deinococcus radiodurans is known for its ability to withstand a large number of genotoxic treatments, including exposure to ionizing or ultraviolet radiation, mitomycin C, desiccation, and oxidative stress. It is able, upon exposure to extreme doses of γ-radiation generating hundreds of DNA breaks, to reconstitute an intact genome in only 2 to 3 hours via an ESDSA mechanism, involving massive DNA synthesis during DNA double strand break repair. Together with efficient DNA repair mechanisms, D. radiodurans possesses a survival kit comprising significant compaction of its nucleoid, protection mechanisms against protein oxidation, an original response to DNA damage and specific proteins induced after irradiation. All of these contribute to the maintenance of genomic integrity and cell survival upon exposure to various genotoxic agents. In spite of the idea that D. radiodurans is an organism with outstanding genomic stability, this bacterium has in its genome a large number of repeat sequences and mobile elements and is also naturally competent. All these factors contribute to the genetic variability of species. I was interested in two processes that can play a role in genetic variability in D. radiodurans: recombination between repeated sequences and natural transformation.The introduction, into the genome of D. radiodurans, of 438 bp direct repeated sequences separated by DNA regions ranging from 1,479 bp to 10,500 bp in length allowed me to demonstrate the major role of Single Strand Annealing (SSA) involving the DdrB protein specific for Deinococcaceae, in the "spontaneous" recombination between the repeated sequences in the absence of the RecA recombinase. The absence of DdrB in strains deficient for recombination further increased the loss of viability observed in these strains, suggesting that SSA is required for the management of blocked replication forks, a major source of genetic instability in the absence of external stress when these forks cannot be rescued by pathways involving recombination proteins.I was also interested in the natural transformation and proteins involved in this process in D. radiodurans. I demonstrated that DprA protein involved in DNA single strand protection and loading of RecA on single-stranded DNA internalized during transformation of many species such as Streptococcus pneumoniae, Helicobacter pylori, or Bacillus subtilis, is also involved in this process in D. radiodurans. I also showed that, in addition to playing a major role in transformation by plasmid DNA, DdrB is also involved in transformation by genomic DNA of cells devoid of the DprA protein

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33

Martinez, Marcos Antonio Rodrigues. ""Estudo das alterações dos microssatélites D6S251 e D6S252 no carcinoma basocelular esporádico"." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5133/tde-10052006-163753/.

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Existe grande interesse na determinação das bases genéticas do carcinoma basocelular (CBC) que expliquem seu fenótipo pouco agressivo e comportamento metastático infreqüente. Investigamos a instabilidade de microssatélites (MSI) e perda de heterozigosidade (LOH) nos microssatélites D6S251 (6q14) e D6S252 (6q16) de CBCs esporádicos de alto e baixo risco histológico através da análise de bandas obtidas pelo gel de poliacrilamida após PCR em comparação com o tecido normal. Não houve alteração do microssatélite D6S252 nas 15 amostras estudadas. Para o microssatélite D6S251, houve alterações em 6 das 26 amostras estudadas (23,07%). MSI e LOH ocorreram em 46,15% das amostras de alto risco (respectivamente 15,38% e 30,76), o que sugere o provável envolvimento da região 6q14 na diferenciação histológica do CBC
A lot of interest lies in determining the genetic basis of basal cell carcinoma (BCC) to explain the lack of aggressive phenotype and infrequent metastatic behavior. We have analyzed the microsatellite instability (MSI) and loss of instability (LOH) in the D6S251 (6q14) and D6S252 (6q16) microsatellites patterns of histological low- high-risk sporadic BCC tumor samples using PCR-based assay in comparison with normal tissue. We have not found any alteration in D6S252 microsatellite 15 samples studied. We have encountered D6S251 alterations in 6 of 26 BCC samples (23.07%).MSI and LOH occurred in 46.15% of high-risk samples (15.38% and 30.76%), These results probably suggests participation of 6q14 region in histological differentiation of BCC

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34

Chen, Xiaomi. "Aberrant DNA Replication at an Ectopic Chromosomal Site in Human Cells." Wright State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=wright1302884072.

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35

Grillo, Giacomo. "The ICF syndrome and emergent players in DNA methylation and development : when studying a rare genetic disease sheds new light on an "old" field." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC300/document.

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La méthylation de l'ADN est un processus vital pour le développement des mammifères. Sa distribution anormale,notamment au niveau des régions répétées du génome, est une signature pathologique. La découverte de maladies héréditaires touchant la stabilité du génome a permis des avancées considérables dans l'identification des acteurs et des mécanismes. Nous avons choisi d'étudier le syndrome ICF (Immunodéficience, instabilité Centromérique et anomalies Faciales), première maladie génétique identifiée avec des défauts de la méthylation de l’ADN, liés à une instabilité chromosomique. Lorsque j'ai commencé ma thèse, des mutations dans les gènes DNMT3B et ZBTB24 avaient été décrites comme causes génétiques du syndrome. Cependant, d'autres causes génétiques restaient inconnues. Nos travaux ont permis d'identifier deux nouveaux gènes, CDCA7 et HELLS, dont les mutations sont responsables du syndrome. J'ai montré que leur perte de fonction dans les cellules somatiques entraîne un défaut de méthylation des répétitions centromériques, suggérant leur rôle dans le maintien de la méthylation de l'ADN. Par conséquent, l'étude de l'étiologie d'une maladie génétique rare a permis d'identifier de nouveaux « gardiens » de la stabilité du génome, avec des fonctions jusqu'alors insoupçonnées dans les processus de méthylation de l'ADN et dans le développement. Au cours de mon doctorat, j'ai établi des cartes de méthylation des cellules de patients ICF afin d'identifier les cibles communes et distinctes de ces facteurs, ainsi que leurs caractéristiques génomiques et épigénomiques. Contrairement aux mutations de DNMT3B,celles de ZBTB24, CDCA7 et HELLS affectent la méthylation dans des régions pauvres en CpG, dans des régions intergéniques et dans des répétitions d'ADN intercalées. Plus généralement, ce sont les régions d'hétérochromatine qui sont les plus touchées et en particulier des clusters des gènes codants et non codants, dont certains sont exprimés de manière monoallélique. Pour mieux caractériser le rôle de ZBTB24 dans le développement et la méthylation de l'ADN,nous avons généré un modèle murin mutant qui nous a permis de monter que ZBTB24 était essentielle pour le développement embryonnaire précoce. De plus, ZBTB24 jouerait un rôle dans l'établissement de la méthylation des séquences répétées de l'ADN, à la fois en tandem ou intercalé. Fait intéressant, ZBTB24 semble être également impliqué dans l'établissement de la marque répressive H3K9me3, suggérant un rôle de la protéine dans le "dialogue" entre la méthylation de l'ADN et celle des histones. Dans l'ensemble, mon travail met l'accent sur la façon dont la méthylation de l'ADN et les marques d'hétérochromatine sont établies et maintenues à des gènes uniques et des répétitions de l'ADN, et fournit de nouveaux acteurs et mécanismes à considérer dans les études sur le maintien de la stabilité du génome
DNA methylation is an essential process for the development of mammals. Its abnormal distribution, particularly at the level of the repeated regions of the genome, is a pathological signature. The discovery of hereditary diseases affecting DNA methylation and the stability of the genome allowed a considerable progress in the identification of their actors and mechanisms. We chose to study the ICF (Immunodeficiency, Centromeric Instability and Facial Abnormalities) syndrome, the first genetic disorder identified with defects in the distribution of DNA methylation, linked to chromosomal instability. When I started my PhD, mutations in two genes had been described to cause the ICF syndrome: DNMT3B and ZBTB24. However, the genetic origin of a subset of ICF patients remained unknown. We identified mutations in CDCA7 and HELLS as causative of the ICF syndrome. I showed that their loss of function in somatic cells results in the loss of DNA methylation at centromeric repeats, strongly suggestive of a role DNA methylation maintenance. Hence, the study of the aetiology of a genetic disease provided new candidate “guardians” of DNA repeats and genome stability, with virtually unknown functions but with exciting potential roles in the DNA methylation machinery and in development. During my PhD, I established methylation maps in ICF patients cells to identify common and distinct targets of these factors, as well as their genomic and epigenomic characteristics. In contrast to DNMT3B mutations, those in ZBTB24, CDCA7 and HELLS affect methylation at CpG-poor regions in intergenic genomic locations and at interspersed DNA repeats, and more generally, at genomic locations with heterochromatic features. Their integrity is required for the methylated status of coding and non-coding clusters of genes, some of which are expressed in a monoallelic manner. To better characterize the role of ZBTB24 in development and DNA methylation pathways, we generated a mouse model carrying mutations in ZBTB24. We showed that ZBTB24 is essential for early development, while it seemed to be dispensable for in vitro differentiation of murine ES cells. We implicated ZBTB24 in the establishment of DNA methylation at DNA repeats, both in tandem or interspersed, in differentiating ES cells. Interestingly, ZBTB24 seems to be also implicated in the establishment of the repressive mark H3K9me3 suggesting that ZBTB24 may indirectly control DNA methylation through an interplay with histone marks. As a whole, our work sheds light on how DNA methylation and heterochromatin marks are established and maintained at unique genes and DNA repeats, and provides new actors and mechanisms to consider in studies of the maintenance of genome stability

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36

Hureau, Thomas. "Mécanismes de régulation du niveau de fatigue périphérique à l'exercice : implications sur la performance motrice et applications à l'exercice réalisé sur plateforme élisphérique." Thesis, Nice, 2015. http://www.theses.fr/2015NICE4011.

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Cette thèse porte sur l’étude des mécanismes de régulation du niveau de fatigue périphérique à l’exercice et sur les conséquences de cette régulation sur la capacité de performance motrice. Nous avons tout d’abord démontré que la puissance est étroitement régulée au cours de sprints répétés – épreuve au cours de laquelle la commande motrice est recrutée à son niveau maximal volontaire – de sorte qu’un seuil critique de fatigue périphérique ne soit jamais dépassé. Nous avons ensuite montré qu’il existe un lien étroit entre l’atteinte de ce seuil critique de fatigue et l’arrivée à une phase de plateau de puissance développée au cours de sprints répétés, indépendamment de la durée de la récupération entre les sprints. Ces résultats ont permis de démontrer que le niveau d'activité de la commande motrice centrale et la puissance sont régulés au cours de sprints répétés dans le but de limiter le niveau de fatigue périphérique à un niveau seuil. Le rôle de ce mécanisme régulateur est cependant dépendant de la nature de la tâche. Nous avons en effet montré que l’arrêt de l’effort d’un exercice de squat isométrique, conduit jusqu'à épuisement, est associé à une défaillance de l’activation centrale volontaire, qui précède l'atteinte du seuil critique de fatigue. Enfin, l'application de ces données et concepts théoriques à l'exercice réalisé sur une plateforme à instabilité servo-assistée (imoove) a permis de montrer que ce type d’outil permet un recrutement accru des muscles posturaux et de l’équilibre comparé à un exercice réalisé sur une surface stable, sans compromettre la fatigue et le recrutement des muscles locomoteurs, déterminants des adaptations positives à l'entraînement
This doctorate thesis focused on the mechanisms involved in the control of peripheral fatigue during exercise and on the consequences of this regulation on exercise tolerance. We first demonstrated that performance during repeated sprints – a trial during which the central motor drive is activated at its maximal voluntary level – is tightly regulated to avoid the development of peripheral fatigue beyond a critical threshold. We then showed that the attainment of the plateau phase of performance, characterized by a constant power output until the last sprint, was closely linked to the attainment of the critical threshold of peripheral fatigue, independently of the recovery duration between sprints. These firsts results demonstrated that central motor drive and power output are regulated during repeated sprints in order to limit the development of peripheral fatigue beyond a critical threshold. However, the role of this regulatory mechanism is task-dependent. Indeed, we showed that time to task failure during the first repetitions of a sustained submaximal isometric contraction is likely associated with failure in central activation of motor units, which precede the attainment of the critical threshold of peripheral fatigue. We then developed applied research protocols on imoove, a servo-assisted instability device for training composed by a board that can move in the three-dimensional plans. Because greater trunk and ankle muscles activity was achieved in imoove without compromising lower limb muscles activity and fatigue, this device may provide sufficient muscle overload to simultaneously develop locomotor, postural and balance muscles

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37

Missirlis, Perseus Ioannis. "CIS-features mediating CAG/CTG repeat instability the Satellog database, and candidate repeat prioritization in schizophrenia." Thesis, 2004. http://hdl.handle.net/2429/15767.

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Polyglutamine repeat expansions in the coding regions of unrelated genes have been implicated in the neurodegenerative phenotype of nine separate diseases. However, little is known about the role of flanking c/'s-sequences in mediating this repeat instability. Brock et al. identified an association between flanking GC content and CAG/CTG repeat instability at many of these disease loci by using a relative measure of repeat instability called 'expandability'. Using this measure, we have extended the analysis of Brock and colleagues and utilized the expandability metric to associate other features theorized to contribute to CAG/CTG repeat instability such as repeat length and purity, proximity to CCCTC-binding factor (CTCF) binding sites, and the nucleosome formation potential of the surrounding DNA. Our results confirmed earlier relationships regarding flanking G C content and CAG/CTG repeat instability and also suggest a novel one involving flanking CTCF binding sites. Conversely, no relationships between expandability and repeat length, purity, and nucleosome formation were detected. Anticipation refers to the progressive worsening of a disease phenotype and earlier age of onset in successive generations. Anticipation has been reported in a number of diseases in which repeat expansion may have a role in etiology. We developed Satellog, a database that catalogs all pure 1-16 repeat unit repeats in the human genome along with supplementary data of use for the prioritization of repeats in disease association studies. For each pure repeat we calculate the percentile rank of its length relative to other repeats of the same class in the genome, its polymorphism within UniGene clusters, its location either within or adjacent to EnsEMBL-defined genes, and its expression profile in normal tissues according to the GeneNote database. By examining the global repeat polymorphism profile, we found that highly polymorphic coding repeats were mostly restricted to trinucleotide repeats, whereas a wider range of repeat unit lengths were tolerated in untranslated sequence. We also found that 3'-UTR sequence tolerates more repeat polymorphisms than 5'-UTR or exonic sequence. Lastly, we use Satellog to prioritize repeats for disease-association studies in schizophrenia. Satellog is available as a freely downloadable MySQL and web-based database.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

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38

Cleary, John. "DNA Replication and Trinucleotide Repeat Instability in Myotonic Dystrophy Type 1." Thesis, 2010. http://hdl.handle.net/1807/24723.

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The expansion of gene-specific trinucleotide repeats is responsible for a growing list of human disorders, including myotonic dystrophy type 1 (DM1). Repeat instability for most of these disorders, including DM1, is characterized by complex patterns of inherited and ongoing tissue-specific instability and pathogenesis. While the mechanistic basis behind the unique locus-specific instability of trinucleotide repeats is currently unknown, DNA metabolic processes are likely to play a role. My thesis involves investigating the contribution of DNA replication to the trinucleotide instability of myotonic dystrophy type 1. Herein I have designed an in vivo primate model system, based on the SV40 replication system, to assess the contribution of DNA replication to DM1 repeat instability. This system allows the assessment, under controlled conditions, and manipulation of variables that may affect replication-associated repeat instability, under a primate cellular system. Using the SV40 model system, I not only confirmed previous observations that repeat length and replication direction affect repeat instability, but also for the first time determined that the location of the replication origin relative to the repeat tract plays an important role in repeat instability. This novel observation allowed for the development of a fork-shift model of repeat instability, in which cis-elements adjacent to the repeat tract affect replication, in turn altering the propensity for repeat instability. To further my study of DNA replication in DM1 repeat instability, I have mapped the origin of replication adjacent to the DM1 locus in human patient cells and the tissues of DM1 transgenic mice actively undergoing repeat instability. The position of the replication origins adjacent to the repeat tract at the DM1 locus places several known cis-elements, including CTCF binding sites, in a position to alter replication as predicted by the fork-shift model. My analysis of the CTCF sites showed them capable of altering replication and repeat instability at the DM1 locus. Taken together these results suggest that the placement of replication origins, repeat tracts and cis-elements, may mark trinucleotide repeat tracts, such as the DM1, for locus-, tissue- and development-specific replication-associated repeat instability.

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39

"Microsatellite instability in the evolution of cervical neoplasm." 2001. http://library.cuhk.edu.hk/record=b5890890.

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Анотація:

Poon Kin-yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 119-147).
Abstracts in English and Chinese.
ACKNOWLEDGMENT --- p.i
ABSTRACT --- p.iii
ABBREVIATIONS --- p.viii
TABLE OF CONTENTS --- p.x
Chapter CHAPTER I --- INTRODUCTION --- p.1
Chapter 1.1 --- Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer --- p.1
Chapter 1.1.1 --- Epidemiology --- p.3
Chapter 1.1.1.1 --- Descriptive Epidemiology --- p.4
Chapter 1.1.1.2 --- Risk Factors --- p.7
Chapter 1.1.2 --- Pathology --- p.22
Chapter 1.1.2.1 --- Macroscopic Appearance --- p.22
Chapter 1.1.2.2 --- Symptoms and Diagnosis --- p.23
Chapter 1.1.2.3 --- Staging Classification --- p.25
Chapter 1.1.2.4 --- Histopathology --- p.29
Chapter 1.2 --- Microsatellite Instability (MSI) --- p.35
Chapter 1.2.1 --- Microsatellite --- p.35
Chapter 1.2.2 --- Mismatch Repair --- p.37
Chapter 1.2.3 --- Microsatellite Instability (MSI) --- p.38
Chapter 1.2.4 --- MSI in Various Cancers --- p.42
Chapter 1.2.5 --- The Role of MSI in Carcinogenesis --- p.49
Chapter 1.2.6 --- MSI as a Diagnostic / Prognostic Tool --- p.50
Chapter CHAPTER II --- AIMS OF THE STUDY --- p.53
Chapter CHAPTER III --- MATERIALS AND METHODS --- p.56
Chapter 3.1 --- Materials --- p.56
Chapter 3.1.1 --- Patients and Specimens --- p.56
Chapter 3.1.2 --- Microsatellite Markers --- p.57
Chapter 3.2 --- Methods --- p.59
Chapter 3.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.59
Chapter 3.2.2 --- Microdissection of Epithelial Cells and Neoplastic Cells from Specimen Sections --- p.60
Chapter 3.2.3 --- DNA Extraction --- p.60
Chapter 3.2.3.1 --- Normal Blood --- p.61
Chapter 3.2.3.2 --- Dissected Cells --- p.62
Chapter 3.2.4 --- DNA Amplification --- p.64
Chapter 3.2.4.1 --- End-labeling of Primers --- p.64
Chapter 3.2.4.2 --- Polymerase Chain Reaction --- p.65
Chapter 3.2.5 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.66
Chapter 3.2.6 --- Autoradiography --- p.67
Chapter 3.2.7 --- Determination of MSI --- p.67
Chapter 3.2.8 --- HPV Detection --- p.68
Chapter 3.2.9 --- Statistical Analysis --- p.69
Chapter CHAPTER IV --- RESULTS --- p.70
Chapter 4.1 --- Incidence of MSI in Cervix --- p.70
Chapter 4.1.1 --- Incidence of MSI in Normal Cervix --- p.70
Chapter 4.1.2 --- Incidence of MSI in CIN --- p.70
Chapter 4.1.3 --- Incidence of MSI in Cervical Carcinoma --- p.71
Chapter 4.1.4 --- Correlation of MSI-positive with the Evolution of Cervical Neoplasm --- p.77
Chapter 4.2 --- Correlation of MSI-positive with Clinicopathological Characteristics in Cervical Carcinoma --- p.77
Chapter 4.2.1 --- MSI and Age --- p.80
Chapter 4.2.2 --- MSI and Clinical Stage --- p.80
Chapter 4.2.3 --- MSI and Histological Grade --- p.80
Chapter 4.2.4 --- MSI and Clinical Status --- p.81
Chapter 4.3 --- Comparison between Two Panels of Microsatellite Markers used in MSI Detection --- p.84
Chapter 4.4 --- Human Papilloma Virus (HPV) Infection in Cervical Neoplasm --- p.89
Chapter 4.4.1 --- HPV Infection and Typing in CIN and Cervical Carcinoma --- p.89
Chapter 4.4.2 --- Correlation of MSI-positive with HPV Infection in Cervical Carcinoma --- p.94
Chapter CHAPTER V --- DISCUSSION --- p.96
Chapter 5.1 --- MSI Detection --- p.96
Chapter 5.1.1 --- Techniques in MSI Assays --- p.98
Chapter 5.1.2 --- Choice of Microsatellite Markers --- p.101
Chapter 5.1.3 --- Diagnostic Criteria of MSI --- p.105
Chapter 5.2 --- The Role of MSI in the Carcinogenesis of Cervical Neoplasm --- p.107
Chapter 5.3 --- The Clinical Significant of MSI in Cervical Carcinoma --- p.111
Chapter 5.4 --- The Interaction between HPV Infection and MSI in Cervical Carcinoma --- p.113
Chapter CHAPTER VI --- CONCLUSION --- p.116
REFERENCES --- p.119

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40

"Microsatellite instability and its significance in cervical and endometrial cancers." 1999. http://library.cuhk.edu.hk/record=b5889973.

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Анотація:

Ip Toi Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 81-105).
Abstracts in English and Chinese.
CONTENTS --- p.i-iii
ACKNOWLEDGEMENT --- p.iv
ABSTRACT --- p.v-vi
Chapter Chapter One --- INTRODUCTION --- p.1-2
Chapter Chapter Two --- LITERATURE REVIEW --- p.3-37
Chapter 2.1 --- Epidemiology and Etiology of Cervical and Endometrial Cancers --- p.3-4
Chapter 2.1.1 --- Epidemiology and Etiology of Cervical cancer --- p.4
Chapter 2.1.1.1 --- Incidence and Mortality --- p.4-6
Chapter 2.1.1.2 --- Etiology --- p.6-8
Chapter 2.1.2 --- Epidemiology and Etiology of Endometrial Cancer --- p.9
Chapter 2.1.2.1 --- Incidence and Mortality --- p.9-11
Chapter 2.1.2.2 --- Rick Factors --- p.11-14
Chapter 2.2 --- Pathology of Cervical and Endometrial Cancers --- p.14
Chapter 2.2.1 --- Pathology of Cervical Cancer --- p.14-15
Chapter 2.2.1.1 --- Macroscopic Appearance --- p.15
Chapter 2.2.1.2 --- Histology --- p.15-18
Chapter 2.2.2 --- Staging of Cervical Cancer --- p.19-21
Chapter 2.2.3 --- Pathology of Endometrial Cancer --- p.21
Chapter 2.2.3.1 --- Macroscopic Appearance --- p.22
Chapter 2.2.3.2 --- Histology --- p.22-24
Chapter 2.2.4 --- Staging of Endometrial Cancer --- p.24-25
Chapter 2.2 --- Introduction to Microsatellite Instability (MI) --- p.25
Chapter 2.3.1 --- DNA structure --- p.25-27
Chapter 2.3.2 --- Microsatellite --- p.27-28
Chapter 2.3.3 --- Mismatch Repair (MMR) --- p.28-29
Chapter 2.3.4 --- Microsatellite Instability (MI) --- p.30-33
Chapter 2.3.5 --- Microsatellite Instability in various cancers --- p.33-37
Chapter Chapter Three --- MATERIALS AND METHODS --- p.38-50
Chapter 3.1 --- Materials --- p.38
Chapter 3.1.1 --- Patients and Specimens --- p.38-39
Chapter 3.1.2 --- Chemicals and Reagents --- p.39
Chapter 3.1.2.1 --- Chemicals --- p.39-40
Chapter 3.1.2.2 --- Solution --- p.40-41
Chapter 3.1.2.3 --- Microsatellite Markers --- p.42
Chapter 3.1.3 --- Major Equipment --- p.43
Chapter 3.2 --- Methodology --- p.43
Chapter 3.2.1 --- DNA Extraction --- p.43-45
Chapter 3.2.2 --- DNA Amplification --- p.45
Chapter 3.2.2.1 --- End-labeling of Primer --- p.45
Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.46
Chapter 3.2.3 --- Electrophoresis of PCR Products and Autoradiography --- p.46-49
Chapter 3.2.4 --- Determination Of Microsatellite Instability (MI) --- p.49
Chapter 3.3 --- Statistical Analyses --- p.50
Chapter Chapter Four --- Result --- p.51-66
Chapter 4.1 --- Microsatellite Instability in Cervical Cancer --- p.51
Chapter 4.1.1 --- Prevalence of MI in Cervical Cancer --- p.51 -54
Chapter 4.1.2 --- MI and Age in Cervical Cancer --- p.55
Chapter 4.1.3 --- MI and Histological Type in Cervical Cancer --- p.55-56
Chapter 4.1.4 --- MI and Histologic Grades in Cervical Cancer --- p.56-57
Chapter 4.1.5 --- MI and Clinical stage in Cervical Cancer --- p.57-58
Chapter 4.1.6 --- MI and Clinical Status in Cervical Cancer --- p.58-59
Chapter 4.2 --- Microsatellite Instability in Endometrial Cancer --- p.59
Chapter 4.2.1 --- Prevalence of MI in Endometrial Cancer --- p.59-62
Chapter 4.2.2 --- MI and Age Groups in Endometrial Cancer --- p.63
Chapter 4.2.3 --- MI and Histological Type in Endometrial Cancer --- p.63-64
Chapter 4.2.4 --- MI and Histologic Grades in Endometrial Cancer --- p.64-65
Chapter 4.2.5 --- MI and Clinical stage of Endometrial Cancer --- p.65
Chapter 4.2.6 --- MI and Clinical Status in Endometrial Cancer --- p.66
Chapter Chapter Five --- Discussion --- p.67-77
Chapter 5.1 --- MI detection --- p.67-71
Chapter 5.2 --- MI of Cervical Cancer --- p.71 -74
Chapter 5.3 --- MI of Endometrial Cancer --- p.74-77
Chapter Chapter Six --- Conclusions --- p.78-80
Reference --- p.81-112
Appendix --- p.113-114

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41

Neto, João Luís Aguiar Martins. "CAG repeat instability in Huntington's disease: insights from HD patients and mouse models." Doctoral thesis, 2017. https://repositorio-aberto.up.pt/handle/10216/108073.

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42

Neto, João Luís Aguiar Martins. "CAG repeat instability in Huntington's disease: insights from HD patients and mouse models." Tese, 2017. https://repositorio-aberto.up.pt/handle/10216/108073.

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43

Liaw, Ying-Hsuan, and 廖盈瑄. "Conservation of human coding mononucleotide repeat mutants in mouse microsatellite instability-high tumors." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/8mr8kw.

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碩士
國立臺灣大學
基因體暨蛋白體醫學研究所
107
DNA mismatch repair mechanism (MMR) plays a critical role in maintaining the stability of the genome. Loss of MMR function leads to the accumulation of mutations and promotes tumorigenesis. Microsatellite instability (MSI) is the hallmark of MMR-deficient cancers. Microsatellites are one to six nucleotide repeats, located in either coding or non-coding regions. These repeats are prone to errors and frequently change in size during DNA replication. Normally, the errors are repaired through MMR. In other words, the lengths of microsatellites can be altered when the MMR pathway is defected. If an alteration of the microsatellite length occurred in the coding region, it may cause a frameshift mutation and result in a premature stop codon. Recent studies indicated that some genetic loci carrying coding mononucleotide repeat (cMNR) mutants are involved in the MSI-H carcinogenesis. However, the sequences of these frequently often reported cMNRs in human are not all conserved in mice. So far, whether those cMNR mutations generally seen in human MSI-H tumors are conserved in the MMR-deficient mouse model is unclear. In this study, I disrupted Trp53, Mlh1, and Msh2 by the ICE CRIM system and successfully generated the MMR-deficient mouse model. In addition, I designed a probe capture panel to enrich the conserved MSI target genes in mice for next-generation sequencing analysis. My result identified some conserved MSI target genes in human patient samples and our mouse MSI-H tumors, including both of those mutated in cMNR regions and non-repetitive sequences of coding regions. Oncogenic mutations, identical to those found in human, such as Kras G12D and G12V were observed in our mouse tumorous tissue. The significant enrichment for mouse orthologous variants of the human predicted MSI target genes involved in tumorigenesis reinforce the adequacy of using mice to study human MSI-H tumors. Our study provided an insight into the MSI target genes in MMR-deficient mice and allowed us to better understand the MMR signatures of possible mutational steps leading to MSI-H tumorigenesis.

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44

"Microsatellite instability and cyclooxygenase-2 expression in gastric carcinogensis." 2001. http://library.cuhk.edu.hk/record=b6073374.

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by Wai-keung Leung.
Thesis (M.D.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (p. 217-232).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.

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45

Ch, Nimilitha. "Probing the role of RNA-DNA hybrids in instigating trinucleotide repeat instability and their interaction with RNase H1." Thesis, 2014. http://raiith.iith.ac.in/68/1/BO12M1001.pdf.

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R-loops are transient intermediates that are formed during the transcription and consist of an RNA-DNA hybrid that is formed between the nascent RNA strand and the template DNA strand and a displaced non-template DNA strand. Formation of R-loops have been detected in organisms from bacteria to humans. If R-loops form more frequently, they impact transcription affecting genome stability, genome integrity and cause a number of diseases. Recently, genome instability caused due to stable RNA-DNA hybrid in R-loop was shown to predominantly associate with expansion of trinucleotide repeats, leading to many incurable neurological and neuromuscular genetic disorders like Myotonic dystrophy1, Fragile X syndrome, Huntington's disease, Friedreich’s ataxia, Spinocerebellar ataxias etc. Till date, the structural information about RNA-DNA hybrids formed by trinucleotide repeat expansions (TREs) are unknown to elucidate the mechanism behind RNA-DNA hybrid in instigating TREs. In this context, we aim here to study the structures of RNA-DNA hybrids consisting of trinucleotide repeats such as dGAA-rUUC, rGAA-dTTC, rCAG-dCTG, rCUG-dCAG, rCGG-dCCG and rCCG-dCGG by employing molecular dynamics (MD) simulation technique. It’s noteworthy that trinucleotide repeat expansion disorders (TREDs) can also be treated at mRNA level, wherein, a short complimentary DNA oligonucleotide is targeted against the mRNA that is followed by the cleavage of the mRNA strand by RNase H1 enzyme. Thus, to understand the efficacy of RNase H1 against TRE containing RNA-DNA hybrid, the interaction of TRE containing RNADNA hybrid with RNase H1 is also probed. Such scenario comes into picture during replication, wherein a transient RNA-DNA hybrid is formed and subsequently, the RNA strand is cleaved by RNase H1. Thus, this investigation is expected to shed light on structural properties of TRE containing RNA-DNA hybrids as well as its complex with RNase H1 to understand the TRE mechanism at replication and transcription levels and their treatment. The study on structure and dynamics of RNA-DNA hybrids consisting of various TREs suggest that these hybrids have both A & B characteristics irrespectively of sequence. The study on HsRNase H1 complexed with TREs containing RNA-DNA hybrids suggest that the protein follow the same cleavage mechanism for all the hybrids irrespective of sequence and thus, antisense strategy can be utilized to treat TREDs at RNA level.

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46

Figueiredo, Ana Sofia Tavares. "Relevance of epigenetics in the pathogenic mechanism of spinocerebellar ataxia type 37." Master's thesis, 2020. http://hdl.handle.net/10362/111133.

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Spinocerebellar ataxia 37 (SCA37) is an autosomal-dominant neurodegenerative disease characterized by cerebellar atrophy, gait and limb incoordination, and dysarthria as the first symptom. SCA37 is caused by an (ATTTC)n insertion within a nonpathogenic (ATTTT)n located in a 5’ UTR intron of DAB1. The age of onset in patients correlates with the number of ATTTCs and there is an increase in repeat insertion size during transmission to the next generation, with larger increases when the father is the transmitting parent. Haplotype analysis suggested that genetic factors flanking the mutant allele act as cis-elements influencing repeat instability. To identify cis-elements of genetic instability involved in the origin of mutant SCA37 chromosomes, we investigated single nucleotide polymorphisms (SNPs) and methylation status in the SCA37 repeat flanking region. SNPs were assessed by Sanger sequencing and DNA methylation by bisulfite sequencing of chromosomes containing nonpathogenic (ATTTT)<50, (ATTTT)>50 and interrupted, and mutant SCA37 alleles. We found a total of nine SNPs and confirmed that the repeat flanking region is highly polymorphic. SNP 7 and SNP 9 were present in all nonpathogenic large, interrupted and mutant SCA37 alleles studied, whereas only a small number of short nonpathogenic chromosomes carried these SNPs; they were associated with an increase in the (ATTTT)n tract, suggesting they might be a factor for repeat instability. These two SNPs are both in CpG dinucleotides, causing their elimination. The location of these SNPs shows an above average occupancy score for CTCF-binding in several cancer and embryonic human cells, which reinforces their involvement in repeat instability. In conclusion, this work allowed the identification of genetic variants that could have modified the epigenetics of the DAB1 (ATTTT)n flanking region and led to repeat instability. The occurrence of variants in important cis-regulatory elements might have created the ideal conditions for the mutational mechanism, originating the SCA37 (ATTTC)n insertion.

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