This dissertation is author version of following the journal article. Takahiro Shimizu, Hiroyuki Marusawa, Yuko Matsumoto, Tadashi Inuzuka, Atsuyuki Ikeda, Yosuke Fujii, Sachiko Minamiguchi, Shin’ichi Miyamoto, Tadayuki Kou, Yoshiharu Sakai, Jean E. Crabtree, Tsutomu Chiba, Accumulation of Somatic Mutations in TP53 in Gastric Epithelium With Helicobacter pylori Infection, Gastroenterology, Volume 147, Issue 2, August 2014, Pages 407-417.e3, ISSN 0016-5085, http://dx.doi.org/10.1053/j.gastro.2014.04.036. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18543号 / 医博第3936号 / 新制||医||1006(附属図書館) / 31443 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 小川 誠司, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22341号 / 医博第4582号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / UFU - Universidade Federal de Uberlândia / UNIPAM - Centro Universitário de Patos de Minas / A metformina (MET) é um fármaco antidiabético utilizado para prevenir a liberação de glicose hepática e aumentar a sensibilidade à insulina nos tecidos. Pacientes diabéticos com câncer têm, em adição, terapia medicamentosa antineoplásica. A doxorrubicina (DXR) é um agente quimioterápico antineoplásico que interfere com enzimas topoisomerase II e gera radicais livres. A MET isolada (2,5; 5,0; 10,0; 25,0 ou 50,0 mM) foi avaliada quanto à mutagenicidade, recombinogenicidade e carcinogenicidade e associado com DXR (0,4 mM) para antimutagenicidade, antirecombinogenicidade e anticarcinogenicidade, utilizando o “Teste para Detecção de Mutação e Recombinação Somática” e o “Teste para Detecção de Clones de Tumores Epiteliais” em Drosophila melanogaster. A MET isolada não induziu mutação ou recombinação, mas foram observados efeitos moduladores da MET sobre as lesões de DNA induzidas pela DXR nas concentrações mais elevadas. Na avaliação da carcinogênese, a MET isolada não induziu tumores, mas quando associado com DXR, MET também reduziu os tumores induzidos por DXR nas concentrações mais elevadas. Sendo assim, nas presentes condições experimentais a MET isolada não apresentou efeitos mutagênicos, recombinogênicos e carcinogênicos, mas foi capaz de modular o efeito da DXR na indução de danos ao DNA e tumores em D. melanogaster. Acredita-se que este efeito modulador esteja relacionado principalmente aos efeitos antioxidantes, anti-inflamatórios e apoptóticos deste medicamento, embora tais efeitos não tenham sido avaliados diretamente. / Metformin (MET) is an anti-diabetic drug used to prevent hepatic glucose release and increase tissue insulin sensitivity. Diabetic cancer patients are on additional therapy with anticancer drugs. Doxorubicin (DXR) is a cancer chemotherapeutic agent that interferes with the topoisomerase II enzyme and generates free radicals. MET (2.5, 5, 10, 25 or 50 mM) alone was examined for mutagenicity, recombinogenicity and carcinogenicity, and combined with DXR (0.4 mM) for antimutagenicity, antirecombinogenicity and anticarcinogenicity, using the Somatic Mutation and Recombination Test and the Test for Detecting Epithelial Tumor Clones in Drosophila melanogaster. MET alone did not induce mutation or recombination. Modulating effects of MET on DXR-induced DNA damage were observed at the highest concentrations. In the evaluation of carcinogenesis, MET alone did not induce tumors. When combined with DXR, MET also reduced the DXR-induced tumors at the highest concentrations. Therefore, in the present experimental conditions, MET alone did not present mutagenic/recombinogenic/carcinogenic effects, but it was able to modulate the effect of DXR in the induction of DNA damage and of tumors in D. melanogaster. It is believed that this modulating effect is mainly related to the antioxidant, anti-inflammatory and apoptotic effects of this drug, although such effects have not been directly evaluated. / Dissertação (Mestrado)

Les LPSNC représentent une localisation rare des lymphomes B diffus à grandes cellules (LBDGC), d’immunophénotype post-GC, dont la tumorigenèse reste mal connue.Notre objectif était de caractériser les altérations moléculaires des LPSNC à l’aide de techniques d’analyse haut débit.Notre projet a montré comme principaux résultats : 1/ la haute fréquence de mutations touchant des gènes impliqués dans la voie de signalisation BCR/TLR/NF-κB, en particulier MYD88, CD79B et TBL1XR1 ; 2/ des déséquilibres chromosomiques récurrents, en particulier la perte du 6q22 et du 6p (locus HLA) ; 3/ des mutations du promoteur de TERT et 4/ des transcrits de fusions ETV6-IGH.Plusieurs altérations semblent être des biomarqueurs pronostiques (i.e perte du 6q22 et délétions homozygotes de CDKN2A), prédictifs de réponse au traitement ou des cibles prometteuses pour des thérapies innovantes.En conclusion, il existe une grande similitude entre le profil moléculaire des LPSNC et celui des LBDGC extra-cérébraux avec néanmoins quelques spécificités. Les LPSNC peuvent résulter d’une tumorigenèse propre mais aussi de son microenvironnement singulier. / PCNSL represent a rare extranodal diffuse large B cell lymphoma (DLBCL) with a post-GC phenotype whose tumorigenesis is still poorly unknown.Our objective was to characterize the molecular genetic alterations of PCNSL using high throughput technologies.Results: We demonstrated 1/ a high incidence of somatic mutations in genes involved in the BCR/TLR/NF-κB pathway, especially MYD88, CD79B and TBL1XR1; 2/ recurrent chromosome imbalances such as 6q22 loss and 6q (HLA locus) homozygous deletions; 3/ TERT promoter mutations and 4/ gene fusions such as ETV6-IGH. Several alterations are associated with a prognostic impact (6q22 loss and CDKN2A homozygous deletions) or are promising targets for novel therapies.To conclude, PCNSL and extracerebral DLBCL share many similarities in terms of molecular genetic profile despite some specificities. PCNSL may result from a specific tumorigenesis but also from its peculiar microenvironment.

Massive parallel sequencing (NGS) is useful in detecting and later classifying somatic driver mutations in cancer tumours. False-positive variants occur in the NGS workflow and they may be mistaken for low frequency somatic cancer mutations in a patient sample. This pushes the need for decreasing the noise rate in the NGS workflow since it may improve the detection of rare allele frequency variants, in particular cancer mutations. In this project, the aim was to reduce the level of false-positive variants in an NGS workflow. The scope was limited to looking at substitution errors and their neighbouring nucleotides. Alongside this, it was also a way to understand how different types of substitution errors are distributed in the data, if their frequencies are affected by neighbouring nucleotides and how data processing may affect these substitution rates. A bioinformatic pipeline was set up where a commercially available genomic DNA sample with known variants was subjected to different trimming and filtering settings. The goal was to reduce the substitution error rate as much as possible, without removing any true variants from the data. The optimised settings were trimming the sequencing reads with 5 bp from the tail and filtering sequencing reads that contained 5 or more substitutions. Three additional samples, whereof two were clinical and the third commercial, were tested with these settings. The results showed that in all samples, C:G>T:A substitutions were of a higher frequency compared to the rest of the substitution types. For all samples, A:T>C:G substitutions, where the neighbouring nucleotide was a C or a G on each side, had a higher frequency compared to A:T>C:G substitutions with other neighbouring nucleotides on both sides. Those substitution types were especially targeted by the trimming. For the two commercial samples, substitutions that resulted in the nucleotide combinations >XAA or >XTT were of a higher frequency compared to the same substitution types that did not result in those nucleotide combinations. Filtering reads with 5 or more substitutions particularly targeted these substitution types. Consequently, filtering had a greater effect on the commercial samples, compared to the clinical samples. Overall, trimming and filtering helped reduce transversions more than the transitions, increasing the transition/transversion ratio after processing the data. The results suggest that trimming and filtering can be a useful method to computationally reduce the transversion errors introduced in an NGS workflow, but transition errors to a lesser extent, in particular A:T>G:C transitions. To confirm these findings, more samples should be tested using this methodology. To better understand the effect of trimming and filtering on variant calling, the scope could in the future be expanded to also look at small insertions and deletions.

No description available.

<p>  </p> <p>Mutations drive evolution, generating variation that selection can act upon. Germline mutations are heritable genetic alterations that occur in the gametes prior to fertilization and until embryogenesis. The study of germline mutations is vital to understanding the genetic basis of heritable diseases and evolution. Somatic mutations are genetic alterations across the body that arise post-fertilization in non-gametic cells. Although somatic mutations in most animals cannot be inherited, they can still significantly affect an organism's reproductive success. In humans, for example, cancers can be the result of somatic mutations. Somatic mutations originate from both exogenous mutagens (e.g. UV radiation) and endogenous processes (e.g. DNA replication, aging). Beyond their origins, we know little about the distribution and frequency of somatic mutations across Animalia. Honey bees provide a unique model for the study of somatic mutations as they are haplodiploid: males come from unfertilized eggs and are haploid, while females come from fertilized eggs and are diploid. It is therefore possible to sequence and robustly identify somatic mutations in haploid males. I have developed a unique exploratory study to elucidate the distribution of somatic mutation accumulation in honey bee drones. With this, I aim to investigate processes generating somatic mutations. Our findings demonstrate that variance in somatic mutational load is better captured across individuals rather than within individuals (across tissues). I provide a comprehensive tissue atlas of somatic mutagenesis in haploids. Our findings drive us to enhance our view of mutagenesis from the tissue level down to the cellular level. </p>

Tesis por compendio / [ES] La domesticación de los cítricos es un proceso en gran parte desconocido. Las investigaciones llevadas a cabo hasta la fecha indican que las variedades de cítricos que se comercializan en la actualidad son, en general, el producto de introgresiones ancestrales intraespecíficas e interespecíficas. Las introgresiones de tipo intraespecífico tuvieron lugar entre las poblaciones de dos subespecies antiguas de mandarinas y aportaron el comportamiento apomíctico que la mayoría de las mandarinas actuales y otros cítricos relacionados conservan todavía hoy. Una vez dichas mezclas genéticas o admixtures quedaron establecidas, otras introgresiones interespecíficas de pummelo establecieron nuevas características en el genoma de mandarina. Durante el proceso de domesticación, la variabilidad genética de estos cítricos se adquirió fundamentalmente a través de mutación espontánea, y mediante la práctica del injerto los genotipos carentes de semillas se extendieron rápidamente, acentuándose el papel de las mutaciones en la selección de cítricos como las naranjas y las mandarinas modernas. En este trabajo, mostramos que las mutaciones somáticas en cítricos se propagan siguiendo un patrón iterativo determinado por un modelo de ramificación simpodial y, en consecuencia, se agrupan en sectores a lo largo del árbol, donde algunas mutaciones quedan permanentemente fijadas. En este escenario, un árbol puede considerarse un mosaico genéticamente compuesto de diferentes genomas, en el que las ramas más jóvenes acumulan un número mayor de mutaciones. En promedio, nuestro árbol experimental de Clementina de 36 años muestra una tasa de mutación de 4.4 × 10-10 bp-1 yr-1, pudiendo contener en total entre 1500 y 5000 variantes y producir 1 mutación somática en cada meristemo axilar. Este número relativamente alto de mutaciones está en línea con el gran número de variedades derivadas de mutaciones espontáneas que se comercializan en cítricos. Para identificar caracteres esenciales adquiridos durante la domesticación, se han analizado los transcriptomas de la pulpa de frutos en desarrollo de la variedad silvestre e incomestible papeda de Ichang, la mandarina ácida Sun Chu Sha Kat y tres segregantes comestibles derivados de un cruce entre mandarinas comerciales. Basándonos en los resultados obtenidos, se propone que durante la transición de las papedas incomestibles a las mandarinas ácidas, la domesticación se caracterizó por una primera fase de cambios radicales en la expresión de los genes que regulan rutas fundamentales tanto del metabolismo primario como del secundario. Esta fase estuvo determinada principalmente por la estimulación de los procesos de crecimiento y la reducción de las defensas químicas, constituidas por compuestos de sabores desagradables. También se sugiere que en una segunda fase los atributos asociados a la palatabilidad de las mandarinas, especialmente la acidez, se mejoraron progresivamente a través de modificaciones específicas. Otros cambios de relevancia se relacionaron con la regulación de genes involucrados tanto en la síntesis de sustancias básicas de agradable aroma y sabor, como en la modulación de transportadores de azúcares. Por lo tanto, la transición entre las papedas incomestibles y las mandarinas de tipo ácido se caracterizó esencialmente por una drástica reprogramación de la expresión génica de rutas metabólicas fundamentales, mientras que las mandarinas modernas evolucionaron posteriormente mediante un refinamiento progresivo de las propiedades relacionadas con la palatabilidad. En su conjunto, estas observaciones sugieren que en cítricos, las tasas de mutación de los tejidos somáticos son consistentes con la idea de que las mutaciones desempeñaron un papel importante en las últimas fases de la domesticación. / [CAT] La domesticació dels cítrics és un procés en gran part desconegut. Les investigacions poratades a terme fins al moment indiquen que les varietats de cítrics comercialitzades hui en dia són, en general, el producte de introgressions ancestrals intraespecífiques i interespecífiques. Les introgressions de tipus intraespecífic van tindre lloc entre les poblacions de dues subespècies antigues de mandarina tot aportant el comportament apomíctic que la majoria de les mandarines actuals i altres cítrics relacionats encara conserven. Una vegada que aquestes barreges genètiques o admixtures es consolidaren, diferents introgressions interespecífiques de pummelo establiren noves característiques en el genoma de mandarina. Durant el procés de domesticació, la variabilitat genètica dels cítrics va ser assolida mitjançant mutació espontània i, per mitjà de la pràctica de l'empelt, genotips sense llavors es van estendre ràpidamente, accentuant el paper de les mutacions en la selecció de cítrics com les taronges i les mandarines modernes. En aquest treball, mostrem com les mutacions somàtiques en cítrics es propaguen seguint un patró iteratiu determinat per un model de ramificació simpodial i, en conseqüència, queden agrupades al llarg de l'arbre, on algunes d'aquestes mutacions romanen fixades. En aquest escenari, un arbre pot ser considerat un mosaic genèticament format per diferents genomes, en el qual les branques més joves acumulen un nombre major de mutacions. De mitjana, el nostre arbre experimental de Clementina mostra una ràtio de mutació de 4.4 × 10&#8722;10 bp&#8722;1 yr&#8722;1, arribant a incloure en total entre 1500 i 5000 variants i produint-hi 1 mutació somática en cada meristem axil·lar. Aquest nombre relativament elevat de mutacions està en línia amb el gran nombre de varietats provinents de mutacions espontànies que es comercialitzen en cítrics. Per tal d'abordar la identificació de trets essencials adquirits durant la domesticació, s'ha analitzat el transcriptoma de fruits en desenvolupament de la varietat silvestre i incomestible papeda d'Ichang, la mandarina àcida Sun Chu Sha Kat i tres segregants comestibles derivats d'un creuament entre mandarines comercials. Basant-nos en els resultats obtinguts, s'ha proposat que durant la transició de les papedes incomestibles a les mandariens àcides, la domesticació es va caracteritzar per una primera fase de canvis radicals en l'expressió dels gens que regulen rutes fonamentals del metabolisme primari i secundari. Aquesta fase va estar determinada principalment per l'estimulació dels processos de creixement i la reducció de les defenses químiques de gust desplaent. També es suggereix que en una segona fase atributs associats a la palatabilitat de les mandarines, especialment l'acidesa, van ser millorats progressivament mitjançant modificacions específiques. Altres canvis de rellevància van estar relacionats amb l'estimulació de gens involucrats tant en la síntesi de substàncies bàsiques d'agradable aroma i sabor, com en la modulació de transportadors de sucres. Per tant, la transició entre les papedes incomestibles i les mandarines àcides va estar caracteritzada essencialment per una dràstica reprogramació de l'expressió gènica de rutes metabòliques fonamentals mentre que les mandarines modernes evolucionaren posteriorment mitjançant un refinament progressiu de propietats relacionades amb la palatabilitat. En conjunt, aquestes observacions suggereixen que, en els cítrics, les ràtios de mutació dels teixits somàtics són consistents amb la idea que les mutacions tingueren un paper important en les últimes fases de la domesticació. / [EN] Citrus domestication is a largely unknown process. Research carried out to date indicates that current commercial citrus varieties are, in general, the product of ancestral intraspecific and interspecific introgressions. Intraspecific introgressions took place between the populations of two antique subspecies of mandarins and contributed to the apomictic behavior that most current mandarins and other related citrus still retain today. Once these genetic admixtures were established, interspecific pummelo introgression brought new traits to the admixtured mandarin genome. During domestication, genetic variability of these citrus occurred mainly through spontaneous mutations and with the practice of grafting, seedless genotypes expanded rapidly, emphasizing the role of mutations in the selection of citrus such as oranges and modern mandarins. In this work, we provide evidence that somatic mutations in citrus propagate following an iterative pattern determined by the sympodial branching model and consequently are grouped in sectors along the tree, where some of them remain fixed. In this scenario, a tree can be considered a mosaic genetically composed of different genomes, in which younger branches accumulate greater number of mutations. On average, our 36-yr-old experimental Clementine tree shows a mutation rate of 4.4 × 10-10 bp-1 yr-1, may carry a total of 1,500 to 5,000 variants and produces 1 somatic mutation per axillary meristem. This relatively high number of mutations is in line with the large number of varieties derived from spontaneous mutations that are commercialized in citrus. To identify key traits elicited by citrus domestication, we analyzed transcriptomes from developing fruit pulp of wild inedible Ichang papeda, Sun Chu Sha Kat sour mandarin and three palatable segregants derived from a cross between commercial mandarins. Based on these results, we propose that during the transition from inedible papedas to sour mandarins, domestication involved a first phase of major changes in the expression of genes regulating central pathways of primary and secondary metabolism. This stage was mainly characterized by both the stimulation of growth processes and the reduction of distasteful chemicals defenses. It is also suggested that in a second phase, edible attributes of mandarins, especially acidity, were progressively improved through specific modifications. Other relevant changes included upregulation of genes involved in the synthesis of key substances of pleasant aroma and flavor, and the modulation of sugar transporters. Thus, the transition from inedible papeda to sour mandarin was essentially defined by a drastic reprogramming of gene expression of fundamental metabolic pathways, while modern mandarins evolved later through progressive refinement of palatability properties. Taken together, these observations suggest that the rates of mutations occurring in citrus somatic tissues are consistent with the idea that they have played an important role during the later steps of citrus domestication. / This research is co-funded by the Ministerio de Ciencia, Innovación y Universidades (Spain) through grant RTI2018-097790-R-100 and by the European Union through the European Regional Development Fund (ERDF) of the Generalitat Valenciana 2014- 2020, through grants IVIA 51915 and 52002 / Pérez Román, E. (2022). Mutation Rate and Gene Expression in Genetic Admixtures of Domesticated Citrus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191452 / Compendio