Objective: Two chromosomal instability (CIN) pathways are described in glioblastoma multiforme (GBM), type 1 and type 2, which can be observed in up to 70% of the cases. Microsatellite instability (MSI) plays a pathogenic role in sporadic cancers such as colon, gastric and endometrial carcinomas with deficient mismatch repair (MMR). We aimed to perform a comprehensive analysis of the relationship between CIN and MSI mechanisms in sporadic glioblastomas. Methods: 129 GBMs were examined (109 newly diagnosed and 20 relapses) investigating MSI, immunohistochemical expression of MMR proteins as well as sequencing and promoter methylation of hMLH1. We characterized the molecular changes frequently correlated with CIN in MSI+ GBMs and compared them with 26 microsatellite-stable tumors. Results: Low-level MSI was observed in 11 of 129 (8.5%) cases and was higher in relapses than in primary GBMs (25 vs. 5.5%, p = 0.027). High-level MSI was not found in any case. A deficient expression of MLH1 and PMS2 without hMLH1 inactivation was observed only in one giant cell GBM. 55% of the MSI+ GBMs showed a profile which did not correspond to one of the known CIN pathways. An inverse association was observed between MSI and mutations of both p53 and PTEN. Conclusions: Our data suggest that CIN and MSI contribute to the genomic instability in GBMs via independent pathways. Since MSI was significantly more frequent in relapses, it might play a role in the malignant progression of GBM. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.

OBJETIVOS: Caracterizar o perfil de imunoexpressão de MLH1, MSH2, MSH6 e PMS2 em adenocarcinomas gástricos, explorando seu desempenho na identificação de características clínicas e patológicas, bem como sua influência prognóstica isolada e em relação aos demais parâmetros. MÉTODOS: Cento e trinta e três casos de adenocarcinomas gástricos esporádicos localmente avançados (pT2a ou mais) operados no Hospital das Clínicas/FMUSP foram incluídos pela ausência de metástases a distância ao diagnóstico (M0) e caracterizados clínica (idade, sexo e sobrevida) e patologicamente (tamanho, local, tipo de Borrmann, tipo histológico, infiltração vascular, perineural e variáveis de estadiamento locorregional). Amostras de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica das enzimas MLH1, MSH2, MSH6 e PMS2, com detecção por sistema de polímeros curtos conjugados a peroxidase. Casos com resultados negativos ou incertos nas amostras de TMA tiveram repetidas as reações em secções convencionais. A associação entre o estado de expressão dos marcadores com variáveis clínicopatológicas foi avaliada através do teste do qui-quadrado ou exato de Fisher. O impacto dos parâmetros clínico-patológicos e estado de expressão das enzimas na sobrevida foi explorado em modelos uni e multivariados de Cox, com construção de curvas de Kaplan-Meyer. Todas as análises estatísticas foram consideradas significativas ao nível de p<0,05. RESULTADOS: Quarenta e cinco casos (33,6%) exibiram perda de expressão de ao menos uma enzima, sendo frequente a perda de duas (9/45: 20%), três (14/45:31,2%) ou quatro enzimas (7/45:15,5%). Anormalidade mais frequente ocorreu com o MLH1 (26,7%), seguida de MSH6 (23%), PMS2 (21%) e MSH2 (20,8%). Quando avaliadas em conjunto, houve correlação entre o estado de expressão de todos os possíveis pares, com destaque para MLH1/PMS2 (rho=0,467, p<0,001) e MSH2/MSH6 (rho=0,666, p<0,001). Perda de MLH1 associou-se a tipos I/II de Borrmann (p<0,001), fenótipo intestinal de Lauren (p=0,005), tubular/túbulo-papilífero (p=0,009), expansivo de Ming (p=0,027) e infiltração em muscular própria (p=0,011). Com relação a perda de MSH2, tipos I/II de Borrmann (p<0,001), padrões tubular/túbulo-papilífero (p=0,008), intestinal (p=0,001), expansivo (p=0,001), infiltração da muscular própria (p=0,025), reação desmoplásica ausente a discreta (p=0,021) e ausência de infiltração perineural (p=0,016) foram mais frequentes. Perda de MSH6 associou-se aos tipos macroscópicos de Borrmann e histológicos de Lauren, OMS e Ming (p<0,001) e ausência de infiltração perineural (p=0,036). Idade mais avançada (p=0,046), tipos I/II de Borrmann (p=0,002), padrão intestinal de Lauren (p=0,021) e menos frequente infiltração perineural (p=0,035) foram identificados nos casos com perda de PMS2. A co-negatividade para os pares MLH1/PMS2 e MSH2/MSH6, além de reproduzir as associações mencionadas, identificou infiltrado linfoplasmocitário intra/peritumoral acentuado (p=0,011 e p=0,013), reação estromal desmoplásica ausente a leve para MSH2/MSH6 (p=0,037) e tamanho maior do tumor primário para MLH1/PMS2 (p=0,021). Pior sobrevida associou-se ao sexo masculino (LogRank: 5,11, p=0,024), tamanho do tumor (3,98, p=0,046), tipos III/IV de Borrmann (4,75, p=0,029), histologia mucinosa/anel-de-sinete da OMS (8,61, p=0,003) e difuso (11,62, p=0,003), infiltração perineural (12,62, p<0,001), metástase linfonodal (23,25, p<0,001) e estadio TNM (35,60, p<0,001) em análises univariadas. Melhor sobrevida associou-se a perda de MLH1, MSH6 e PMS2 isoladamente (5,46, p=0,019; 6,08, p=0,014; 7,46, p=0,006) e dos pares MLH1/PMS2 (7,89, p=0,005) e MSH2/MSH6 (5,29, p=0,021). Em modelos multivariados compostos pelos parâmetros clínicopatológicos, apenas o sexo masculino (HR=2,42, p=0,047), tipo histológico difuso (4,94, p=0,037) e estadios II, IIIA e IV (2,23, p=0,088; 3,12, p=0,022; 33,24, p=0,005), constituíram variáveis independentes de determinação prognóstica. Nas análises multivariadas incluindo o estado de expressão das enzimas, evidenciou-se que as perdas de PMS2 e do par MLH1/PMS2 associaram-se significativamente a maior sobrevida (3,84, p=0,029 e 9,82, p=0,028).CONCLUSÕES: O presente estudo demonstra o valor da imunohistoquímica na identificação de alterações na expressão de enzimas MMR, sendo a mais frequentemente negativa a MLH1. A frequente co-negatividade aponta para a importância da dimerização na funcionalidade do sistema de reparo. A perda isolada destes marcadores, e especialmente do par MLH1/PMS2, define perfil clínico-patológico característico, permitindo avanços no conhecimento previamente atribuído a fenótipo microssatélite instável conforme determinado em métodos moleculares. Em análises multivariadas, o estado de expressão de PMS2 isoladamente ou do par MLH1/PMS2 constitui fator independente de determinação prognóstica / OBJECTIVES: To characterize immunoexpression profile of MLH1, MSH2, MSH6 e PMS2 in gastric adenocarcinomas, exploring its performance to identify distinctive clinico-pathological features, as well as their prognostic implications in univariate and multivariate analyses. METHODS: A hundred and thirty three cases of locally advanced (pT2a or higher) sporadic gastric adenocarcinomas operated on Hospital das Clínicas/FMUSP were included due to absence of distant metastases at diagnosis (M0). Clinical (age, gender and survival) and pathological features (size, local, Borrmann´s type, histological type, vascular and perineural infiltration and locorregional staging parameters) were characterized. One millimeter samples were placed on tissue microarray blocks (TMA) and immunohistochemical detection of MLH1, MSH2, MSH6 and PMS2 performed on obtained sections, with revelation developed by peroxidase conjugated short-polymer based reagents. Negative or equivocal results obtained with TMA samples were repeated on conventional sections. Association between expression status for these markers and clinico-pathological features were evaluated by chisquare or Fisher´s exact test when appropriate. Impact of clinico-pathological features and expression status on disease specific survival were explored by Cox uni and multivariate models, with Kaplan-Meyer curves being fitted to illustrate these. All statistical results were considered significant at p<0,05. RESULTS: Forty five cases (33.6%) showed loss of at least one mismatchrepair enzyme, being frequent loss of two (9/45: 20%), three (14/45:31.2%) or even the four evaluated enzymes (7/45:15.5%). The most frequent abnormality addressed MLH1 (26.7%), followed by MSH6 (23%), PMS2 (21%) and MSH2 (20.8%). When analyses were performed in conjunction, correlation was identified for the expression status of all possible pairs, mainly the functional heterodimers MLH1/PMS2 (rho=0.467, p<0.001) and MSH2/MSH6 (rho=0.666, p<0.001). MLH1 loss was associated to Borrmann´s types I/I (p<0.001), Lauren´s intestinal phenotype (p=0.005), tubular/tubulo-papillary architecture (p=0.009), Ming´s expansile type (p=0.027) and infiltration limited by muscular propria (p=0.011). Among cases showing MSH2 loss, Borrmann´s I/I (p<0.001), tubular/tubulo-papillary (p=0.008), intestinal (p=0.001), expansile (p=0.001), muscular propria infiltration (p=0.025), absent to mild stromal desmoplasia (p=0.021) and absent perineural infiltration (p=0.016) were more frequent. MSH6 loss was associated to Borrmann´s gross type and Lauren, WHO and Ming´s histological types (p<0.001), as well as absent perineural infiltration (p=0.036). More advanced age (p=0.046), Borrmann´s types I/I (p=0.002), Lauren´s intestinal morphology (p=0.021) and less frequent perineural infiltration (p=0.035) were identified as associated to PMS2 loss. Conegativity for MLH1/PMS2 and MSH2/MSH6 pairs resumed all the above mentioned associations and additionally identified heavy lymphoplasmacytic infiltrate (p=0.011 e p=0.013), absent to mild stromal desmoplasia for MSH2/MSH6 (p=0.037) and increased primary tumor size for MLH1/PMS2 (p=0.021). In univariate analyses, decreased disease-specific survival was associated to male gender (LogRank: 5.11, p=0.024), tumor size (3.98, p=0.046), Borrmann´s types III/IV (4.75, p=0.029), mucinous/signet-ring cell morphology according to WHO (8.61, p=0.003) and Lauren´s diffuse morphology (11.62, p=0.003), perineural infiltration (12.62, p<0.001), lymph node metastases (23.25, p<0.001) and TNM staging (35.60, p<0.001). Better survival was seen in cases showing loss of MLH1, MSH6 and PMS2 when individually analyzed (5.46, p=0.019; 6.08, p=0.014; 7.46, p=0.006), as well as MLH1/PMS2 (7.89, p=0.005) and MSH2/MSH6 heterodimeric pairs (5.29, p=0.021). In multivariate models addressing clinico-pathological features, only male gender (HR=2.42, p=0.047), diffuse histological type (4.94, p=0.037) and stages II, IIIA and IV (2.23, p=0.088; 3.12, p=0.022; 33.24, p=0.005, respectively) were independent prognostic features. Multivariate analyses including status of MMR enzymes and the most significant clinicopathological features disclosed that PMS2 and MLH1/PMS2 losses were independent predictors of increased disease-specific survival (3.84, p=0.029 e 9.82, p=0.028). CONCLUSIONS: The present study demonstrates immunohistochemical detection of mismatch-repair enzymes as a tool to identify losses of these markers, being the most frequently negative MLH1. The frequently observed co-negativity points toward the importance of heterodimerization of these proteins in functional activity of mismatch-repair system. Losses of these markers, mainly MLH1/PMS2 pair, define a distinctive clinico-pathological profile and add knowledge to the previously reported associations with microsatellite instability as defined by molecular approach. In multivariate analyses, expression status of PMS2, as well as MLH1/PMS2 pair, revealed independent prognostic impact on diseasespecific survival

Objective: Two chromosomal instability (CIN) pathways are described in glioblastoma multiforme (GBM), type 1 and type 2, which can be observed in up to 70% of the cases. Microsatellite instability (MSI) plays a pathogenic role in sporadic cancers such as colon, gastric and endometrial carcinomas with deficient mismatch repair (MMR). We aimed to perform a comprehensive analysis of the relationship between CIN and MSI mechanisms in sporadic glioblastomas. Methods: 129 GBMs were examined (109 newly diagnosed and 20 relapses) investigating MSI, immunohistochemical expression of MMR proteins as well as sequencing and promoter methylation of hMLH1. We characterized the molecular changes frequently correlated with CIN in MSI+ GBMs and compared them with 26 microsatellite-stable tumors. Results: Low-level MSI was observed in 11 of 129 (8.5%) cases and was higher in relapses than in primary GBMs (25 vs. 5.5%, p = 0.027). High-level MSI was not found in any case. A deficient expression of MLH1 and PMS2 without hMLH1 inactivation was observed only in one giant cell GBM. 55% of the MSI+ GBMs showed a profile which did not correspond to one of the known CIN pathways. An inverse association was observed between MSI and mutations of both p53 and PTEN. Conclusions: Our data suggest that CIN and MSI contribute to the genomic instability in GBMs via independent pathways. Since MSI was significantly more frequent in relapses, it might play a role in the malignant progression of GBM. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La méiose est un processus de ségrégation des chromosomes essentiel pour la gamétogenèse chez tous les organismes qui présentent une reproduction sexuée. Ce mécanisme nécessite des connections entre chromosomes homologues et des structures intermédiaires d’ADN appelées Jonction de Holliday. Ces jonctions sont résolues principalement par complexe MutLγ (Mlh1-Mlh3). Des mutations sur les gènes impliqués en méiose s’accompagnent chez l’homme de problèmes allant de la stérilité à des réarrangements chromosomiques comme la trisomie. Mlh1-Mlh3 joue aussi un rôle dans la voie de réparation des mésappariements de l’ADN (MisMatch Repair - MMR). Notre laboratoire a révélé la première structure cristalline de la région C-terminale du complexe MutLα (Mlh1-Pms1) qui est l’endonucléase majeure de la voie MMR. Mon projet de thèse s’insère dans le cadre de l’étude des différentes fonctions des MutL eucaryotes et plus particulièrement sur le mécanisme moléculaire de MutLγ (Mlh1-Mlh3). Au cours de ma thèse, nous avons déterminé la structure cristalline du domaine C-terminale du complexe Mlh1-Mlh3 qui contient le site d’endonucléase et caractérisé 3 états du site actif. Nous avons montré le rôle de Mlh1 dans le site endonucléase. Nous avons caractérisé la spécificité de ce domaine pour les Jonctions de Holliday et proposé un modèle du site de fixation de l’ADN sur le complexe entier. Ce modèle a permis de proposer un nouveau mutant de séparation de fonction de Mlh1-Mlh3, appelé KERE, qui a été analysé par gel retard et génétique. / Meiosis is key process in sexual reproduction, where chromosomes are segregated. During this process, a parental diploid cell divides into haploid gametes. This mechanism requires connections between homologous chromosomes and intermediate DNA structures called Holliday Junctions. These junctions are mainly resolved by MutLγ (Mlh1-Mlh3) complex. Mutations of genes involved in meiosis are associated with human diseases including sterility and chromosomal rearrangements such as trisomy. Mlh1-Mlh3 plays also a role in DNA mismatch repair (MMR). Our laboratory has characterized the first crystal structure of the C-terminal region of the MutLα complex (Mlh1-Pms1) which is the major endonuclease in MMR. My thesis aims at understanding the molecular mechanism of MutLγ (Mlh1-Mlh3) mainly involved in meiosis and to compare it with Mlh1-Pms1 mainly involved in MMR.We determined the crystal structure of the C-terminal domain of the MutLγ complex which contains the endonuclease site. We characterized the structure of three different states of the active site. We showed how Mlh1 is an integral part of the Mlh3 endonuclease site. We characterized the specificity of this domain for Holliday Junctions and proposed a model of the full-length complex and its DNA binding sites. Finally, we design new separation of function allele of Mlh1-Mlh3, called KERE, which was analyzed by EMSA and genetic experiments.

No description available.

No description available.

En génomique comparative, on considère classiquement trois forces déterminant l'évolution des séquences : la mutation, la sélection et la dérive génétique. Récemment, lors de l'étude de l'origine évolutive des variations de la composition en base des génomes, un quatrième agent a été identifié : la conversion génique biaisée (BGC). Le BGC est intimement lié à la recombinaison méiotique et semble présent chez la plupart des eucaryotes. Ce phénomène introduit une surreprésentation de certains allèles dans les produits méiotiques aboutissant à une augmentation de la fréquence de ces variants dans la population. Ce processus est capable de mimer et d'interférer avec la sélection naturelle. Il est donc important de le caractériser afin de pouvoir le distinguer efficacement de la sélection dans l'étude de l'adaptation à l'échelle moléculaire. C'est ce que nous nous attachons à faire dans le cadre de ce travail. Pour cela nous utilisons deux espèces modèles. Premièrement la levure Saccharomyces cerevisiae pour laquelle une carte de recombinaison haute résolution permettant l'analyse du processus de conversion, est disponible. L'étude approfondie de cette carte nous a permis de lever le voile sur les mécanismes moléculaires qui sous-tendent le BGC. Deuxièmement, grâce à des découvertes récentes sur la détermination des patrons de recombinaison via la protéine PRDM9 chez les mammifères, nous avons quantifié la dynamique et l'intensité de ce processus dans l'histoire évolutive récente de l'homme. Ces résultats nous ont permis de confirmer la place du BGC comme quatrième force d'évolution moléculaire, mais aussi de discuter de l'origine évolutive de ce phénomène

Abstract Colorectal cancer is the third most common cancer in the developed countries. Originally, development of CRC was thought to proceed by a sequence of steps known as an adenoma-carcinoma sequence. At present CRC is recognized as a disease developing through diverse pathways. Serrated adenocarcinoma represents an endpoint of tumors developing from serrated pathway. This thesis focuses on studying the molecular alterations in serrated adenocarcinoma. Microsatellite instability, hypermethylation of promoter region in DNA repair genes hMLH1 and MGMT, frequency of KRAS and BRAF mutations and mutation spectrum of PTCH1 was determined in serrated adenocarcinomas (n=42) and compared to non-serrated adenocarcinomas (n=75). MSI, particularly low level of MSI (p=0.02) and methylation of both hMLH1 and MGMT promoters (p=0.004, p=0.026) were found to be more prevalent for serrated CRC. BRAF mutation was frequent and specific to serrated adenocarcinomas (p&lt;0.001) and KRAS mutations were more frequent in serrated adenocarcinomas than in non-serrated cancers (p=0.002). A significant association between BRAF mutation, hMLH1 and MGMT methylation and MSI-H phenotype was found in serrated carcinomas. KRAS mutation was seen in association with MSS/MSI-L phenotype; in fact, if serrated adenocarcinoma presents with MSI-L there always seems to be a KRAS mutation as well. Negative immunohistochemical staining of the hMLH1 enzyme was in association with methylation of the gene and proved reliable in the detection of MSI-H phenotype (p&lt;0.0001). Sequencing analysis of the whole coding regions of the PTCH1 gene did not reveal any truncating mutation to explain the previously detected downregulation of the gene in serrated CRCs. In conclusion, serrated adenocarcinomas proved to be an independent, but heterogeneous subtype of CRCs. High combined mutation rate (79–82%) of KRAS and <I>BRAF</I> in serrated adenomas and adenocarcinomas indicates that MAPK activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma, and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. High frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors. / Tiivistelmä Paksu- ja peräsuolisyöpä eli kolorektaalisyöpä on Suomessa kolmanneksi yleisin syöpätyyppi. Syöpää edeltävien muutosten tunnistaminen on tärkeää, jotta sen ehkäisy ja seuranta olisi tehokasta. Tavallisia adenoomapolyyppeja on pidetty tärkeimpinä kolorektaalisyövän esiastemuutoksina. 2000-luvulla on havaittu, että nk. sahalaitapolyypit edustavat tärkeää osaa esiastemuutoksista, ja näistä kehittyvää syöpää kutsutaan sahalaitaiseksi syöväksi. Sahalaitaisen syövän kehittymismekanismit eroavat huomattavasti tavallisesta kolorektaalisyövästä. Tässä väitöskirjassa keskityttiin tutkimaan sahalaitaiselle syövälle tyypillisiä morfologisia piirteitä sekä geneettisiä muutoksia. Työssä selvitettiin DNA mikrosatelliitti-instabiliteetin sekä DNA korjausgeenien hMLH1 ja MGMT promoottorialueiden hypermetylaation esiintyminen, nk. MAPK –signaalinsiirtoreitin komponenttien, KRAS ja BRAF -geenien, mutaatioiden yleisyys sekä PTCH1 geenin mutaatiokirjo sahalaitaisissa (n=42) ja tavallisissa kolorektaalisyövissä (n=75). DNA:n mikrosatelliitti-instabiliteetti, erityisesti matala-asteisena (MSI-L) (p=0.02) sekä <I>MLH1</I> ja hMGMT -geenien metylaatio (p=0.004, p=0.026) olivat yleisempiä sahalaitaisissa syövissä. <I>BRAF</I> mutaatio oli yleinen sekä spesifinen sahalaitasyöville (p&lt;0.001). Myös KRAS -mutaatiot olivat yleisempiä sahalaitaisissa syövissä (p=0.002). BRAF mutaatio, hMLH1 sekä MGMT metylaatio ja korkea-asteinen mikrosatelliitti-instabiliteetti (MSI-H) esiintyivät hyvin usein yhdessä sahalaitaisissa syövissä. Sahalaitaisissa syövissä KRAS –mutaatiot liittyivät MSI-L fenotyyppiin. hMLH1 geenin ilmentyminen tutkittiin myös immunohistokemiallisesti. Sahalaitaisissa syövissä MLH1 –proteiinin häviäiminen oli yhteydessä metylaatioon ja liittyi spesifisesti MSI-H:n esiintymiseen (p &lt; 0.0001). PTCH1 geenin sekvensointi ei paljastanut proteiinin toimintaa vahingoittavia muutoksia, eikä tuloksen perusteella pystytä selittämään aikaisemmin havaittua geenin ilmentymisen häviämistä sahalaitaisessa syövässä. Tulosten perusteella sahalaitainen syöpä on oma, mutta heterogeeninen kolorektaalisyövän alatyyppi. KRAS ja BRAF –geenien aktivoivien mutaatioiden yleisyys (79–82%) osoittaa, että MAPK -reitin aktivaatio on tärkeää sahalaitaisen syövän kehityksessä. BRAF -mutaatiot ovat spesifisiä sahalaitaisille syöville, ja yhdessä metylaation sekä MSI-H:n kanssa identifioi osan sahalaitasyövistä omaksi ryhmäkseen. <I>KRAS</I> –mutaatioiden yleisyys sahalaitaisissa syövissä antaa aiheen epäillä, että merkittävä osa KRAS –mutaation sisältävistä kolorektaalisyövistä kehittyy sahalaitapolyypeista.

The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability. One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability. In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice.