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The mismatch repair system (MMR) is an important pathway for maintaining genome stability because it can remove the errors generated while the cell is replicating. If these errors are left uncorrected, they can lead to genomic mutations. Thus, the deficient mismatch repair system is associated with the development of sporadic cancers and degenerative diseases such as Lynch Syndrome. MMR involves a set of proteins including MutSα, MutLα, MutLβ, and MutLγ. MutSα and MutLα play a major role in MMR whereas MutLβ and MutLγ provide minor contributions to this pathway. Recent studies have suggested that MutLβ and MutLγ are involved in the triplet DNA repeat expansion pathway. Our study in Saccharomyces cerevisiae shows important preliminary data of Mlh3 in maintaining genomic stability. We studied its interactions with other proteins involved in the mismatch repair system. Interestingly, Mlh3 interactions with Msh3 and Pol 35 DV suggest that there is a predilection of MutSβ and MutLγ to work in the lagging strand. Additionally, Top1, Mlh3and Msh3our results have shown that these genetic interactions could lead to an increase in mistakes in the MMR pathway. Moreover, it could lead to the suggestion that they are also involved in another pathway such as transcription. Finally, we confirm the involvement of Mlh3 in the resolution of frameshift mutations in the His-7A locus. Even though they are interesting results is too early to make final conclusions. Further analysis needs to be done.

Escherichia coli possède une grande plasticité génomique comme en témoigne la diversité des souches à l’intérieur de cette espèce bactérienne. Bien que la majorité des souches soient inoffensives ou à tout le moins opportunistes, plusieurs ont acquis des facteurs de virulence spécifiques leur procurant un pouvoir pathogénique. Les souches pathogènes comme E. coli O157 :H7 sont responsables de cas de morbidité, mortalité et pertes économiques importantes dans l’industrie agro-alimentaire dans le monde entier. L’évolution bactérienne est un mécanisme continuel qui se fait via l’échange d’éléments génétiques mobiles, de mutations ponctuelles et autres réarrangements génétiques. Ces changements génétiques peuvent procurer des avantages sélectifs permettant une adaptation bactérienne rapide face aux stress et changements environnementaux et favorisant le développement de pathogènes émergents. Dans la première partie de ce projet, nous avons étudié la région intergénique mutS-rpoS, qui est une des plus grandes sources de polymorphisme chromosomique chez les entérobactéries. Notre analyse génomique comparative a permis de confirmer le polymorphisme à l’intérieur même d’un ensemble de souches Escherichia spp., Salmonella spp. et Shigella spp. De plus, nous avons pu confirmer que certains types de polymorphismes dans la région mutS-rpoS étaient fortement associés à certains types de pathogènes chez E. coli. Dans notre analyse, nous avons ressorti un groupe de gènes à l’intérieur de la région mutS-rpoS qui pourraient sevir comme marqueur chromosomique intéressant pour les E. coli extra-intestinaux (ExPEC), un groupe comprennant des souches hautement pathogènes et difficiles à définir par les tests actuelllement disponibles. Dans notre analyse bio-informatique, nous avons isolé ce groupe de gènes associé aux ExPEC et nous l’avons caractérisé in sillico. Nous avons également inclus dans l’analyse deux souches hypermutables du genre Escherichia spp. de notre collection, isolées d’animaux de ferme. L’hypermutabilité ou la capacité d’acquérir des mutations plus rapidement que la normale accélère le processus d’évolution et la capacité d’adaptation de ces souches. La région mutS-rpoS est reliée au système de réparation de l’ADN bactérien (MMRS) et pourrait être impliquée dans l’apparition du phénotype d’hypermutabilité. Durant les dernières années, de plus en plus d’espèces du genre Escherichia ont été isolées de cas cliniques d’animaux et d’humains. Ces souches atypiques ont un potentiel de virulence très élevé, des combinaisons de gènes de virulence et des variants génétiques différents des souches typiques, et certaines souches ont même évolué en tant que pathogènes. Les souches de l’espèce E. albertii ont été isolées récemment et ont un grand potentiel de virulence autant chez les humains que chez les oiseaux. Ces souches sont souvent confondues avec d’autres organismes pathogènes comme E. coli dans les tests biochimiques, et le manque de connaissances sur E. albertii rend son identification difficile. Dans la deuxième partie de ce projet, nous avons identifié des gènes spécifiques aux souches d’E. albertii ainsi que des gènes de virulence présents chez E. albertii par comparaisons génomiques, ce qui a permis de développer et optimiser un test PCR (réaction en chaîne par polymérase) visant l’identification génomique rapide et fiable d’E. albertii.

The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events. / Master of Science / The CRISPR/Cas9 system is the newest generation of genetic engineering tool for genome editing. Genetic modifications can be introduced while repairing double strand breaks (DSBs) on DNA, induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing pig fibroblast under low amount of nutrient could enrich the cells in G0/G1 phase of cell cycle, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.

Syfte: Syftet med föreliggande in-vitro studie är att undersöka olika kompositbaserade reparationssystem och dess bindningsstyrka till porslin för metallkeramiska konstruktioner. Material och metod: Metallstavar tillverkades via CAD/CAM i CoCr (5.0x5.0x15.0mm) kortsidan förbehandlades genom sandblästring (aluminumoxid, 250 µm), ångblästring, oxidation, sandblästring och ångblästring. Fältspatporslin applicerades enligt följande: opak, dentin, emalj, till höjden av 1.2 ± 0.2 mm. Porslinsytorna slipades med sandpapper, 600-, 800-, 1200-kornstorlek kiselkarbid. Fem grupper (n=15) gav totalt 75 provkroppar. På vardera provkropp applicerades adhesiv samt kompositmaterial enligt fabrikanternas anvisningar (Kuraray, Kulzer, Voco, Ivoclar) inklusive kontrollgrupp som etsades med fluorvätesyra (Ultradent). Samtliga provkroppar termocyklades (5000 cykler, mellan 5°C och 55°C) före/efter applicering av kompositmaterial. Skjuvkraftstest utfördes på alla provkroppar med belastningshastighet 0.5 mm/min. Frakturanalys utfördes där brottytorna delades in i adhesiv-, kohesiv- eller mixfraktur. Resultaten analyserades med one-way ANOVA, Tukey´s test med signifikansnivån α=0.05. Resultat: Fluorvätesyra påvisade en signifikant högre bindningsstyrka 11.32 ± 4.28  MPa i jämförelse med övriga grupper (p<0.01). Övriga gruppers medelvärde var mellan 2.61 ± 1.36 till 3.96 ± 2.85 MPa. Det fanns ingen signifikant skillnad mellan de övriga grupperna. Samtliga grupper fick adhesiva frakturer, men Voco-gruppen uppvisade även mixfrakturer.  Slutsats: Samtliga reparationssystem visar på låg bindningsstyrka och baserat på resultaten kan det inte rekommenderas. Förbehandling av porslinsyta med fluorvätesyra i kombination med silan resulterar i högst bindningsstyrka till komposit. Värdet är trots allt lågt för en god långtidsprognos, men det enda som kan rekommenderas. Det faktum att fluorvätesyra har giftiga egenskaper vore det lämpligt att fortsätta utvecklingen av reparationssystem för att hitta ett lämpligt alternativ. / Purpose: The purpose of this study is to investigate different repair systems and their bond strength to porcelain for metal-ceramic constructions. Material and method: Metal were manufactured by CAD / CAM in CoCr (5.0x5.0x15.0mm), was pretreated with sandblasting (alumina, 250 µm), steam blasting, oxidation, sandblasting and steam blasting. Feldspar porcelain was applied according to the following steps: opaque, dentin, enamel, selfglaze to a final porcelain height of 1.2 ± 0.2 mm. The porcelain surfaces were groud with silicon carbide sandpaper, 600-, 800-, 1200-grain size. Five groups (n = 15) in a total of 75 specimens. Adhesives and composite materials were applied to each group according to the instructions of the various manufacturers  (Kuraray, Kulzer, Voco, Ivoclar, Ultradent, Etching with hydrofluoric acid). All specimens were thermocycled (5000 cycles) in two water baths between 5 ° C and 55 ° C before and after application of composite material. Shear bond strength test was performed on all specimens with a load speed of 0.5 mm / min and the bond strength was reported in MPa. Fracture analysis was performed where the fracture surfaces were divided into adhesive, cohesive or mixed fracture. The results were analyzed with one-way ANOVA, Tukey´s test with significance level α= 0.05. Result: The result clearly showed that hydrofluoric acid had a significantly higher bond strength of 11.32 ± 4.28 MPa compared to the other groups (p <0.01). The mean value of the other groups was between 2.61 ± 1.36 to 3.96 ± 2.85 MPa, with  no significant difference between the groups. All groups had adhesive fractures, but the Voco-group also showed mixed fractures. Conclusion: The study concludes that all repair systems have a low bond strength and based on the results can’t be recommended. Pretreatment of the porcelain surface with hydrofluoric acid in combination with silan results in the highest bond strength to composite, the bond strength is still too low for long-term use, but the only one that can be recommended. However hydrofluoric acid is highly toxic which require further research in the field in order to develop reparation systems with suitable properties.

In a bacterium's environment, life conditions are subject to constant changes. One of the proposed mechanisms of adaptation to these changes is the increase in mutation rate. Bacterial mutability is generally kept very low by action of various mechanisms of control and repair, one of the most important ones being the Mismatch Repair, which is the master regulator of genetic stability of organisms. When its function is impaired, larger amounts of mutations occur in cells. In adverse conditions, these might be beneficial for cells' adaptation. The role of these repair mechanisms in adaptive processes in Bacillus subtilis has not yet been definitely resolved. The previous work in our lab focused on establishing an experimental system to measure the extent of mutagenesis in B. subtilis, and the influence of several stresses on mutation rate was assessed. No significant increase in mutability was found to be triggered by nutrient limitation in stationary growth phase, hyperosmotic stress or increased cultivation temperature. Furthermore, a system to monitor the expression of mismatch repair proteins was constructed, which has not revealed significant differences between stressed and nonstressed growth conditions. This thesis follows the results of previous experiments, expanding the range of stresses...

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A queilite actínica (QA) é o resultado da exposição crônica dos lábios à radiação ultravioleta do sol. É considerada uma lesão cancerizável e calcula -se que cerca de 10% a 20% evoluirão para carcinoma epidermóide. Já foi sugerido que virtualmente todos os carcinomas epidermóides de lábio (CEL) iniciem como QA. Sabe-se que as radiações solares têm a capacidade de alterar o DNA e que, nesse contexto, os genes de reparo de DNA têm papel fundamental em reparar essas alterações e limitar os danos causados. Porém, estes genes também podem ser vítimas das radiações ultravioletas do sol. O objetivo deste trabalho foi estudar a participação dos genes de reparo de mau pareamento (RMP) do DNA em QA de diferentes graus de atipia e comparar os achados ao carcinoma de lábio relacionado à QA. Para tanto, foram analisadas nestas lesões a expressão e distribuição das proteínas codificadas por dois destes genes, hMLH1 e hMSH6, através da imunoistoquímica. Foi observado que a expressão das duas proteínas diminuía de acordo com o agravamento da indiferenciação celular das lesões. A partir disto, pode-se concluir que a diminuição na expressão de hMLH1 e hMSH6 parece estar relacionada com a progressão do grau de atipia nas QA, e, conseqüentemente, com a tumorigênese do CEL. Além disso, a proteína hMSH6 não pareceu ter um papel importante no reparo do DNA do epitélio labial normal. / Actinic cheilitis (AC) results from chronic and excessive exposure of the lips to the ultraviolet radiation in sunlight. AC is recognized as a potentially malignant condition and it is estimated that 10% to 20% will become lip squamous cell carcinoma (LSCC). It has been suggested that virtually every LSCC was initially AC. It is well known that solar radiation causes DNA damage and, in this context, the DNA repair systems play an extremely important role in restoring the injuries. The purpose of this study was to evaluate the participation of the mismatch repair (MMR) system in AC with all grades of dysplasia, as well as, LSCC related to AC. The protein expression and distribution of two of these genes, hMLH1 and hMSH6 were analyzed by means of immunohistochemistry. The results have shown an association between decreased expression of hMLH1 and hMSH6 proteins and the progression of the dysplasia in AC and, therefore, LSCC carcinogenesis. In addition, the hMSH6 protein does not seem to have a primary role in DNA repair of the normal lip epithelium.

A queilite actínica (QA) é o resultado da exposição crônica dos lábios à radiação ultravioleta do sol. É considerada uma lesão cancerizável e calcula -se que cerca de 10% a 20% evoluirão para carcinoma epidermóide. Já foi sugerido que virtualmente todos os carcinomas epidermóides de lábio (CEL) iniciem como QA. Sabe-se que as radiações solares têm a capacidade de alterar o DNA e que, nesse contexto, os genes de reparo de DNA têm papel fundamental em reparar essas alterações e limitar os danos causados. Porém, estes genes também podem ser vítimas das radiações ultravioletas do sol. O objetivo deste trabalho foi estudar a participação dos genes de reparo de mau pareamento (RMP) do DNA em QA de diferentes graus de atipia e comparar os achados ao carcinoma de lábio relacionado à QA. Para tanto, foram analisadas nestas lesões a expressão e distribuição das proteínas codificadas por dois destes genes, hMLH1 e hMSH6, através da imunoistoquímica. Foi observado que a expressão das duas proteínas diminuía de acordo com o agravamento da indiferenciação celular das lesões. A partir disto, pode-se concluir que a diminuição na expressão de hMLH1 e hMSH6 parece estar relacionada com a progressão do grau de atipia nas QA, e, conseqüentemente, com a tumorigênese do CEL. Além disso, a proteína hMSH6 não pareceu ter um papel importante no reparo do DNA do epitélio labial normal. / Actinic cheilitis (AC) results from chronic and excessive exposure of the lips to the ultraviolet radiation in sunlight. AC is recognized as a potentially malignant condition and it is estimated that 10% to 20% will become lip squamous cell carcinoma (LSCC). It has been suggested that virtually every LSCC was initially AC. It is well known that solar radiation causes DNA damage and, in this context, the DNA repair systems play an extremely important role in restoring the injuries. The purpose of this study was to evaluate the participation of the mismatch repair (MMR) system in AC with all grades of dysplasia, as well as, LSCC related to AC. The protein expression and distribution of two of these genes, hMLH1 and hMSH6 were analyzed by means of immunohistochemistry. The results have shown an association between decreased expression of hMLH1 and hMSH6 proteins and the progression of the dysplasia in AC and, therefore, LSCC carcinogenesis. In addition, the hMSH6 protein does not seem to have a primary role in DNA repair of the normal lip epithelium.