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Das COP9 Signalosom ist ein hochkonservierter Proteinkomplex bestehend, aus acht Untereinheiten. In der vorgelegten Promotionsarbeit konnte ein bislang unbekannter Regulationsmechanismus der Biogenese des COP9 Signalosoms identifiziert werden. Die siRNA-vermittelte Reduktion der CSN1-Expression führte zu einer Reduktion der Expression aller CSN-Untereinheiten. Die Transfektion von His-CSN1 in siCSN1-Zellen induzierte die CSN-Neusynthese und ferner einen Anstieg der c-Myc- und STAT1 Expression. Durch die Stimulation der Zellen mit IFN alpha bzw. IFN gamma konnte die de novo Synthese des CSN-Komplexes induziert werden. Die siRNA-vermittelte Inhibition von STAT1, c-Myc bzw. Lin28B führte ebenso wie die Behandlung der Zellen mit AG9 bzw. AG490, pharmakologischen Inhibitoren der JAK-Kinasen, zu einer Reduktion der Proteinexpression der CSN-Untereinheiten. Dabei standen signifikanten Veränderungen auf der Proteinebene geringfügige Änderungen auf der mRNA-Ebene gegenüber. Daher wurde ein post-transkriptioneller Mechanismus zur Regulation der Expression der CSN-Untereinheiten unter Beteiligung von miRNAs postuliert. Diese Regulation wird vermutlich durch die Aktivität von c-Myc und Lin28B verstärkt. Dies stellt einen neuen, bislang unbekannten Mechanismus für die Regulation der Biogenese des COP9 Signalosoms, vermutlich über den c-Myc/Lin28B/let-7-Weg, dar. Die Co-Transfektion der siCSN1-Zellen mit spezifischen komplementären Inhibitoren dieser miRNAs führte zu einer Induktion der Proteinexpression der CSN-Untereinheiten. Die Transfektion von let-7 miRNA-Mimics bewirkte eine Reduktion der Expression der CSN-Untereinheiten in den siCSN1-Zellen. Ferner konnten im Rahmen dieser Arbeit mittels der miRBase Sanger Datenbank und der Software MicroInspector Bindestellen für let-7 miRNAs an den mRNAs der CSN- und der proteasomalen Lid-Untereinheiten identifiziert werden. Der gezeigte Regulationsmechanismus könnte auch für die Biogenese des proteasomalen Lids von Bedeutung sein. / The COP9 signalosome is a highly conserved protein complex composed of eight subunits. In this study a novel, regulatory mechanism of CSN biogenesis was identified. We used stable transfected siCSN1 cells in which the protein and the mRNA expression of CSN subuntis were downregulated. Transfection of His-CSN1 in those siCSN1 cells led to the induction of the de novo Synthesis of the whole CSN complex. In addition the expression of the transcription factors STAT1 and c-Myc was elevated. The cells were treated with IFN alpha or IFN gamma, respectively. This resulted in the induction of the CSN de novo synthesis. Moreover, the siRNA-mediated inhibition of STAT1, c-Myc, Lin28B as well as treatment with the pharmacological inhibitors AG9 or AG490 led to a reduced protein expression of the analysed CSN subunits. We found that in all experiments there was a significant change on protein level in contrast to a marginal change on the RNA level. Based on our study we hypothesized that the CSN biogenesis ist regulated post-transcriptionally by miRNAs. The participation of miRNAs in the regulation of CSN biogenesis was further analysed. The siCSN1 cells were transfected with complementary hairpin inhibitors of let-7 miRNAs, leading to an induction of the CSN synthesis. The transfection of let-7 miRNA-mimics, which enhance the impact of miRNA on target mRNAs, resulted in an decrease of the CSN expression. These findings prove the involvement of miRNAs in CSN biogenesis, presumably via the c-Myc/Lin28B/let-7 pathway. Furthermore, using miRBase Sanger database and MicroInspector software, potential binding sites for let-7 miRNAs were detected within the mRNA sequences of CSN subunits as well as of subunits of the proteasomal lid. Therefore, there is evidence to suggest that this mechanism is crucial for the regulation of the biogenesis of the proteasomal lid as well.

La fibrillation atriale (FA) est un problème de santé publique majeur dans le monde entier. Le remodelage électrique, structurel et neuronal est sous-jacent à la myopathie atriale. La pharmacothérapie actuelle est souvent inefficace en raison du manque de connaissance de la pathophysiologie de la FA.Pour comprendre comment se réalise le remodelage atrial, une approche Omique qui explore le transcriptome, l'épigénome (méthylome et microOme) et le génome de patients atteints de FA a été réalisée. Parallèlement, le phénotype de vieux rats spontanément hypertendus (SHRs) a été caractérisé et une étude pharmacologique avec la décitabine (5-Aza-2'-deoxycitidine) a été menée. Les patients atteint de FA présentent un profil transcriptomique et d'expression de miRNA alteré dans l'oreillete gauche (OG), soulignant le rôle important d'un processus de "œmorphogénèse de la structure anatomique". L'expression réduite de Pitx2 était inversement corrélée à la taille de l'OG et ne pouvait pas être expliquée ni par le facteur de transcription ni par la surexpression de Smyd2, une cible de miR-519b. Les SHRs, similairement aux observations chez l'homme, ont développé des arythmies dépendantes de l'âge associées au remodelage atrial et ventriculaire gauche. La FA a été trouvée associée à l'hyperméthylation du promoteur de Pitx2 à la fois chez l'homme et chez les SHRs. L'agent hyperméthylant décitabine a amélioré le profil arhytmique de l'ECG et les activités SOD, et la réduction de la fibrose dans le ventricule gauche des SHRs. En utillisant une approche NGS basée sur un panel personnalisé de 55 gènes candidats à la myopathie atriale dans une cohorte de 94 patients atteints de FA, 11 nouvelles variantes faux-sens potentiellement pathogènes impliqués dans le remodelage structurel ont été identifiés. Des études fonctionnelles de ces variants ont débuté. Trois patients sont également des porteurs de variantes dans les gènes connus de FA. Les résultats actuels suggèrent que 1) la régulation épigénétique peut jouer un rôle dans la pathophysiologie de la FA 2) les agents hypométhylants doivent être considérés comme une nouvelle thérapie de la FA 3) une approche Omique peut aider à découvrir de nouveaux mécanismes sous-jacents à la myopathie atriale / Atrial fibrillation (AF) is a major public health care problem worldwide. Electrical, structural, and neural remodeling underlie atrial myopathy. Current pharmacotherapy is often ineffective due to the lack of knowledge of AF pathophysiology. To understand how atrial remodeling occurs, an Omic approach that explore the transcriptome, epigenome (methylome and microOme) and genome of AF patients was performed. In parallel, ageing spontaneously hypertensive rats (SHRs) were phenotypically characterised and a pharmacological study with decitabine (5-Aza-2’-deoxycitidine) was conducted. AF patients presented an altered transcriptomic and microRNA expression profile in the left atria (LA), emphasizing the important role of an "anatomical structure morphogenesis" process. The Pitx2 reduced expression was inversely correlated with LA size, and could not be explained by transcriptor factor. Smyd2 is a target of miR-519b-3p. SHRs, similar to what is observed in humans, developed age-dependent arrhythmias associated with left atrial and ventricular remodeling. AF was found to be associated with Pitx2 promoter hypermethylation both in humans and in SHRs. The hypomethylating agent decitabine improved ECG arrhythmic profiles and superoxide dismutase activities, and reduced fibrosis in the left ventricle of SHRs. Using a next-generation sequencing approach based on a custom panel of 55 atrial myopathy candidate genes in a cohort of 94 AF patients, 11 novel potentially pathogenic missense variants involved in structural remodeling were identified. Functional studies of these variants have started. Three patients were also carriers of variants in known AF-causing genes. The present results suggest that 1) epigenetic regulation may play a role in the pathophysiology of AF 2) hypomethylating agents have to be considered as a new AF therapy 3) an Omic approach may help to uncover new mechanisms underlying atrial myopathy

A metamorfose em insetos é um dos mais complexos e belos eventos biológicos conhecidos, dirigido por sucessivas alterações morfo-fisiológicas. Este intricado processo é coordenado por componentes moleculares como ecdisteroides (20E) e hormônio juvenil (HJ), fatores de transcrição e microRNAs (miRNAs). Os miRNAs regulam a expressão de genes-alvo, que por sua vez orquestram alterações fisiológicas e anatômicas necessárias para o completo desenvolvimento do organismo. Apesar do enorme esforço, os circuitos genéticos e endócrinos que regulam a metamorfose em insetos sociais, como a abelha Apis mellifera, estão longe de serem completamente esclarecidos. Os miRNAs são importantes componentes da maquinaria celular e parecem ser ubíquos no controle de processos biológicos. Desvendar novas interações miRNA-mRNAs alvo envolvidas com a metamorfose e a regulação das cascatas de 20E e HJ lançará uma luz sobre esse complexo evento. Em nosso estudo nós investigamos os papéis de miR-34, miR-281, miR-252a e miR-252b, conhecidos como reguladores da metamorfose em insetos, no modelo A. mellifera. Todos estes miRNAs revelaram alto grau de conservação filogenética, bem como responderam ao tratamento com 20E, sofrendo flutuações na abundância de transcritos. Usando as informações disponíveis e nossos bancos de dados, nós identificamos interações envolvendo estes miRNAs e genes participantes nas cascatas de HJ e 20E: ultraspiracle (Usp), fushi tarazu-transcription factor 1 (ftz-f1), ecdysone receptor (EcR), calponin (chd64), insulin receptor 2 (inr2), e Krüppel homolog 1 (Krh1). A predição das interações miRNA-mRNAs alvo revelou que os receptores de ecdisteroides EcR e Usp, bem como o fator de transcrição ftz-f1 são alvos importantes dos miRNAs estudados, apresentando sítios para os quatros miRNAs investigados. Observamos também que os seis genes codificadores de proteína são putativamente alvejados por miR-34. Por meio do ensaio da luciferase, pudemos validar as interações entre miR-34 e os alvos Kr-h1, chd64 e inr2; miR-252a e os alvos ftz-f1 e EcR; miR-252b e os alvos chd64 e ftz-f1; miR-281 e os alvos ftz-f1, EcR e Usp. A investigação dos perfis de expressão dos miRNAs ao longo do desenvolvimento larval (L3-PP3) e pupal (Pw), contrastados com os perfis de seus respectivos alvos, apontou muitos casos de relações positivas miRNA-mRNA. Estes resultados complementaram os resultados de validação, e expuseram a regulação exercida pelo miRNA sobre seus alvos. Juntos, os nossos resultados apontam para novas interações miRNA-mRNAs, envolvidas com a metamorfose em A. mellifera. As regulações por nós propostas e validadas bem como suas caracterizações e relações com os hormônios reguladores da metamorfose, são inéditas e acrescentam muito ao conhecimento sobre a regulação da metamorfose em A. mellifera. Nesse contexto, nossa pesquisa definitivamente contribui para uma melhor compreensão dos eventos moleculares envolvidos com a metamorfose de abelhas. / Insect metamorphosis is one of the most complex and beautiful of known biological events; it consists of successive morphological and physiological alterations. This intricate process is coordinated by various molecular components, including ecdysteroids (20E), juvenile hormone (JH), transcription factors and microRNAs (miRNAs). The miRNAs regulate gene expression, which in turn orchestrates physiological and anatomical changes necessary for successful insect ontogeny. Despite enormous efforts, the endocrine and genetic circuits that regulate metamorphosis in social insects, such as honey bees (Apis mellifera), are far from being completely elucidated. The miRNAs are a substantial component of this molecular machinery and seem to be ubiquitously involved in the control of biological processes. Disclosing new miRNA-target interactions involved in metamorphosis and in the regulation of 20E and JH cascades can shed light on these poorly understood events. In this study, we provide new pieces to this puzzle. We investigated the roles of miR-34, miR-281, miR-252a and miR-252b, known to be important regulators of insect metamorphosis, in the A. mellifera model. All of these miRNAs revealed a high degree of phylogenetic conservation and responded to treatment with 20E, which altered transcript abundance. Using available information and our databases, we identified interactions involving these miRNAs and the component genes of JH and 20E pathways: ultraspiracle (Usp), fushi tarazu-transcription factor 1 (ftz-f1), ecdysone receptor (EcR), calponin (chd64), insulin receptor 2 (inr2), and Krüppel homolog 1 (Kr-h1). Prediction of miRNA-target interactions revealed that the ecdysteroid receptors EcR and Usp and the transcription factor ftz-f1 are highly targeted by miRNAs involved in metamorphosis; they presented binding sites for all four miRNAs. We also observed that all six-protein coding genes are putatively targeted by miR-34. Using the luciferase assay, we were able to validate the interactions of miR-34 with the targets Krh1, chd64 and inr2; miR-252a with the targets ftz-f1 and EcR; miR-252b with the targets chd64 and ftz-f1; and miR-281 with the targets ftz-f1, EcR and Usp. Investigation of miRNA expression profiles during larval (L3-PP3) and pupal (Pw) development, as a function of the profiles of their respective targets, demonstrated many cases of positive miRNA-mRNA relationships. These results complemented the validation results, showing how the miRNAs regulate their targets. In conclusion, we identified various previously unknown miRNA-mRNA interactions involved in the metamorphosis of A. mellifera. The regulatory pathways proposed and validated by us, as well as their characterizations and relationships with metamorphosis regulator hormones, are unique and add to the understanding of the regulation of metamorphosis in A. mellifera. In this context, our research contributes to a better understanding of the molecular events involved in honey bee metamorphosis.

O início e a progressão do carcinoma espinocelular oral (CEO) são caracterizados pela aquisição de alterações genéticas e epigenéticas. A proteína SET é descrita como uma oncoproteína e, recentemente, o seu acúmulo foi mostrado em CEO. Diversas funções têm sido atribuídas à SET, tais como controle do ciclo celular, sobrevivência celular, migração celular, acetilação de histonas, e resposta ao estresse oxidativo. Este contexto sugere a SET como alvo terapêutico, mas primeiramente, é essencial entender a sua ação na tumorigênese e progressão em CEO. A hipótese central no presente estudo refere-se ao papel da SET na instabilidade genômica e reparo de DNA, bem como na regulação epigenética da expressão de miRNA, com impacto no desenvolvimento e progressão de CEO. O silenciamento estável de RNA foi realizado usando plasmídeo contendo short hairpin RNA contra SET (shSET) em linhagens de CEO in vitro (HN12 e Cal27) e in vivo (tumores xenoenxerto de HN12). Efeitos da redução da SET em CEO, in vitro e in vivo, foram avaliados no perfil de metilação (MSP, methylation specific PCR), expressão de miR-9 (qRT-PCR) e reparo de DNA (reparo mismatch e de quebra de fita dupla - DSB). A instabilidade genômica foi abordada por meio de cinco microssatélites (PCR convencional) para avaliar a instabilidade de microssatélites (MSI), e ensaio cometa para avaliar danos ao DNA (SSB, DSB, cross-link, etc.). O status de proteínas envolvidas em reparo de DSB (ATM, BRCA1 e MLH1) foi avaliado por imunofluorescência e Western blotting (WB). A resposta aos danos no DNA (DDR) induzidos por radioterapia (raios-X) foram analisados nas células HN12 por meio de ensaios clonogênico e de ciclo celular; proteínas associadas à apoptose, autofagia, ciclo celular e reparo foram avaliadas por WB. A redução da SET nas células HN12 modificou a transcrição dos loci codificantes do miR-9 por meio da reversão parcial de hipermetilação, tanto in vitro quanto in vivo, para o locus miR-9-1, e in vitro para o miR-9-3, com aumento nos níveis de miR-9 e miR-9*. A análise de 5 microssatélites mostrou alteração no perfil alélico de dois marcadores, D5S346 e D2S123, nas células HN12 shSET e nos tumores xenoenxerto da HN12 shSET (in vivo) em relação aos respectivos controles, o que sugere a presença de MSI e é um indício do papel da SET no reparo de DNA tipo mismatch. A linhagem HN12 shSET apresentou diminuição de danos no DNA e aumento das proteínas de reparo de DSB, MLH1, ATM, p-ATM e BRCA1 em relação a células HN12 shCTRL. Na DDR induzida por raios-X as células HN12 shSET apresentaram nas primeiras 48 horas uma menor perda de viabilidade (menor % em sub G0/G1), com parada em G2/M, maior nível de ATM ativa, aumento dos níveis de p21 e LC3B-II, além de menor clivagem de PARP e caspase-8 em relação as células HN12 shCTRL; isto sugere uma melhor resposta a danos de DSB e ativação de vias de sobrevivência nas células HN12 shSET. Entretanto, após 12 dias de radioterapia as células HN12 shSET mostraram uma tendência a menor sobrevivência. Portanto, os nossos resultados indicam o envolvimento da SET na regulação transcricional do miR-9, nas vias de reparo do tipo mismatch e de DSB em CEO, com potenciais implicações tanto na tumorigênese quanto na progressão da doença. / Oral squamous cell carcinoma (OSCC) onset and progression are characterized by acquisition of genetic and epigenetic alterations. SET protein is known as an oncoprotein and, recently, its accumulation was demonstrated in OSCC. Several functions have been attributed to SET, such as cell cycle control, cell survival, cell migration, histone acetylation, and response to oxidative stress. This context outstands SET as a therapeutic target, but first, it is essential to understand its role in tumorigenesis and progression in OSCC. The central hypothesis of this study refers to SET role in genomic instability and DNA repair, as well as miRNA epigenetic regulation, with impact in OSCC development and progression. Stable SET knockdown (shSET) was achieved using short hairpin RNA against SET mRNA in vitro (HN12 and Cal27, OSCC cell lines) and in vivo (HN12 xenografts tumors). Effects of SET knockdown were assessed in OSCC, in vitro e in vivo, regarding DNA methylation (MSP, methylation specific PCR) and expression of miR-9 (qRT-PCR), and DNA repair (mismatch and double-strand breaks/DSB repair). Genomic instability was addressed by means of five microsatellites (conventional PCR) to assess microsatellite instability (MSI), and comet assay to assess DNA damage (SSB, DSB, cross-link, etc.). The status of proteins involved in DSB repair (ATM, BRCA1 and MLH1) was assessed by immunofluorescence and Western blotting (WB). The DNA damage response (DDR) induced by ionizing radiation (X-rays) was assessed in HN12 cells through clonogenic and cell cycle assays; proteins associated with apoptosis, autophagy, cell cycle and repair were assessed by WB. SET knockdown in HN12 cells modified miR-9 transcription through hypermethylation partial reversal for miR-9-1 locus, both in vitro and in vivo, and for miR-9-3 in vitro, with increase of miR-9 and miR-9* levels. Analysis of 5 microsatellites showed changes in the allelic profile of two markers, D5S346 and D2S123, in HN12 shSET cells (in vitro) and HN12 shSET xenografts tumors (in vivo) compared to their controls, suggesting MSI and it\'s a clue of SET role in mismatch repair. HN12 shSET cells showed decreased DNA damage and increased DSB repair protein levels (MLH1, ATM, p-ATM and BRCA1) compared to HN12 shCTRL. In the first 48 hours, HN12 shSET X-rays-induced DDR showed lower loss of viability (lower % in subG0/G1), increased G2/M checkpoint, higher levels of active ATM, p21, LC3B-II, and less PARP and caspase-8 cleavage than HN12 shCTRL. These results suggest a higher efficiency of DSB damage response and activation of survival pathways in the presence of SET knockdown. However, 12 days after radiotherapy, HN12 shSET cells presented a tendency for higher intrinsic radiosensitivity in relation to control. Therefore, our findings indicate a SET involvement in miR-9 transcriptional regulation, and in mismatch and DSB repair, with potential implications in oral tumorigenesis and progression.

O início e a progressão do carcinoma espinocelular oral (CEO) são caracterizados pela aquisição de alterações genéticas e epigenéticas. A proteína SET é descrita como uma oncoproteína e, recentemente, o seu acúmulo foi mostrado em CEO. Diversas funções têm sido atribuídas à SET, tais como controle do ciclo celular, sobrevivência celular, migração celular, acetilação de histonas, e resposta ao estresse oxidativo. Este contexto sugere a SET como alvo terapêutico, mas primeiramente, é essencial entender a sua ação na tumorigênese e progressão em CEO. A hipótese central no presente estudo refere-se ao papel da SET na instabilidade genômica e reparo de DNA, bem como na regulação epigenética da expressão de miRNA, com impacto no desenvolvimento e progressão de CEO. O silenciamento estável de RNA foi realizado usando plasmídeo contendo short hairpin RNA contra SET (shSET) em linhagens de CEO in vitro (HN12 e Cal27) e in vivo (tumores xenoenxerto de HN12). Efeitos da redução da SET em CEO, in vitro e in vivo, foram avaliados no perfil de metilação (MSP, methylation specific PCR), expressão de miR-9 (qRT-PCR) e reparo de DNA (reparo mismatch e de quebra de fita dupla - DSB). A instabilidade genômica foi abordada por meio de cinco microssatélites (PCR convencional) para avaliar a instabilidade de microssatélites (MSI), e ensaio cometa para avaliar danos ao DNA (SSB, DSB, cross-link, etc.). O status de proteínas envolvidas em reparo de DSB (ATM, BRCA1 e MLH1) foi avaliado por imunofluorescência e Western blotting (WB). A resposta aos danos no DNA (DDR) induzidos por radioterapia (raios-X) foram analisados nas células HN12 por meio de ensaios clonogênico e de ciclo celular; proteínas associadas à apoptose, autofagia, ciclo celular e reparo foram avaliadas por WB. A redução da SET nas células HN12 modificou a transcrição dos loci codificantes do miR-9 por meio da reversão parcial de hipermetilação, tanto in vitro quanto in vivo, para o locus miR-9-1, e in vitro para o miR-9-3, com aumento nos níveis de miR-9 e miR-9*. A análise de 5 microssatélites mostrou alteração no perfil alélico de dois marcadores, D5S346 e D2S123, nas células HN12 shSET e nos tumores xenoenxerto da HN12 shSET (in vivo) em relação aos respectivos controles, o que sugere a presença de MSI e é um indício do papel da SET no reparo de DNA tipo mismatch. A linhagem HN12 shSET apresentou diminuição de danos no DNA e aumento das proteínas de reparo de DSB, MLH1, ATM, p-ATM e BRCA1 em relação a células HN12 shCTRL. Na DDR induzida por raios-X as células HN12 shSET apresentaram nas primeiras 48 horas uma menor perda de viabilidade (menor % em sub G0/G1), com parada em G2/M, maior nível de ATM ativa, aumento dos níveis de p21 e LC3B-II, além de menor clivagem de PARP e caspase-8 em relação as células HN12 shCTRL; isto sugere uma melhor resposta a danos de DSB e ativação de vias de sobrevivência nas células HN12 shSET. Entretanto, após 12 dias de radioterapia as células HN12 shSET mostraram uma tendência a menor sobrevivência. Portanto, os nossos resultados indicam o envolvimento da SET na regulação transcricional do miR-9, nas vias de reparo do tipo mismatch e de DSB em CEO, com potenciais implicações tanto na tumorigênese quanto na progressão da doença. / Oral squamous cell carcinoma (OSCC) onset and progression are characterized by acquisition of genetic and epigenetic alterations. SET protein is known as an oncoprotein and, recently, its accumulation was demonstrated in OSCC. Several functions have been attributed to SET, such as cell cycle control, cell survival, cell migration, histone acetylation, and response to oxidative stress. This context outstands SET as a therapeutic target, but first, it is essential to understand its role in tumorigenesis and progression in OSCC. The central hypothesis of this study refers to SET role in genomic instability and DNA repair, as well as miRNA epigenetic regulation, with impact in OSCC development and progression. Stable SET knockdown (shSET) was achieved using short hairpin RNA against SET mRNA in vitro (HN12 and Cal27, OSCC cell lines) and in vivo (HN12 xenografts tumors). Effects of SET knockdown were assessed in OSCC, in vitro e in vivo, regarding DNA methylation (MSP, methylation specific PCR) and expression of miR-9 (qRT-PCR), and DNA repair (mismatch and double-strand breaks/DSB repair). Genomic instability was addressed by means of five microsatellites (conventional PCR) to assess microsatellite instability (MSI), and comet assay to assess DNA damage (SSB, DSB, cross-link, etc.). The status of proteins involved in DSB repair (ATM, BRCA1 and MLH1) was assessed by immunofluorescence and Western blotting (WB). The DNA damage response (DDR) induced by ionizing radiation (X-rays) was assessed in HN12 cells through clonogenic and cell cycle assays; proteins associated with apoptosis, autophagy, cell cycle and repair were assessed by WB. SET knockdown in HN12 cells modified miR-9 transcription through hypermethylation partial reversal for miR-9-1 locus, both in vitro and in vivo, and for miR-9-3 in vitro, with increase of miR-9 and miR-9* levels. Analysis of 5 microsatellites showed changes in the allelic profile of two markers, D5S346 and D2S123, in HN12 shSET cells (in vitro) and HN12 shSET xenografts tumors (in vivo) compared to their controls, suggesting MSI and it\'s a clue of SET role in mismatch repair. HN12 shSET cells showed decreased DNA damage and increased DSB repair protein levels (MLH1, ATM, p-ATM and BRCA1) compared to HN12 shCTRL. In the first 48 hours, HN12 shSET X-rays-induced DDR showed lower loss of viability (lower % in subG0/G1), increased G2/M checkpoint, higher levels of active ATM, p21, LC3B-II, and less PARP and caspase-8 cleavage than HN12 shCTRL. These results suggest a higher efficiency of DSB damage response and activation of survival pathways in the presence of SET knockdown. However, 12 days after radiotherapy, HN12 shSET cells presented a tendency for higher intrinsic radiosensitivity in relation to control. Therefore, our findings indicate a SET involvement in miR-9 transcriptional regulation, and in mismatch and DSB repair, with potential implications in oral tumorigenesis and progression.

<p>In summary there currently exist techniques to discover miRNA however both require many calculations to be performed during the identification limiting their use at a genomic level. Machine learning techniques are currently providing the best results by combining a number of calculated and statistically derived features to identify miRNA candidates, however almost all of these still include computationally intensive secondary-structure calculations. It is the aim of this project to produce a miRNA identification process that minimises and simplifies the number of computational elements required during the identification process.</p>

Barley and wheat are the two most important crop species in Turkey. Molecular studies for increasing crop yield of these species are very important for the economic benefits of Turkey. Powdery mildew and yellow rust are the two main pathogens, infecting barley and wheat, respectively in our country and causing a great amount of yield loss each year. Till now, classical genetics studies were performed in order to develop resistant barley and wheat cultivars, but these studies have not been succesful. Therefore, molecular plant-pathogen interactions studies are starting to become the new tool to fight against pathogens. In this thesis, two important aspects of plant microbe interactions were investigated. In the first part, the role of microRNAs (miRNAs) in powdery mildew-barley pathosysytem, and yellow rust-wheat pathosystem were studied. The expression levels of miRNAs and their putative targets were investigated via miRNA microarray analysis and qRT-PCR, respectively, in response to virulent and avirulent pathogen infections. These data were used to establish a new model for powdery mildew-barley and yellow rust-wheat pathosystems. In the second part, functional analysis of a novel F-box gene, which was a ZTL-type F-box, was performed by using Barley Stripe Mosaic Virus mediated Virus Induced Gene Silencing. This F-box gene (HvDRF) (Hordeum vulgare Disease Related F-box) was induced upon yellow rust infection and we studied its role in powdery mildew infection. The results confirmed HvDRF as a positive regulator of race specific immunity and enlarged the roles of ZTL-type F-box proteins to biotic stress responses.

No description available.

<p>In summary there currently exist techniques to discover miRNA however both require many calculations to be performed during the identification limiting their use at a genomic level. Machine learning techniques are currently providing the best results by combining a number of calculated and statistically derived features to identify miRNA candidates, however almost all of these still include computationally intensive secondary-structure calculations. It is the aim of this project to produce a miRNA identification process that minimises and simplifies the number of computational elements required during the identification process.</p>