O glioblastoma (GBM) é considerado um dos tumores mais agressivos do sistema nervoso central (SNC). Mesmo com o uso de protocolos modernos de tratamento o prognóstico se mantém bastante reservado, sendo que crianças com GBM apresentam uma sobrevida média de 12 a 15 meses. Mecanismos epigenéticos podem interferir no processo de carcinogênese, sendo que a acetilação do DNA pode modular a expressão de genes que atuam no controle do ciclo celular, contribuindo assim para o desenvolvimento e progressão de neoplasias. Estudos clínicos demonstram que inibidores de histonas deacetilases (HDACs), em monoterapia ou combinados a outros agentes antineoplásicos, são clinicamente ativos e bem tolerados no tratamento de uma ampla variedade de tumores. Estes inibidores podem sensibilizar a resposta celular à irradiação ionizante, possibilitando uma redução nas doses-padrão utilizadas, minimizando os efeitos colaterais a curto e longo prazo. A radiação ionizante induz dano no DNA e é geralmente aceito que quebras da dupla-fita (DSBs) é o tipo de lesão mais severa relacionada à sobrevivência celular e preservação da integridade genômica. No presente estudo, avaliamos o potencial efeito radiosensibilizante do PCI-24781, um novo e potente pan-inibidor de HDAC nas linhagens celulares de GBM pediátrico SF188 e KNS42. Foram comparadas as taxas de proliferação celular, clonogenicidade e apoptose das linhagens SF188 e KNS42 com ou sem tratamento com PCI-24781. Também foram comparadas as taxas de clonogenicidade das linhagens SF188 e KNS42 que foram irradiadas com ou sem tratamento prévio com PCI-24781. Adicionalmente, foram avaliados os efeitos do PCI-24781 na expressão de algumas das principais proteínas responsáveis pelo reparo de quebras da dupla-fita ocasionadas pela irradiação. Para os ensaios de proliferação celular foram utilizados os tempo de 24, 48, 72 e 96h, para apoptose, 48h e para capacidade clonogênica sem irradiação o tempo de 48h, em diferentes doses de PCI-24781 (0,25 - 16 M). O inibidor bloqueou significativamente a proliferação celular (p<0,05), induziu morte por apoptose (p<0,05) e reduziu a capacidade na formação de colônias (p<0,001) em ambas as linhagens. No ensaio para avaliação da radiosensibilidade, foram utilizadas as doses do IC30 11 de cada linhagem do ensaio clonogênico seguida de diferentes doses de irradiação. Ambas as linhagens apresentaram uma significativa (p<0,001) diminuição na formação de colônias em todas as doses de irradiação. A linhagem mais resistente à droga, SF188 foi escolhida para estudo do reparo de quebras da dupla-fita ocasionadas pela irradiação. As expressões da proteína Rad51, importante na via de reparo por recombinação homóloga (HR), e das proteínas DNA-PKcs, Ku70 e Ku86, importantes na via de reparo por união terminal não-homóloga (NHEJ) apresentaram uma maior diminuição quando a linhagem irradiada foi previamente tratada com PCI-24781 em comparação à radioterapia exclusiva. Estes achados demonstram que o inibidor de histona PCI-24781 apresenta um importante papel como agente radiosensibilizante, comprometendo o reparo das quebras de dupla-fita em células de GBM pediátrico tratadas com radioterapia. / Glioblastoma (GBM) is considered one of the most aggressive tumors to affect the central nervous system (CNS). Even employing modern treatment protocols the prognosis remains very poor, with children affected by GBM presenting a median survival rate of 12 to 15 months. Epigenetic mechanisms may interfere with the process of tumorigenesis, and DNA acetylation can modulate the expression of genes that contribute in cell cycle control and participate to the development and progression of cancer. Clinical studies demonstrate that histone deacetylase inhibitors (HDACs), alone or in combination with other antineoplastic agents, are clinically active and well tolerated in the treatment of a wide variety of tumors. These inhibitors may sensitize the cellular response to ionizing radiation, enabling the reduction in standard doses of radiation, ultimately minimizing both short and long-term side effects. Ionizing radiation induces DNA damage and it is generally accepted that the double-stranded breaks (DSBs) is the most severe type of injury related to cell survival and preservation of genomic integrity. In the present study, we evaluated the potential radiosensitizer effect of PCI-24781, a novel potent pan-HDAC inhibitor in the pediatric GBM cell lines SF188 and KNS42. We compared the cell proliferation rates, apoptosis of clonogenicity of KNS42 and SF188, with or without treatment with PCI-24781. Moreover, clonogenicity rates were compared between cell lines that were irradiated with or without prior treatment with PCI-24781 Additionally, we evaluated the effects of PCI-24781 in the expression of some of the major proteins responsible for the repair of double-stranded breaks caused by the irradiation. For the cell proliferation assays, the times of 24, 48, 72 and 96 hours were used, for apoptosis, the time of 48h and clonogenic capacity without irradiation, the time of 48h, and different doses of PCI-24781 (0,25 - 16 M). The inhibitor significantly blocked cell proliferation (p<0,05), inducing cell death by apoptosis (p<0,05) and reducing the colony forming ability (p<0,001) of both lineages. In the assays to evaluate the radiosensitivity , the IC30 doses of the clonogenic assays were used for each cell-line after different doses of irradiation. Both lineages showed a significant decrease (p<0,001) in colony formation at all doses of irradiation. The most resistant cell-line to the drug, SF188, was 13 chosen to study the double-strand breaks repair caused by irradiation. The Rad51 protein levels, critical for homologous recombination (HR), and the DNA-PKcs proteins Ku70 and Ku86, important for DNA repair through non-homologous end joining (NHEJ) showed significant decrease in expression when cell-line was treated with PCI-24781 prior to radiotherapy. These data demonstrates that the histone deacetylase inhibitor PCI-24781 plays an important role as a radiosensitizer agent, compromising the repair of double-strand breaks in pediatric GBM cells following irradiation.

Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.

Numerous deaths, tragedies, and underreported drug side effects occur in outsourced clinical trials. Total quality system breakdowns occur even though quality agreement contracts and quality management systems are used by pharmaceutical organizations. The purpose of this single case study was to explore strategies clinical quality assurance managers use to avoid breakdowns in quality with outsourced clinical trials in Asia-Pacific countries. The study included a purposeful sample of 15 clinical quality assurance managers from 1 pharmaceutical organization located in the Northeast region of the United States. The conceptual framework was von Bertalanffy's general systems theory. Face-to-face semistructured interviews or e-mail questionnaires containing open-ended questions were used to gather data from clinical quality assurance managers who had a minimum of 5 years of experience with outsourced clinical trials. Coded data and themes were identified through the modified van Kaam method. The three emergent themes were the following: vendor quality management, building quality in outsourced clinical trials, and quality management systems. Results of the study may contribute to social change by helping pharmaceutical organizations' leaders develop strategies and tools to improve the quality of outsourced clinical trials.

Au cours de la réplication, la réparation des cassures double brin (CDB) par recombinaison homologue (RH), basée sur la synthèse d’ADN à partir de la chromatide sœur, permet le maintien de la stabilité du génome. La religature d’extrémités (EJ) éloignées de CDB peut quant à elle générer des réarrangements menaçant son intégrité. Nous avons étudié le mécanisme de réparation par EJ en fonction de la distance séparant deux cassures double brin. En utilisant des substrats intra-chromosomiques permettant la mesure de l’efficacité et de la fidélité du EJ après ligature d’extrémités éloignées ou proximales, nous avons mis en évidence l’implication du complexe de cohésion dans l’inhibition du EJ d’extrémités distales. Le complexe de cohésion joue donc un rôle central dans l’interface réplication/réparation ; la cohésion des chromatides sœurs favorise la réparation par RH et permet l’inhibition spécifique du EJ d’extrémités éloignées, probablement en limitant la mobilité de la chromatine endommagée et la formation d’une synapse propice au rapprochement des extrémités. La religature d’extrémités éloignées est également nécessaire aux mécanismes de diversification des gènes des immunoglobulines tels que la recombinaison V(D)J et la commutation de classe. L’étude de souris Rad21+/- a également démontré une implication du complexe de cohésion dans ces mécanismes essentiels à la diversité de l’information génétique. Le complexe de cohésion étant impliqué dans ces mécanismes et dans l’inhibition des réarrangements complexes tels que les translocations et insertions il est un acteur essentiel de la diversité et de la stabilité génomique. / DNA double-strand breaks (DSBs) repair is essential for genome stability/diversity, but can also generate genome rearrangements. Although non-homologous end-joining (NHEJ) is required for genome stability maintenance, the joining of distant double strand ends (DSE) should inexorably lead to genetic rearrangements. We analyzed the efficiency and accurency of close or distal EJ repair. Our data show that global end-joining is more efficient on close ends (34bp) compared to distal ends (3200bp) and that C-NHEJ is favored on close ends, resulting in more accurate outcome, compared to distal ends where more mutagenic A-EJ events takes place. In addition, the joining of distal ends favors the insertion/capture of DNA sequences. These data show only few kb distances between two DSEs are sufficient to jeopardize DSB repair efficiency and accuracy, leading to complex scars at the re-sealed junctions, and cell response is sufficiently sensitive to differently process such distal ends. We next addressed the question of the mechanisms preventing the joining of distant DSE. We show that depletion of the cohesin complex proteins specifically stimulates the end-joining of I-SceI-induced DSBs distant of 3200bp, while the joining of close DSEs (34bp) remained unaffected. Consistently, exome sequencing and cytogenetic analysis revealed that RAD21 ablation generates large chromosome rearrangements and a strong induction of replication stress-induced chromosome fusions. These data reveal a role for the cohesin complex in the protection against profound genome rearrangements arising through ligation of distant DSEs.

Abstract Social communication skills play a central role in the developmental outcomes for young children with autism. Due to deficits in social communication skills, many young children with autism develop problem behavior. Providing these children with the right tools to communicate properly may decrease their problem behavior. This study examines the impact of contingency mapping intervention on problem behavior and functional communication skills in three children with autism, using a multiple-baseline design. Before implementation of contingency mapping, verbal contingency was implemented in the first phase of intervention, which was associated with minimal increases in communication skills and decreases in problem behavior across children. Further increases in communication skills and decreases in problem behavior in the contingency mapping condition indicate that the use of the contingency map as a visual aid may be an effective way to augment the effects of contingency instruction.

I min undersökning studerar jag elevernas interaktion face-to-face på raster där den bärbara datorn finns med. Syftet är att undersöka hur den bärbara datorn påverkar interaktionen mellan eleverna på raster. Mina frågeställningar är hur och när elever är socialt responsiva respektive asocialt responslösa på raster samt hur elevernas interaktionsritualer ser ut?   Jag har använt en kvalitativ metod för att kunna besvara mitt syfte och mina frågeställningar. Jag intervjuade fem elever i årskurs ett på en gymnasieskola i Karlstad och genomförde tre observationstillfällen.   Studien visar att den egna datorn både kan ses som en tillgång och hinder på raster för elever på gymnasiet vid interaktion. Många av eleverna använder datorerna för att visa varandra saker som de sen diskuterar kring. Den sociala responsiviteten visade sig tydligt vid dessa tillfällen. Datorn som en gemensam symbol och en gemensam aktivitet verkade vara en betydelsefull interaktionsritual för eleverna. Vid andra tillfällen verkade datorn istället vara ett hinder för interaktionen. Eleven uppträdde då passivt i interaktionen och deltog i mindre utsträckning eller inte alls. De blev asocialt responslösa. Interaktionsritualen blev i dessa fall misslyckad.   Nyckelord: Bärbar dator, raster, social responsivitet, asocial responslöshet, interaktionsritualer / In this study I examine pupils´ face-to-face interaction when the laptop is there. The purpose is to see how the laptop impacts the interaction between students at breaks. My research questions are how and when pupils are social responsive respectively asocial responseless and also what the rituals of interactions look like.   I have used a qualitative method to answer my purpose and questions. I interviewed five pupils in year one of upper secondary school in Karlstad and also conducted three observations.   The study shows that the laptop is an impediment but also an asset in the interaction between students at breaks. Many pupils use their laptops to show each other things which could start a discussion. Social interaction was very evident at these moments. The laptop as a symbol and a common activity seemed to be an important interaction of ritual for the pupils. At other occasions the laptop seemed to be an impediment for the interaction. The pupils then became passive and participated less or not at all. He or she became asocial responseless. The interacion ritual then failed.   Keywords: Laptop, breaks, social response, asocial responseless, interaction ritual

Theoretical modeling of biological response to radiation describes qualitatively and quantitatively the results of radiobiological effects at the molecular, chromosomal, and cellular level. The repair-misrepair (RMR) model is the radiobiological model chosen for our study. It models deoxyribonucleic acid (DNA) damage formation and lesion repair through linear and quadratic processes. Double strand breaks (DSB) are a critical lesion in DNA. With increasing LET, the number of DSB per track traversing the cell nucleus increases. Using a compound Poisson process (CPP), we describe DNA damage formation. Three models were considered: a simple CPP using constant LET, a CPP using a chord length distribution, and a CPP using specific energy distribution. In the two first cases, and for low LET radiation the initial distribution of DSB was well approximated by a Poisson distribution, while for high LET radiation the initial distribution of DSB deviated slightly from a Poisson distribution. In the last case, DSB distribution was much broader than a pure Poisson distribution. Datasets from the literature for seven human cell lines, exhibiting various sensitivities to radiation were analyzed. We compared stochastic, CPP, and CPP using chord length distribution, with deterministic RMR models. For low LET radiation and at high dose rates the stochastic survival results agree well with the deterministic survival results. Also the stochastic model allows for non-linearity at low doses due to the accumulation of sub-lethal damage. At low dose rates deterministic results overestimate the surviving fraction compared to stochastic results. For high LET radiation stochastic and deterministic survival results agree. Stochastic survival results using specific energy distribution diverged from deterministic results by underestimating the surviving fraction at low and high LET radiation. The dose rate sparing curve, representing surviving fraction at a dose of 10Gy vs. dose rate shows that deterministic survival results are consistent with stochastic survival results, using CPP, or CPP with chord length distribution, for low and high dose rate values. Compared to deterministic aspects of DNA damage formation we concluded that stochastic aspects of DNA damage formation and repair using CPP or CPP with chord length distribution are not as prominent as reported in the earlier studies.

DNA double-strand breaks (DSBs) are lethal in eukaryotic cells if left unrepaired. In Saccharomyces cerevisiae the Mre11/Rad50/Xrs2 (MRX) complex is required for repair of DSBs through homologous recombination and nonhomologous end joining. Although Mre11 complexes exhibit 3'[rightwards arrow]5' exonuclease activity and endonuclease activity on DNA hairpin and single-stranded DNA overhang substrates in vitro, the role of the MRX complex in homologous recombination in vivo is not well understood. It has been shown to be specifically required for the processing of protein-conjugated DNA ends at DSBs during meiosis and hairpin-capped DSBs in mitotic cells and has been suggested that the Mre11 nuclease functions to remove damaged DNA ends. Recently, the Sae2 protein has been demonstrated to be involved in hairpin-capped DSBs and DNA end processing along with MRX in vivo. However, the Sae2 protein has no known homologs outside of fungi and no obvious motifs to suggest the function(s) of the Sae2 protein. We have purified recombinant Sae2 and MRX and report that the Sae2 protein itself is a single-stranded DNA endonuclease. The Sae2 protein stimulates the 3[rightwards arrow]5' exonuclease activity of the MRX complex. Also, the MRX complex can stimulate Sae2 nuclease activity to cleave ssDNA adjacent to DNA hairpin structures. The Sae2 protein also binds independently to double-stranded DNA and forms higher order protein-DNA complexes with MRX. These results provide biochemical evidence for functional cooperatively between MRX and Sae2 on DSBs and hairpin-capped DNA ends. / text

Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

Στην παρούσα μελέτη εξετάζεται ο εντοπισμός διαρθρωτικών μεταβολών εφαρμόζοντας έναν έλεγχο τύπου σωρευτικών αθροισμάτων τετραγώνων και συγκεκριμένα τη στατιστική των Kokoszka και Leipus σε σειρές αποδόσεων του Χρηματιστηρίου Αθηνών. Η ανάλυση στηρίζεται στην εφαρμογή ενός GARCH (1,1) υποδείγματος. / -