In S. cerevisiae initiation of replication occurs from discrete sites in the genome, known as origins and these display a characteristic temporal profile of activation during S phase of the cell cycle. The genomic context of origins has been demonstrated to be important to determine the time of firing, more specifically histone acetylation levels surrounding origins can influence their activation time. How increased acetylation is translated into earlier firing of specific origins is currently unknown. Bromodomains are known to bind acetylated histones in vivo. The bromodomain-containing Yta7p has been identified in a complex with various remodelers of chromatin and subunits of DNA polymerase ǫ. It is also a target of cell cycle and checkpoint kinases. Therefore, Yta7p makes an excellent candidate to bind acetylated histones surrounding replication origins and affect an alteration in the chromatin structure that could influence time of firing. Deletion of the histone deacetylase RPD3 results in a rapid S phase phenotype due to increased histone acetylation at “late-firing” origins. Increased acetylation at “late” origins leads to an advance in the time of firing of those specific origins. The aim of this study was to investigate the hypothesis that the bromodomain-containing protein Yta7p binds to histones with increased acetylation near to replication origins and subsequently influences origin firing. Hence, deletion of YTA7 would abolish the rapid S phase of a ∆rpd3 strain. Indeed the S phase of the ∆rpd3∆yta7 strain was reverted to WT duration. A role for Yta7p in DNA replication is also inferred by two additional lines of evidence presented in this thesis. Synthetic growth defects are evident when YTA7 and RPD3 deletion is combined with mutation of a third replication protein. In addition, ∆rpd3∆yta7 mutants are sensitive to HU, which is a phenotype shared by many strains with deletions in genes that encode proteins involved in DNA replication. Evidence to support a direct role of Yta7p in DNA replication events is provided by identification of an S phase specific binding of Yta7p to replication origins. Moreover, levels of Yta7p bound to early-firing origins are increased compared with their later-firing counterparts. Levels of Yta7p that are bound to “late-firing” origins are only increased in conditions of RPD3 deletion, where the resulting increase in histone acetylation at the “late-firing” origins is associated with advanced time of firing. Time of Yta7p binding at these “late” origins is also advanced concomitantly. This data supports the hypothesis that Yta7p provides a functional link between histone acetylation and time of origin activation. In searching for a specific replication linked function of Yta7p it was observed that recruitment of the FACT subunit Spt16p to replication origins was increased in conditions of YTA7 deletion. A second function for Yta7p in the S phase checkpoint was also demonstrated and the two roles of Yta7p, in DNA replication and S phase checkpoint, were separated depending upon their requirement for the bromodomain. The data produced in this thesis adds to our knowledge of DNA replication events and highlights the importance of histone modifications and chromatin remodeling to the replication field. This thesis describes the direct involvement of a protein, which was previously unassociated, with DNA replication and S phase checkpoint function and provides good ground work for future investigation.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

Degenerate Oligonucleotide Primed-PCR (DOP-PCR) can potentially enhance analysis of low copy number DNA samples. Theoretically, this procedure replicates fragments of the genome that can then be used for downstream multiplex STR analysis. The objective of this study is to optimize DOP-PCR by examining ramplelongation times and cycle numbers in the non-specific amplification portion of DOP-PCR, and by modifying the degenerate primer. Additionally, other methods such as Multiple Displacement Amplification (MDA) and Low Copy Number PCR (LCN PCR) were examined for their ability to create accurate DNA profiles from low DNA input amounts. Increasing the ramplelongation times showed no effect on downstream STR amplification success. An increase of cycle number increased DNA yield, but STR amplification success was undetermined. Although modifying the degenerate primer to one with a higher degeneracy decreased DNA yield, it ultimately improved STR amplification success. In comparison studies, LCN PCR produced higher STR amplification success than MDA.

The relation of English Language Arts (ELA) to the economy has played a historic role in educational policy, persisting to today’s corporate reform movement. It is, however, an area that remains under-researched. This study builds upon the limited existing literature base with a systematic replication of the College Board’s National Commission on Writing for America’s Families, Schools, and Colleges’ (NCW) 2004 report, “Writing: A Ticket to Work…Or a Ticket Out.” The guiding research questions for this study are 1) How important is writing in the workplace? 2) Is writing an important hiring consideration? 3) What kind of writing is expected on the job today? 4) Do employees have the writing skills employers seek? 5) Is writing a promotion criterion? 6) Do American companies provide writing training; if so, what is the cost? To answer these questions, human resources executives of Business Roundtable, Fortune 500 (with no redundancy), and Inc. 5000 companies were surveyed regarding the use and importance of writing in their respective organizations. To establish validity evidence, the College Board’s original instrument was pretested and piloted prior to full administration, and a principal components factor analysis was conducted to explore potential latent variables that may explain variance related to respondents’ perceptions regarding the use and importance of writing. Responses were analyzed descriptively and compared to the College Board’s findings, and results suggest that modern employers utilize writing differently and value it more highly now than in 2004. ELA curricula and workforce development initiatives may consequently benefit from updates in order to allow for more equitable economic opportunity.

Foot-and-mouth disease (FMD) is a highly infectious disease of wild and domestic cloven–hoofed animals such as cattle, swine and deer. It is caused by one of the most contagious animal diseases known; FMD virus (FMDV). Since the disease is endemic in many countries, transmission by international travel/trade presents an on-going potential threat to the UK. Very little is known at the molecular level about how FMDV replicates within host cells. In this study, FMDV replicons have been used to investigate FMDV RNA replication and to improve our understanding of the viral life cycle: a process which will aid in the production of a new generation of live-attenuated vaccine candidate strains. Sequences encoding the capsid coding region of the genome have been replaced with green fluorescent protein (GFP) such that replication can be monitored in live cells via GFP fluorescence. This provides a rapid, non-invasive screen for replicative fitness that can be used outwith high disease security facilities. Differences between replicating and non-replicating forms could easily be distinguished, highlighting the potential of this system to screen for attenuated genomes. The FMDV replicon system was improved through a series of construct modifications until an optimal system was produced. A range of different methods were used to attenuate the replication of these genomes. Of major significance is the finding that increasing dinucleotide frequencies were shown to decrease growth kinetics of Echovirus 7 – as opposed to altering the codon-pair bias - and the application of this finding to construction of further replicon systems (and RNA viruses in general) is described.

La kinase Cdk8 est impliquée dans la régulation de la transcription. Pourtant, cette protéine est retrouvée sur la chromatine lors de la réplication dans le modèle d'extrait d'œufs de xénope, où la transcription n'est pas active. Mon projet de thèse avait pour but de caractériser les fonctions de Cdk8 sur la chromatine en cours de réplication.Les résultats de ma thèse montrent que Cdk8 est impliquée dans la réplication de l'ADN. Le recrutement de Cdk8 sur la chromatine est concomitant avec le recrutement de certaines protéines du complexe de pré-réplication, bien qu'elle ne soit pas accumulée au niveau des foyers de réplication.Cependant, la déplétion de Cdk8 induit des défauts de réplication de l'ADN.Ces défauts ne sont pas induits par une collision entre le réplisome et la machinerie transcriptionnelle. En effet, l'ARN polymérase II, engagée sur la chromatine mais inactive en condition normale, est moins abondante sur la chromatine en absence de Cdk8.La déplétion de Cdk8 conduit à la diminution du recrutement des complexes de pré-réplication et des complexes de pré-initiation de la réplication. Cette diminution conduit à une baisse du taux de réplication sans activation du checkpoint intra-S. L'ensemble de mes résultats montrent que Cdk8 est nécessaire à une réplication normale de l'ADN. Plusieurs mécanismes semblent être mis en jeu, à savoir, un défaut de recrutement de la machinerie de réplication, l'accumulation de la protéine Adenomatous polyposis coli (APC) sur l'ADN ainsi que des modifications post-traductionnelles des histones. / The Cdk8 kinase is involved intranscriptional regulation.This protein is found on chromatin during DNA replication in xenopus egg extract model when transcription is not active. My PhD project was to characterize Cdk8 functions on chromatin during replication.My results show that Cdk8 is involved in DNA replication.Cdk8 is not found at replication foci , but its recruitment on chromatin occurs at the same time as several components of the pre-replication complex.Moreover, Cdk8 depletion leads to DNA replication defects.These defects are not induced by collision between the replisome and transcriptional regulators (RNA polymerase II and transcription factors). Indeed, RNA polymerase II, which is on chromatin in an inactive form under normal conditions, is less abundant on chromatin in absence of Cdk8.Cdk8 depletion leads to a decrease in pre-replication complexes and pre-initiation complexes recruitment. This decrease induces a reduction in DNA replication rate without activating the intra-S checkpoint.My data show that Cdk8 is necessary for proper DNA replication. It seems that Cdk8 depletion involves several mechanisms : altered replication machinery recruitment, presence of Adenomatous Polyposis Coli (APC) protein on DNA, and post-traductional modifications of histones.

This paper demonstrates that even regression results obtained by techniques close to the standard ordinary least squares (OLS) method can be difficult to replicate if a stochastic model fitting algorithm is employed. / Series: Research Report Series / Department of Statistics and Mathematics

De 50 à 100 millions de cas de maladie de dengue sont recensés chaque année dans le monde. Le virus de la dengue présente aujourd’hui un problème de santé publique avec son émergence en Europe et particulièrement en France. L’infection par le virus peut être asymptomatique ou être responsable de trois pathologies: l’une avec des symptômes grippaux (DF), une autre avec des hémorragies modérées (DHF) et une dernière avec des hémorragies sévères entraînant un syndrome de choc (DSS).Les facteurs de l’hôte jouent un rôle important dans le développement de formes sévères mais les facteurs viraux impliqués restent peu décrits. Le but de ce travail de thèse était de mieux comprendre ces facteurs viraux au travers de l’étude des dynamiques de circulation de souches de dengue 3 génotype III en Afrique et de la caractérisation de trois souches de dengue de sérotype I du Cambodge. Ce travail nous a permis de mettre en évidence la circulation de variants pendant les épidémies, permettant de supposer que la présence de variants permet une meilleure dissémination, ainsi que des caractéristiques génotypiques et phénotypiques particulières in vitro aux souches associées aux formes hémorragiques et aux formes avec syndrome de choc chez l’homme. Ces travaux ont été complétés par le développement d’un système original de détection du virus de la dengue et des autres virus du genre Flavivirus. Ce travail de thèse a permis d’identifier de potentiels facteurs de virulence propres au virus, ouvrant la voie à la recherche sur le rôle de certaines protéines virales dans la pathogénicité. / From 50 to 100 million cases of dengue illness occurred every year in the world. Today, dengue virus is a public health problem with its emergence in Europe, particularly in France. DENV infection can be asymptomatic or be responsible for three distinct pathologies: one with flu-like symptoms (DF), another with moderate hemorrhage (DHF) and the last one with severe bleeding leading to shock syndrome (DSS). Host factors have an important role in the development of severe forms but implicated viral virulence factors stay not well described. The aim of this research work was to better understand these viral factors through study of dengue serotype 3 genotype III dynamics of circulation in Africa and through the characterization of three dengue serotype 1 strains in Cambodia. This work highlighted the circulation of variants during epidemics, allowing us to suppose that the presence of variants permits a better dissemination, as well as specific phenotypic and genotypic characteristics in vitro of strains associated with hemorrhagic forms or forms with shock syndrome in humans. These works were completed by the development of an original system of detection of dengue virus and other viruses of genus Flavivirus. This research work allowed identifying potential virulence factor specific to virus, opening the way for research on the role of certain viral proteins in pathogenicity.

Thesis advisor: Ellen Winner / Previous research reported that people can successfully determine the winner of a musical competition when viewing a six second film clip of the performer without sound (Tsay, 2013, 2014); in contrast, when given an audio-only film clip or a clip that combined auditory and visual information, people perform at chance. Given the well-known publication bias in psychology (Ioannidis, 2005), this surprising and counterintuitive finding begs replication. In Study 1, 112 participants were randomly assigned to a sound, video, or video-plus-sound condition and were asked to select the winning musician after viewing five pairs of clips, one showing the winner and the other showing a non-winning musician. Clips were presented for 60 instead of six seconds, with the goal of giving participants more information about the performance, a modification we predicted would enhance performance in the audio and audio-visual conditions. Contrary to Tsay (2013), participants performed at chance in all three conditions. To more directly replicate Tsay (2013), in Study 2, 69 additional participants were randomly assigned to either a sound, video, or sound plus video condition and were asked to select the winning musician after viewing five pairs of 6-second clips showing the winner and another, non-winning musician. Here again the results did not replicate Tsay (2013): Participants performed significantly above chance in only one condition – when only hearing the performance and not seeing it. These results suggest that previous findings showing increased performance in rating musical performances without sound may be spurious and due to sampling error, issues in experimental design, low power, publication bias, or some combination of these. This also shows the strong importance of replication studies. / Thesis (BA) — Boston College, 2014. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Psychology Honors Program. / Discipline: Psychology.

A replicação do DNA tem início nas origens de replicação que são licenciadas na transição das fases M/G1, pelo complexo de pré-replicação (CPR), e ativadas apenas na fase S. Existem diversas origens de replicação no genoma, mas apenas parte destas origens é disparada em diferentes momentos de S, havendo assim origens early (disparadas no início de S) e late (disparadas mais tardiamente). Em trypanosomas as origens de replicação são reconhecidas por um CPR formado por Orc1/Cdc6 e pelo complexo MCM2-7. Em T. cruzi observamos que existem dois mecanismos diferentes para controlar a replicação do DNA. Durante o ciclo celular da forma epimastigota, as proteínas do CPR são sempre expressas e ligadas ao DNA, mas durante o ciclo de vida Orc1/Cdc6 se liga ao DNA apenas nas formas que replicam, e Mcm7 não é expressa nas que não replicam. Também foi analisado o perfil das forquilhas de replicação em T. brucei utilizando a técnica de SMARD onde vimos que a velocidade da forquilha é semelhante a dos demais eucariontes, além de encontrarmos a primeira origem de replicação late. / The DNA replication starts at the origins of replication, which are licensed at M/G1 transition, by the pre replication complex (PRC), and are activated just at S phase. There are many origins of replication along genome, but some of them are fired at different moments of S phase. So there are early and late origins fired at the beginning or later in S phase, respectively. The PRC of trypanosomes is composed of Orc1/Cdc6 and Mcm2-7. We could observe that in T. cruzi there are two distinct ways to control DNA replication. Whereas in epimastigote cell cycle the PRC are expressed and bound to DNA in all phases, during T. cruzi life cycle Orc1/Cdc6 is bound to DNA only in replicative forms and Mcm7 is absent in the non-replicative forms. We also analyzed the fork profile in T. brucei through SMARD technique. We found that the speed of replication fork is similar from other eukaryotes and that different replication origins are fired every cell cycle. Finally, we found a new origin of replication that is the first late origin described in this organism.