Note: no title page

Note:

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with group 3 (G3) MB representing the most aggressive subgroup. Despite MYC amplification representing an independent poor prognostic factor in G3 MB, efforts to target the MYC pathway have met with limited therapeutic success. As such, alternative mediators of G3 MB continue to be sought. The RNA binding protein and neural stem cell determinant Musashi-1 (MSI1) has been implicated in a number of adult stem cells in various organs (e.g., brain, gut, ovaries/testes) with mounting evidence that MSI1 is an essential regulator of cancer stem cells (e.g., brain, gut, lung). Early studies in MB have shown MSI1 to be essential for tumour maintenance, however the direct interactions and specific mechanisms conferring tumours with high MSI1 expression (i.e., G3 MB) are yet to be determined. Here, I show MSI1 is an essential moderator of G3 MB in both a MYC amplified and p53 mutated (MP) mouse model of G3 MB and patient-derived xenograft (PDX) models. MSI1 inhibition resulted in an abrogation of tumour initiation in both models, translating to a significantly prolonged survival. To determine how MSI1 regulates the post-transcriptional landscape of human G3 MB, an unbiased multiplatform approach was undertaken, using enhanced cross-linking and immunoprecipitation (eCLIP), and differential analyses post-MSI1 inhibition at the transcriptome-, proteome-, and translatome-wide scale, revealing MSI1's key role in moderating G3 MB-associated cancer driving genes. In summary, employing innovational multi-platform integrative approach to stem cell cancer biology, I show the neural RNA binding protein MSI1, an essential master stem cell regulator, is hijacked from its normal neural developmental function to orchestrate the aberrant translational landscape of G3 MB. / Thesis / Doctor of Philosophy (PhD) / Brain tumours are the leading cause of childhood cancer death with medulloblastoma (MB) representing the most frequent malignant childhood brain tumour. Analysis of the data retrieved from multiple genetic studies of MB, we have determined that there are 4 genetic subgroups of MB: Wnt, Shh, Group 3 (G3) and Group 4 (G4). The subgroup with the worse prognosis is Group 3, and unique to this subgroup is the overproduction of the MYC gene products (i.e., MYC amplification). In fact, MYC amplification alone is associated with a poor prognosis in these children. As such many researchers and clinicians have been working together to find a way to target MYC. Although many pre-clinical experimental studies have cured MYC-amplified G3 MB using gene-targeting therapy, these results unfortunately have not translated into early clinical trials. Therefore, alternative targets that mediate the aggressiveness of MYC-amplified G3 MB is being sought. As cancer stem cells (CSC) have been implicated in tumour development and maintenance, a gene worthy of investigation in a neurodevelopmental tumour such as MB, is Musashi-1 (MSI1). MSI1 protein has been identified in high levels in many human cancers, been observed to play a crucial role in promoting normal stem cell features, and is also implicated in driving cancer. The protein that the MSI1 gene produces binds to genes and modifies them to either stabilize or destabilize their path to becoming a protein. By manipulating MSI1 in both NSC and MB CSC, I will observe how these cells either display greater or less cancer associated features. Further, with a new technology allowing researchers to identify MSI1 binding sites, we aim to determine how MSI1 modifies cancer causing and normal neural stem cell genes. Moreover, I will be studying both the gene-, pre-protein- and protein-level changes after experimentally manipulating MSI1 gene levels to tease out its’ main cancer associated function. Altogether, we found a core list of genes that MSI1 modulates with functional significance giving us clues for a therapeutic targeting strategy for G3 MB.

Using a qualitative multiple case study design, this study investigated the ways that teachers’ pedagogical beliefs about learning: 1) affect students’ access to and use of one-to-one technologies and 2) shape one-to-one learning environments. Results indicate that teachers’ pedagogical beliefs are not a predictor of student access to computing devices, but do impact how students use the devices in the classroom. Also, teachers’ use of technology resources reflects their pedagogical beliefs. Teachers in the study reported that the increased access to technology via a one-to-one program positively impacted the learning environment by: facilitating better communication with students; empowering students; providing better feedback to students; making the teachers’ job easier; providing teachers and students with better access to resources; allowing more ways to differentiate student learning; developing 21st century technology skills; providing variety; and helping motivate students to learn. Teachers’ perceived negative impacts of the one-to-one program centered on the difficulties of shifting to new classroom management methods that accommodate technology devices and the distractions associated with the computing devices. Teachers also reported that they need better professional development to successfully integrate technology into their classrooms. Future professional development for one-to-one programs should be directed towards developing student-centered pedagogies with a focus on collaboration and integration of technology into the educational curriculum.

La vitesse de réplication du HIV-1(Human Immunodeficiency Virus type 1), qui semble corrélée de manière directe à la vitesse de progression de la maladie vers le stade SIDA, est essentiellement contrôlée au niveau transcriptionnel. La transcription du HIV-1 est régulée par la structure chromatinienne, des éléments agissant en cis localisés dans les LTRs, des facteurs de transcription agissant en trans et par la protéine virale trans-activatrice Tat (revu dans Quivy et al. 2007, Bisgrove et al. 2005, Rohr et al. 2003, Rabson and Graves 1997). En plus de l’enhancer localisé dans le LTR5’ du HIV-1, un enhancer intragénique, localisé dans le gène pol du HIV-1, inductible par le phorbol 12-myristate 13-acétate (PMA) a été identifié. La localisation progressive de l’activité enhancer a permis de définir deux domaines distincts et indépendants dans cet enhancer intragénique : les fragments 5103 et 5105 localisés respectivement dans la partie centrale du gène pol et dans une région couvrant la fin du gène pol, le gène vif, le gène vpr et le premier exon codant des gènes tat et rev (Verdin et al. 1990). Les fragments 5103 et 5105 se comportent tous deux comme des enhancers inductibles par le PMA lorsqu’ils sont clonés en amont du promoteur de la thymidine kinase dans un vecteur rapporteur en cellules HeLa. Notre laboratoire a précédemment identifié trois sites de liaison pour les facteurs de transcription AP-1 dans le fragment 5103 (Van Lint et al., 1991). Au cours de notre thèse, nous avons poursuivi la caractérisation de ces sites de liaison AP-1 et avons montré que les facteurs c Fos, JunB et JunD interagissent in vitro avec ces motifs. Pour chaque site, nous avons identifié des mutations qui abolissent la liaison des facteurs AP-1 sans altérer la séquence en acides aminés sous-jacente de la transcriptase inverse. Par des expériences de transfection transitoire, nous avons démontré que les sites AP 1 intragéniques sont entièrement responsables de l’activité enhancer PMA-dépendante du fragment 5103. De plus, l’activité PMA-inductible du fragment 5103 est inhibée par le mutant dominant négatif A-Fos à condition que les sites ne soient pas mutés. A l’inverse, l’expression ectopique de dimères forcés AP-1 affecte positivement l’activité enhancer du fragment 5103. Enfin, nous avons étudié le rôle biologique des sites AP-1 intragéniques dans la réplication virale et avons montré que ces sites contribuent positivement à l’infectivité du virus. Durant la seconde partie de notre thèse, nous avons entamé la caractérisation physique et fonctionnelle du fragment 5105. Nos résultats de transfection transitoire montrent que l’activité PMA inductible du fragment 5105 est localisée dans le dernier tiers de ce dernier : le sous fragment 5105.3. L’analyse bioinformatique de cette région a permis de mettre en évidence un site de liaison pour les facteurs AP-1 in vitro. Des mutations ponctuelles permettent d’abolir la liaison des facteurs à leur site mais altèrent la séquence en acides aminés sous-jacente codant pour les protéines Tat et Rev. Nous avons montré que ce site est impliqué dans l’activité transcriptionnelle de ce fragment. L’expression ectopique du mutant dominant négatif A-Fos inhibe l’activité transcriptionnelle PMA-inductible du fragment 5105. Une analyse bioinformatique plus large nous a ensuite permis d’identifier in vitro, par retard de migration sur gel, 5 sites de liaison pour le facteur YY1 et 2 sites de liaison pour le facteur PU.1 dont les implications pour le virus restent encore à déterminer.

No description available.

No description available.

With increasing emphasis on technology in schools, the importance of technology policies is great. This study investigated policies for four 1:1 secondary schools in Texas (schools with a ratio of one computing device per student), particularly with respect to the relationship of those policies to practice. The purpose of the study was to determine the current status of the National Education Technology Standards (NETS) essential conditions as reflected in policy and the relationship of those conditions to practice as measured through levels of technology usage and teaching innovation. Schools were selected through purposive, criterion sampling. Open-ended interviews were conducted with twelve participants (principals, technology directors, and superintendents). Policies were rated by campus principals and the researcher using a rubric based on the NETS essential conditions. Finally, surveys of proficiency and readiness measures were collected from 156 teachers using the School Technology and Readiness (STaR) instrument and the Levels of Teaching Innovation (LoTI) instrument. Interviews were transcribed and coded using structural and frequency coding. Policies were analyzed using magnitude coding and policy ratings. A qualitative analysis determined patterns between policy and practice. Quantitative data collected from surveys were measured against policy ratings and magnitude coding using bivariate correlation methods in SPSS. Quantitative analysis revealed two statistically significant relationships between policy and reported levels of practice in the classroom. Qualitative elements of the study from interviews and policy ratings revealed six findings that may explain a lack of correlation between policy and practice: a lack of ability for leadership to identify 1:1 program policy; lack among school leaders of perceived relationship between policy and practice; a belief among leaders that they are communicating policy to stakeholders even though they demonstrated difficulty in articulating policy; an inability to identify specific research-based theoretical foundations in policy; a lack of meaningful measurement of practices; and a lack of leadership at the same school to interpret policy similarly. A seventh finding revealed potential patterns between conditions that are addressed extensively in policy and evidence of those conditions in practice. Qualitative findings, in particular, contribute insights into disconnections between policy and practice in 1:1 settings.

Sauerstoffmangel spielt in der Pathophysiologie zahlreicher Erkrankungen mit gro§er klinischer Relevanz wie z.B. Herzinfarkte und SchlaganfŠlle eine zentrale Rolle. Eine Anpassung an Hypoxie wird durch die Familie der Hypoxie-induzierbaren Faktoren (HIF) vermittelt. Als Transkriptionsfaktoren sind HIF-1alpha und HIF-2alpha entscheidende Regulatoren von Genen, die durch niedrige Sauerstoffspannung aktiviert werden und Anpassungen wie z.B. Angiogenese (VEGF) und Umstellung auf anaeroben Energiemetabolismus (glykolytische Enzyme) bewirken. HIF ist ein Heterodimer, bestehend aus einer Alpha- und Beta-Untereinheit. WŠhrend die Beta-Untereinheit konstitutiv prŠsent ist, reprŠsentiert die Alpha-Untereinheit die sauerstoffregulierte Komponente von HIF, welche unter Normoxie proteasomal degradiert wird. Unter hypoxischen Bedingungen werden beide Faktoren in Zellinien stabilisiert. Das gleiche gilt fŸr HIF-1alpha in vivo. Bisher waren die Organe und Zellen der Expression von HIF-2alpha im lebenden Organismus unbekannt. Die vorliegende Arbeit konzentrierte sich daher auf die Charakterisierung der HIF-2alpha -Expression in vivo. Unter normoxischen Kontrollbedingungen war HIF-2alpha nicht nachweisbar. Es zeigte sich jedoch nach Stimulation mit 0,1 % CO (funktionelle AnŠmie) eine Induktion von HIF-2alpha in allen untersuchten Organen einschlie§lich Hirn, Leber, Lunge, Darm, Herz und Niere. Sowohl der zeitliche Verlauf des Anstiegs (1-6 Stunden) als auch die IntensitŠt der Induktion variierten in den einzelnen Organen, was durch Immunoblotting nachgewiesen werden konnte. Durch immunhistochemische Untersuchungen konnte gezeigt werden, in welchen Zellen der verschiedenen Gewebe eine nukleŠre Akkumulation von HIF-2alpha erfolgte. In Niere und Hirn erfolgte eine Induktion in nicht-parenchymatšsen Zellen, in Leber und Darm hingegen fand sich eine vorwiegende Expression in parenchymatšsen Zellen. Eine gleiche Verteilung zwischen den beiden verschiedenen Zelltypen fand sich im Herz. Durch Untersuchungen der mRNA mittels RNA Protection Assay konnte bestŠtigt werden, dass die Regulation von HIF-2alpha posttranslational erfolgt, da keine Hochregulation der mRNA festgestellt werden konnte. Die Daten zeigen, dass HIF-2alpha eine wichtige Rolle bei der transkriptionellen Antwort auf verminderte SauerstoffverfŸgbarkeit in vivo spielt und dass seine Rolle zu der von HIF-1alpha verschieden ist. / Deficiency of oxygen plays a central role in the pathophysiology of diseases with great clinical relevance like heart failure and stroke. An adaptation to hypoxia is mediated by the family of hypoxia-inducible factors (HIF). As transcription-factors HIF-1alpha and HIF-2alpha are regulators of genes that are activated by low oxygen tension and that cause adaptations to hypoxia like angiogenesis (VEGF) and change to anaerobic energy-metabolism (glycolytic enzymes). HIF is a heterodimer, consisting of an Alpha- and Beta-subunit. Whereas the Beta-subunit is constitutively present, the Alpha-subunit represents the oxygen-regulated component of HIF, which is degraded in the proteasom under normoxia. Under hypoxic conditions both subunits are stabilized in cell lines and HIF-1alpha is widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo were unknown. Therefore this work concentrates on the characterisation of the expression of HIF-2alpha in vivo. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, in brain, liver, kidney, lung, intestine and heart after stimulation with 0,1% CO (functional anaemia). Time course and amplitude varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in kidney and brain, predominantly parenchymal in liver and intestine or equally distributed in the heart. These data indicate that HIF-2alpha plays an important role in the transcriptional response to hypoxia in vivo and is complementary to rather than redundant with HIF-1alpha.

A hipóxia ocorre quando a demanda de oxigênio molecular necessário para gerar ATP é insuficiente. Os genes ativados por hipóxia compreendem o gene Hif-1a (Hipóxia-fator induzível 1a), Vegf-a (fator de crescimento endotelial vascular a), Arnt e Vhl (von Hippel-Lindau). Neste estudo foram utilizadas linhagens de camundongos geneticamente selecionados para alta (AIRmax) ou baixa (AIRmin) resposta inflamatória aguda (AIR). Foram realizados testes biológicos para caracterizar as reações inflamatórias produzidas por Biogel e TPA, bem como o tipo PAH cancerígeno. Testamos a expressão de mRNA de genes de hipóxia e caracterização de polimorfismo da região codificadora do Hif-1a no cromossomo 12. Camundongos AIRmax demonstraram uma maior reação inflamatória que os AIRmin para biogel e TPA enquanto o inverso foi observado com o DMBA. Os conjuntos de dados de fenótipos, expressão gênica e polimorfismo candidatam a região do cromossomo 12, que contém, entre outros, o gene Hif-1a, como participante da regulação da AIR. / Hypoxia occurs when the demand for molecular oxygen necessary to generate ATP is insufficient. Genes activated by hypoxia comprise the Hif-1a gene (Hypoxia-Inducible Factor 1a), Vegf-a (Vascular Endothelial Growth Factor a), Arnt and Vhl (von Hippel-Lindau). In this study we used lines of mice genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory response (AIR). We conducted biological tests to characterize the inflammatory reactions produced by Biogel and TPA, and the type PAH carcinogen. We tested the mRNA expression of genes of hypoxia and characterization of polymorphism of the coding region of Hif-1a gene on chromosome 12. We found that the mice AIRmax had greater intensity of the inflammatory reaction that AIRmin to biogel and TPA while the reverse was observed with the DMBA. The data sets of phenotypes, gene expression and polymorphism applying the region of chromosome 12 that contains, among others, the gene Hif-1a, as part of the regulation of AIR.