3,412 research outputs found
Regulation of Mec1 (ATR) signaling in budding yeast
No full textCells are continuously challenged by various sources of DNA damage that can contribute to cancer formation if not appropriately repaired. To cope with this threat, cells have conserved mechanisms called the DNA damage checkpoints that sense damaged DNA, stop the cell cycle, and upregulate DNA repair. Central players in these checkpoints are the PI3K-like kinases ATM and ATR (S.c. Tel1 and Mec1). Mec1 senses single stranded DNA (ssDNA) that is exposed at stalled replication forks and activates the S phase checkpoint. However, ssDNA, which is generated at the lagging strand during normal replication, does not cause detectable checkpoint activation. It is unknown how Mec1 is regulated in S phase. To study this, we took advantage of a mutant allele of MEC1, mec1-100, which is proficient for the G2 DNA damage checkpoint, but is compromised in G1-S and intra-S-phase checkpoints.
In the first part of this thesis we aimed at identifying regulatory factors. We screened for spontaneous survivors on a lethal dose of the replication fork-stalling agent hydroxyurea (HU) for mec1-100 cells. We mapped additional mutations in mec1-100 or mutations in either PPH3 or PSY2, which form a highly conserved phosphatase (PP4) complex. In a second, more unbiased, high-throughput screen we combined mec1-100 with a collection of 1525 gene deletions involved in chromatin processes and scored double mutants for HU sensitivity. pph3Δ and psy2Δ were among the top mec1-100 suppressor hits, confirming a strong genetic interaction. Suppression by pph3Δ was correlated with the phosphorylation of the downstream kinase Rad53. However, it did not depend exclusively on Rad53, because residual suppression of mec1-100 by pph3Δ could also be observed in rad53Δ cells. We tested whether Psy2-Pph3 might regulate Mec1 directly, and found a physical interaction between Psy2 and Ddc2-Mec1. Moreover, we found that a phosphorylation site within Mec1 (S1991) is downregulated in mec1-100 cells and restored when Pph3 is also lost. However, we were unable to demonstrate that Pph3 dephosphorylates Mec1 directly in vitro. Phosphorylation required both Mec1 kinase activity and Rad53. Thus, we speculate that Mec1 phosphorylation is achieved through Rad53, which is in turn regulated by Pph3, indicating the existence of a feedback loop from Rad53 back to Mec1. Mutation of the phosphorylation site renders cells sensitive to the radiomimetic drug Zeocin, indicating an important role in the survival of DNA damage. Finally, we applied quantitative phosphoproteomics to identify Mec1 and Pph3 targets. We found that the levels of the majority of the phosphopeptides that are affected by a tel1Δ mec1-100 mutation but not by rad53Δ can be rescued due to additional deletion of PPH3. The data presented here support a model in which Pph3 is a major regulator of Mec1 signaling.
In a second part mec1-100 was further characterized in order to understand the mechanism by which its two point mutations outside of the catalytic domain (F1179S, N1700S) cause defects in the replication checkpoint. We find that the mutations leave kinase activity in vitro, oligomerization and Ddc2-Mec1 interaction intact. Genetic analysis shows that mec1-100 is additive, rather than epistatic with mutation or deletion of any of the canonical checkpoint activating proteins Ddc1, Dna2, Dpb11, Rad24, Mrc1, Rad9, Tel1 or Chk1. Thus, we conclude that mec1-100 does not impair function of any of these proteins. We hypothesized that the mutated region might constitute a regulatory domain that is bound by a yet unknown factor. IP experiments followed by mass spectrometry analysis did not show reproducibly decreased interaction of any protein. Additional detailed biochemical analysis is needed to fully understand the mechanism of the two mec1-100 mutations.
We further characterize intragenic mec1-100 suppressor mutations by mapping them to a homology model. While some mutations reside within the kinase domain, and could influence catalytic activity, others might as well be involved protein-protein interactions. We asked whether suppression would involve Rad24 dependent Mec1 activation. Interestingly, we find that suppression by mutations in residues that might make protein-protein contacts completely requires Rad24. Other suppressor mutations relied less on Rad24. Thus, we conclude that intragenic suppression of mec1-100 HU sensitivity employs at least two different mechanisms: one that is Rad24-dependent and a second that is Rad24–independent. These unpublished results will help in understanding Mec1 function and regulation once structural data is available.
The third experimental part resolves the role of the RecQ helicase Sgs1 in replication checkpoint signaling. It was shown before that Sgs1 and Mec1 synergistically contribute to replication fork stabilization under replication stress. Both interact with the ssDNA binding protein RPA. Here, we created a mutant, sgs1-r1, which lacks the RPA interaction domain. While sgs1-r1 is proficient to stabilize stalled forks under replication stress, it is synthetic lethal with mus81Δ, slx4Δ, slx5Δ and slx8Δ. These could provide alternative means to recover stalled forks by resolving crossover structures, DNA repair or break induced replication. . Sgs1 was previously shown to promote Rad53 activation in a manner independent of its helicase activity. We show here that Sgs1 checkpoint function requires the R1 domain. Mec1 phosphorylates Sgs1 in this domain and Sgs1 phosphorylation allows its binding to Rad53 in vitro and in vivo. We thus propose that Sgs1 serves as a mediator in checkpoint signaling by recruiting Rad53 to stalled replication forks for activation.
This work provides new insights into Mec1 signaling by elucidating the checkpoint function of Sgs1 and defining Psy2-Pph3 as a major regulator of this pathway
Olitsky, Peter K. -- 1935-64 -- Correspondence, Individual -- letter, 1953-05-06
No full textLetter from Gasser, Herbert S. to Schlesinger, R. Walter dated 1953-05-06.Sabin Collection Fair Use Policy</a
[Claudius Schobinger und Rudolf Gasser]
No full textLinks im Bild Claudius Schobinger (konvertiert 1684) mit einer Waagschale, rechts Rudolf Gasser, im Hintergrund das Grossmünster. Der reformierte Pfarrer vertreibt den katholischen Geistlichen, in der Waagschale wiegt die "BIBLIA SACRA" mehr als die KultgegenständeAnonyme/r Künstler/inErschien als Titelkupfer in: Claudius Schobinger: Schriftmässige Waag-Schale, Zürich 1695Text ober- und unterhalb des Bildes: "... Man hat dich in einer Wagschüssel gewogen, und du bist zu leicht erfunden worden. Mein leicht erfunder Schatz / Macht dass ich räum den Platz, / und dass ich meinen Glatz / so kläglich reib und kratz.
Grosser Catechismus, das ist, Newe Predigen nach dem kleinen Catechismo R. P. Canisii : uber vier Hauptstuck christlicher catholischer Lehr, als über den apostolischen Glauben, uber die Hoffnung, und Liebe, wie auch über die siben hh. Sacramenten, und zehen Gebott Gottes, sehr nutzlich, und dienstlich allen Predigeren, Kinden-Lehreren, und Beichtvätteren
No full textzusamen getragen, und gestellet durch P.F. Rudolphum von Schweytz ...Titelseite zweifarbig (schwarz-rot) gedruckt
Cambios en la presión intraocular en pacientes con neuralgia trigeminal sometidos a radiofrecuencia del ganglio de Gasser
No full textABSTRACT Introduction: Trigeminal neuralgia is characterized by paroxysmal attacks of intense pain, lancinating as electric shocks, accompanied by burning sensation and periodic appearance in the distribution of one or more trigeminal branches. The anatomical relationship between the Gasser ganglion and the cavernous sinus could produce an increase in the intraocular tension after the application of the radiofrequency of the trigeminal ganglion when modifying the venous drainage of the eye, a situation that until now has not been studied. The objective was to determine if the application of conventional radiofrequency in the Gasser ganglion produces changes in the intraocular tension in patients with primary trigeminal neuralgia of second and / or third branch. Material and methods: Longitudinal study that included patients diagnosed with trigeminal neuralgia of second and / or third branch; In the operating room and under general anesthesia, radiofrequency procedure was performed in Gasser ganglion, with generator Neurotherm R 500, needles of 100 mm in length and 22 G, with active tip of 5 mm, sensory stimulation at 50 Hertz with 0.3 - 0.5 V applying 80º C for 60 seconds in 2 or 3 phases. Intraocular pressure was measured 24 hours before the procedure, 24 hours after the procedure and seven days after the procedure. The analysis of the intraocular pressure, as well as the EVA scale of pain was determined by an ANOVA analysis of repeated measures. Results: Thirty patients were included of which 26 were women, the average age was 59.6 ± (SD 11.16). Intraocular tension 24 h before, one day after and one week after performing radiofrequency of the Gasser ganglion, did not show significant changes with ANOVA for repeated samples (p 0.916). Conclusions: Intraocular tension was not modified in the short term after radiofrequency of the Gasser ganglion.RESUMEN Introducción: La neuralgia del trigémino es un cuadro caracterizado por ataques paroxísticos de dolor intenso, lancinante como descargas eléctricas, acompañado de sensación urente y de aparición periódica en la distribución de una o más ramas trigeminales. La relación anatómica entre el ganglio de Gasser y el seno cavernoso pudiera producir un incremento en la tensión intraocular después de la aplicación de la radiofrecuencia del ganglio trigeminal al modificar el drenaje venoso del ojo, situación que hasta el momento no se ha estudiado. El objetivo fue determinar si la aplicación de radiofrecuencia convencional en el ganglio de Gasser produce modificaciones en la tensión intraocular en pacientes con neuralgia trigeminal primaria de segunda y/o tercera rama. Material y métodos: Estudio longitudinal que incluyó a pacientes con diagnóstico de neuralgia del trigémino de segunda y/o tercera rama; en quirófano y bajo anestesia general, se les realizó procedimiento de radiofrecuencia en Ganglio de Gasser, con generador Neurotherm R 500, agujas de 100 mm de longitud y 22 G, con punta activa de 5 mm, estimulación sensitiva a 50 Hertz con 0,3-0,5 V aplicando 80 °C por 60 segundos en dos o tres fases. La presión intraocular se midió 24 horas previas al procedimiento, 24 horas posteriores al procedimiento y siete días después del mismo. El análisis de la presión intraocular, así como la escala EVA de dolor, se determinó mediante un análisis de ANOVA de medidas repetidas. Resultados: Se incluyeron 30 pacientes de los cuales 26 fueron mujeres, la edad promedio fue de 59,6 ± (DE 11,16). La tensión intraocular 24 h antes, un día después y una semana después de la realización de la radiofrecuencia del ganglio de Gasser no mostró modificaciones significativas con ANOVA para muestras repetidas (p = 0,916). Conclusiones: La tensión intraocular no se modificó a corto plazo después de realizar radiofrecuencia del ganglio de Gasser
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Digitally connected: global perspectives on youth and digital media
No full text“Digitally Connected: Global Perspectives on Youth and Digital Media,” is a first-of-its kind collection of essays that offers reflections from diverse perspectives on youth experiences with digital media and with focus on the Global South. It creatively combines adult voices with written and visual contributions by young people from around the world.
In this unique ebook, more than 30 academics, practitioners, government officials, tech industry representatives and activists team up with 25 youth contributors to share their views and opinions about digital technologies and the impact the Internet has on young people’s lives. Collectively, the contributors address a series of big questions related to youth and digital media by exploring key topics such as safety and wellbeing; identity, privacy and reputation; skills, literacies, and cultures of learning; creativity; innovation and entrepreneurship; participation and civic engagement; and youth participation and policy.
“By making these diverse reflections and youth contributions available to the public, we hope to continue and further stimulate the global conversation about both the challenges and opportunities children and youth face online,” said Urs Gasser, executive director of the Berkman Center and the book’s co-editor.
The ebook is an output of Digitally Connected, an initiative incubated by the Berkman Center in collaboration with UNICEF that brings together a network of people from around the world who, together, are addressing the challenges and opportunities children and youth encounter in the digital environment.
“The heart and soul of Digitally Connected is this amazing group of people from all around the globe. Together we aspire to make the Internet an even better place and experience for all children and youth,” said Sandra Cortesi, director of Youth and Media and the book’s co-editor.
Digitally Connected was launched in April 2014 at a first-of-its-kind international symposium on children, youth, and digital media co-hosted by the Berkman Center and UNICEF, in collaboration with PEW Internet, EU Kids Online, the Internet Society (ISOC), the Family Online Safety Institute (FOSI), and YouthPolicy.org. This event was followed by “Conectados al Sur,” a regional (Latin America and Caribbean) symposium on child and youth digital citizenship co-hosted by Argentina’s Ministry of Justice and Human Rights through the National Directorate for Personal Data Protection, UNICEF Argentina (with the support of the Division of Communication at UNICEF headquarters), and the Berkman Center. The goal of both events was to debate the challenges and opportunities youth encounter online, map and explore the global and regional state of relevant research and practice, share and discuss global insights and ideas, and encourage collaboration between participants across regions and continents. With a particular focus on voices and issues from the Global South, the events addressed topics such as inequitable access, risks to safety and privacy, skills and digital literacy, spaces for participation, civic engagement, and innovation
Ten polymorphic microsatellite loci for Allobates femoralis, an Amazonian dendrobatoid frog
No full textJehle R, Gasser H, Pfunder M, Amezquita A, Lima AP, Hoedl W. Ten polymorphic microsatellite loci for Allobates femoralis, an Amazonian dendrobatoid frog. MOLECULAR ECOLOGY RESOURCES. 2008;8(6):1326-1328
Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets
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