Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

Debriefing after resuscitations in a pediatric emergency department

Multiple guidelines recommend debriefing of actual resuscitations to improve clinical performance. We implemented a novel standardized debriefing program using a Debriefing In Situ Conversation after Emergent Resuscitations Now (DISCERN) tool. Following the development of the evidence-based DISCERN tool, we conducted an observational study of all resuscitations (intubation, CPR, and/or defibrillation) at a pediatric emergency department (ED) over one year. Resuscitation interventions, patient survival, and physician team leader characteristics were analyzed as predictors for debriefing. Each debriefing's participants, time duration, and content were recorded. Thematic content of debriefings was categorized by framework approach into Team Emergency Assessment Measure (TEAM) elements. There were 241 resuscitations and 63 (26%) debriefings. A higher proportion of debriefings occurred after CPR (p<0.001) or ED death (p<0.001). Debriefing participants always included an attending and nurse; the median number of staff roles present was six. Median interval (from resuscitation end to start of debriefing) & debriefing durations were 33 (IQR 15,67) and 10 minutes (IQR 5,12), respectively. Common TEAM themes included co-operation/coordination (30%), communication (22%), and situational awareness (15%). Stated reasons for not debriefing included: unnecessary (78%), time constraints (19%), or other reasons (3%). Debriefings with the DISCERN tool usually involved higher acuity resuscitations, involved most of the indicated personnel, and lasted less than 10 minutes. This qualitative tool could be adapted to other settings. Future studies are needed to evaluate for potential impacts on education, quality improvement programming, and staff emotional well-being.^

Development and implementation of a dynamic heterogeneous proton equivalent anthropomorphic thorax phantom for the assessment of scanned proton beam therapy

DEVELOPMENT AND IMPLEMENTATION OF A DYNAMIC HETEROGENEOUS PROTON EQUIVALENT ANTHROPOMORPHIC THORAX PHANTOM FOR THE ASSESSMENT OF SCANNED PROTON BEAM THERAPY by James Leroy Neihart, B.S. APPROVED: ______________________________David Followill, Ph.D. ______________________________Peter Balter, Ph.D. ______________________________Narayan Sahoo, Ph.D. ______________________________Kenneth Hess, Ph.D. ______________________________Paige Summers, M.S. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston DEVELOPMENT AND IMPLEMENTATION OF A DYNAMIC HETEROGENEOUS PROTON EQUIVALENT ANTHROPOMORPHIC THORAX PHANTOM FOR THE ASSESSMENT OF SCANNED PROTON BEAM THERAPY A THESIS Presented to the Faculty of The University of Texas Health Science Center at Houston andThe University of TexasMD Anderson Cancer CenterGraduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE by James Leroy Neihart, B.S. Houston, Texas Date of Graduation August, 2013 Acknowledgments I would like to acknowledge my advisory committee members, chair David Followill, Ph.D., Peter Balter, Ph.D, Narayan Sahoo, Ph.D., Kenneth Hess, Ph.D., Paige Summers M.S. and, for their time and effort contributed to this project. I would additionally like to thank the faculty and staff at the PTC-H and the RPC who assisted in many aspects of this project. Falk Pӧnisch, Ph.D. for his breath hold proton therapy treatment expertise, Matt Palmer and Jaques Bluett for proton dosimetry assistance, Matt Kerr for verification plan assistance, Carrie Amador, Nadia Hernandez, Trang Nguyen, Andrea Molineu, Lynda McDonald for TLD and film dosimetry assistance. Finally, I would like to thank my wife and family for their support and encouragement during my research and studies. Development and implementation of a dynamic heterogeneous proton equivalent anthropomorphic thorax phantom for the assessment of scanned proton beam therapy By: James Leroy Neihart, B.S. Chair of Advisory Committee: David Followill, Ph.D Proton therapy has been gaining ground recently in radiation oncology. To date, the most successful utilization of proton therapy is in head and neck cases as well as prostate cases. These tumor locations do not suffer from the resulting difficulties of treatment delivery as a result of respiratory motion. Lung tumors require either breath hold or motion tracking, neither of which have been assessed with an end-to-end phantom for proton treatments. Currently, the RPC does not have a dynamic thoracic phantom for proton therapy procedure assessment. Additionally, such a phantom could be an excellent means of assessing quality assurance of the procedures of proton therapy centers wishing to participate in clinical trials. An eventual goal of this phantom is to have a means of evaluating and auditing institutions for the ability to start clinical trials utilizing proton therapy procedures for lung cancers. Therefore, the hypothesis of this study is that a dynamic anthropomorphic thoracic phantom can be created to evaluate end-to-end proton therapy treatment procedures for lung cancer to assure agreement between the measured and calculated dose within 5% / 5 mm with a reproducibility of 2%. Multiple materials were assessed for thoracic heterogeneity equivalency. The phantom was designed from the materials found to be in greatest agreement. The phantom was treated in an end-to-end treatment four times, which included simulation, treatment planning and treatment delivery. Each treatment plan was delivered three times to assess reproducibility. The dose measured within the phantom was compared to that of the treatment plan. The hypothesis was fully supported for three of the treatment plans, but failed the reproducibility requirement for the most aggressive treatment plan.

Regulation of mRNA splicing in Moloney murine sarcoma virus mutants

Cells infected with the conditionally defective MuSVts110 mutant of Moloney murine sarcoma virus are transformed at 33$\sp\circ$C but appear morphologically normal at 39$\sp\circ$C. The molecular basis for this phenotype is as follows: MuSVts110 contains a 1487 nucleotide central deletion that has truncated the 3$\sp\prime$ end to the gag gene and the 5$\sp\prime$ end of the mos gene. The resulting gag-mos junction is out-of-frame and the v-mos protein is not expressed. At 33$\sp\circ$C or lower, a splicing event is activated such that a 431 base intron is removed to realign the gag and mos gene in-frame, allowing the expression of a transforming protein P85$\sp{gag-mos}$. Temperature-dependent splicing appeared to be an intrinsic property of MuSVts110 transcripts and not a general feature of pre-mRNA splicing in 6m2 cells since splicing activity of a heterologous transcript in the same cells did not vary with temperature. The possibility that the splice event was not temperature-sensitive, but that the accumulation of spliced transcript at the lower growth temperatures was due to its selective thermolability was ruled out as stability studies revealed that the relative turnover rates of the unspliced and spliced MuSVts110 transcripts were not affected by temperature.^ The consensus sequences containing the splice sites activated in the MuSVts110 mutant (5$\sp\prime$ gag and 3$\sp\prime$ mos) are present, but not utilized, in wild-type MuSV-124. To test the hypothesis that it was the reduction of the 1919 base intervening sequence in MuSV-124 to 431 bases in MuSVts110 which activated splicing, the identical 1487 base deletion was introduced into cloned wild-type MuSV-124 DNA to create the MuSVts110 equivalent, ts32.^ To examine conditions permissive for splicing, we assayed splice site activation in a series of MuSV-124 "intron-modification" mutants. Data suggest that splicing in wild-type MuSV-124 may be blocked due to the lack of a proximal branchpoint sequence, but can be activated by those intron mutations which reposition a branch site closer to the 3$\sp\prime$ splice site. (Abstract shortened with permission of author.) ^

Altered tumorigenic phenotype in mice with a hypomorphic p53 mutant allele

Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^

La educación especial a debate

En este texto y en consonancia con propuestas similares de otros autores, se cuestiona el sentido y la utilidad de la educación especial, como ámbito de conocimiento y para la intervención educativa. En opinión de unos y otros, continuar manteniendo dicho enfoque, lejos de contribuir significativamente a la mejora de la calidad de vida de aquellos a los que dice servir, y al pensar e intervenir desde perspectivas y políticas sociales y educativas “sectorizadas", se refuerza el estatus quo de las perspectivas dominantes y se limitan los esfuerzos por conseguir una auténtica igualdad de derechos y oportunidades de quienes se encuentran en situación de vulnerabilidad. En el mismo también se plantean los contenidos más relevantes de la inclusión educativa como marco de referencia común para mejorar la educación de todo el alumnado, sin exclusiones.

Liberdade - ponto de chegada de uma criação contínua : os portuguez (1814-1826)

Razão de uma escolha: a importância do periódico O Portuguez e do seu redactor João Bernardo da Rocha Loureiro no desencadear da Revolução Liberal portuguesa de 1820, que deu início ao processo que colocou fim à monarquia absoluta e desencadeou a desconstrução do Antigo Regime em Portugal. Nesta panorâmica, e tendo como horizonte de chegada a fruição da liberdade, pode dizer-se que, enquanto sistema de significações colectivas, o imaginário do redactor Rocha Loureiro parece actuar como reacção ao poder dissolvedor da inteligência, como regulador que satisfaz a busca apaixonada de um grupo ou sociedade que persegue a sua identidade da qual a liberdade é elemento constituinte, fundamental e fundamentador. A liberdade define-se pela ausência de limitação às garantias necessárias para o harmonioso desenvolvimento do indivíduo. Antes de mais a liberdade é, na sua essência, uma ausência de limitação, sendo simultaneamente uma determinação positiva e espontânea da vontade de liberdade em que cada um procura atingir o fim racional de que está possuído. O indivíduo deseja a liberdade com vista a alcançar o melhor possível do seu ser. Na essência, a vontade de liberdade de cada um confunde-se com a vontade colectiva que encontra a sua expressão no poder político.

La liquidación de la Guerra Civil

El tema de la guerra civil española no puede comprenderse cabalmente sin estudiar tanto el clima social y político anterior al enfrentamiento bélico como el proceso de liquidación de este trascendente hecho histórico. El bando triunfante pretendió que el cese de las operaciones militares ponía fin a la guerra, pero ésta no terminó entonces para la mayoría de los españoles. Durante muchos años continuó el uso de la fuerza, en forma unilateral bajo la forma de una sangrienta represión en el orden interno y un largo exilio de los vencidos. Otros hechos son los sufrimientos morales y psicológicos impuestos, los perjuicios culturales ocasionados, el destierro y su repercusión sobre millones de hogares. También pesan los quebrantos económicos y sociales sobre el bienestar general Para apreciar la forma elegida por el régimen franquista para liquidar la guerra civil española resulta ilustrativo detallar algunos aspectos de la modalidad puesta en práctica, cuyo fracaso pone de manifiesto la mezquindad con que actuó el régimen dictatorial.

Theoretical frames for the new tasks in career guidance and counseling

Personal and career development interventions aim to help people find answers to personal and career development issues that stem from the societal context in which they live. Societal definitions of these career issues have a double consequence. On the one hand, these issues differ from one culture to another; and, on the other, they evolve along with the contexts in which they are expressed. Implementation of rigorous career development interventions requires, first, a scientific reconstruction of these societal issues and, second, a clear definition of these interventions' goals and ends. Our current view of the societal issues relating to personal and career development interventions may be phrased thus--"How can we help individuals direct their lives, in the (human) society where they interact?" It may be turned into the following scientific question: "What are the factors and processes of life-long self-construction?" An articulation of three major propositions (sociological, cognitive and dynamic) seems to be needed to answer this question. Such a theoretical frame does not allow for a definition of personal and career development interventions ends. In the world of today, the adoption by everyone of a personal ethic of responsibility towards all life on Earth (H. Jonas) could well be a fundamental end to these interventions.

Naturaleza e historia en la obra de Denis Diderot

En los últimos años los críticos han derribado varios estereotipos que habían sido utilizados para caracterizar a la filosofía de la Ilustración. Esos clichés en muchos casos se encontraban muy alejados del pensamiento de los filósofos iluministas. No obstante, varios temas no han sido aún suficientemente revisados. En este trabajo se intenta demostrar que la reflexión de Denis Diderot sobre el sentido de la historia, en la medida que desborda los diferentes esquemas interpretativos esbozados hasta el momento por los especialistas, es uno de ellos

La liquidación de la Guerra Civil

El tema de la guerra civil española no puede comprenderse cabalmente sin estudiar tanto el clima social y político anterior al enfrentamiento bélico como el proceso de liquidación de este trascendente hecho histórico. El bando triunfante pretendió que el cese de las operaciones militares ponía fin a la guerra, pero ésta no terminó entonces para la mayoría de los españoles. Durante muchos años continuó el uso de la fuerza, en forma unilateral bajo la forma de una sangrienta represión en el orden interno y un largo exilio de los vencidos. Otros hechos son los sufrimientos morales y psicológicos impuestos, los perjuicios culturales ocasionados, el destierro y su repercusión sobre millones de hogares. También pesan los quebrantos económicos y sociales sobre el bienestar general Para apreciar la forma elegida por el régimen franquista para liquidar la guerra civil española resulta ilustrativo detallar algunos aspectos de la modalidad puesta en práctica, cuyo fracaso pone de manifiesto la mezquindad con que actuó el régimen dictatorial.

Theoretical frames for the new tasks in career guidance and counseling

Personal and career development interventions aim to help people find answers to personal and career development issues that stem from the societal context in which they live. Societal definitions of these career issues have a double consequence. On the one hand, these issues differ from one culture to another; and, on the other, they evolve along with the contexts in which they are expressed. Implementation of rigorous career development interventions requires, first, a scientific reconstruction of these societal issues and, second, a clear definition of these interventions' goals and ends. Our current view of the societal issues relating to personal and career development interventions may be phrased thus--"How can we help individuals direct their lives, in the (human) society where they interact?" It may be turned into the following scientific question: "What are the factors and processes of life-long self-construction?" An articulation of three major propositions (sociological, cognitive and dynamic) seems to be needed to answer this question. Such a theoretical frame does not allow for a definition of personal and career development interventions ends. In the world of today, the adoption by everyone of a personal ethic of responsibility towards all life on Earth (H. Jonas) could well be a fundamental end to these interventions.

Naturaleza e historia en la obra de Denis Diderot

En los últimos años los críticos han derribado varios estereotipos que habían sido utilizados para caracterizar a la filosofía de la Ilustración. Esos clichés en muchos casos se encontraban muy alejados del pensamiento de los filósofos iluministas. No obstante, varios temas no han sido aún suficientemente revisados. En este trabajo se intenta demostrar que la reflexión de Denis Diderot sobre el sentido de la historia, en la medida que desborda los diferentes esquemas interpretativos esbozados hasta el momento por los especialistas, es uno de ellos

Theoretical frames for the new tasks in career guidance and counseling

Personal and career development interventions aim to help people find answers to personal and career development issues that stem from the societal context in which they live. Societal definitions of these career issues have a double consequence. On the one hand, these issues differ from one culture to another; and, on the other, they evolve along with the contexts in which they are expressed. Implementation of rigorous career development interventions requires, first, a scientific reconstruction of these societal issues and, second, a clear definition of these interventions' goals and ends. Our current view of the societal issues relating to personal and career development interventions may be phrased thus--"How can we help individuals direct their lives, in the (human) society where they interact?" It may be turned into the following scientific question: "What are the factors and processes of life-long self-construction?" An articulation of three major propositions (sociological, cognitive and dynamic) seems to be needed to answer this question. Such a theoretical frame does not allow for a definition of personal and career development interventions ends. In the world of today, the adoption by everyone of a personal ethic of responsibility towards all life on Earth (H. Jonas) could well be a fundamental end to these interventions.