1,721,048 research outputs found
Unravelling RecQ helicase function in genome stability using Strand-seq
No full textHelicases are a highly conserved family of motor proteins responsible for interacting with and unwinding canonical and non-canonical DNA and RNA structures. The RecQ class of helicases, known to suppress illegitimate recombination, are implicated in aging and cancer with four of the five human RecQ helicases directly linked to genome instability syndromes characterized in some cases by strong cancer predisposition or premature aging. While no human disease has been associated with the RECQL5 helicase, loss of this gene in cells is known to result in elevated double strand breaks (DSBs) and sister chromatid exchange events (SCEs), a phenotype of genome instability similar to what is observed in RecQ helicase-linked diseases of strong cancer predisposition. Until recently, studying SCEs has been limited to cytogenetic assays that map at megabase resolution. I used single cell template strand sequencing (Strand-seq) to map SCEs as changes in template strand orientation before and after loss of RECQL5 at kilobase resolution. I generated over 20 single and double knockout models for RECQL5 as well as BLM, WRN and RECQL1 helicases using CRISPR-Cas9 in the human haploid cell line, KBM7, and mapped SCEs to the genome using custom bioinformatic approaches to improve resolution and accuracy of SCE detection. I performed enrichment analysis to show SCEs are frequently occurring near actively transcribed genes with guanine quadruplexes (G4s) and common fragile sites further supporting the role of these helicase genes in suppressing inappropriate recombination at specific genomic elements. I also developed novel bioinformatic approaches to generate genotype-specific call sets for copy number alterations (CNAs), inversions, and translocations. Uncovering the role of DNA helicases in DNA repair and replication pathways is critical for understanding their significance in cancer and aging. Stand-seq offers a unique method to study helicases by mapping the location of SCEs arising in their absence.Science, Faculty ofGraduat
Analysis of single telomeres using fluorescence microscopy
No full textEukaryotic chromosome ends are protected by special DNA structures known as telomeres. In mammals the DNA of telomeres consist of TTAGGG repeats which, in cooperation with specialized proteins, “cap” the ends of chromosomes to protect the chromosomes from end-to-end fusion and erosion. Thus, telomeres are important to maintain chromosome stability and play a vital role in preserving the information in our genome. A key factor of telomeric function is the length of the telomeres. Short and dysfunctional telomeres with less than a few dozen repeats are associated with genomic instability and tumorigenesis. Furthermore, loss of telomere function is implicated in numerous diseases like bone marrow failure, hematological malignancies and other cancers.
There is a substantial body of evidence indicating that the average length of telomeres can provide prognostic information in human diseases. However, limitations in the currently available technologies for detecting and measuring the length of telomeres has hampered progress in translating telomere length assays into clinical practice. Additionally, many questions about the relation between telomere length and telomere function remain to be answered. Consequently, novel approaches to study single telomeres are of significant interest.
In this study, I investigated two cutting-edge technologies to assess properties of telomeres. I used quantitative fluorescence in situ hybridization (Q-FISH) to identify the length of telomeres based on fluorescence values. Using Q-FISH, I was able to generate DNA measurements using plasmids with different size telomeric inserts that served as a reference for length quantifications taken on different platforms.
Also, I initiated explorations of a novel high-throughput method to study physical properties of single telomeres and, potentially, measure the length of telomere repeats. Convex Lens-induced Confinement technique (CLiC) is a technique developed to image individual biological molecules and study their dynamics. Using the CLiC platform, I sought to define the length of plasmid DNA based on diffusion coefficient values.
My work has set the stage for others to explore the CLiC platform to study properties of telomeres including biological properties as well as their length. The latter can possibly be used as a prognostic tool in bone marrow failure, hematological malignancies and other disorders.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Understanding the impact of senescence on CAR-T cell manufacturing
No full textChimeric antigen receptor (CAR)-T cell therapy has proven efficacious in hematological
malignancies, with success in CD19-targeted therapy for leukemia/lymphoma and B cell maturation antigen (BCMA)-targeted therapy in multiple myeloma. However, a significant portion
of patients still relapse post-therapy. Factors such as T cell exhaustion and T cell memory
phenotypes have been thought to contribute, given that they may impact persistence which has
been correlated with poor outcomes after CAR-T cell therapy. In this project, we explore whether
telomere maintenance plays a role in CAR-T cell persistence, given that shortened telomeres result in a loss of proliferative capacity. During CAR-T cell manufacturing, the T cells undergo a rapid expansion phase, which may lead to telomere shortening. Thus, methods were explored to measure telomerase activity, telomere length, cell cycle progression, senescence-associated -galactosidase (SA -gal) activity, and various cell surface markers. Through repetitive stimulations, telomerase activity decreased with each round of stimulations, and cells reached a plateau in growth. SA -gal increased with each round of stimulation and an increased proportion of cells were observed in G₀/G₁. To address this, an 8-day and 12-day manufacturing process was assessed, to determine if this may better preserve cell phenotype. The 8-day process had an increased proportion of the cells in G₂/M compared to the 12-day manufacture, however had increased levels of SA -gal. Lastly, epigenetic age has been explored as a potential predictor of patient outcomes and was assessed in healthy donor samples and patient samples from a CD19-directed CAR-T cell clinical trial. Responders were found to have a lower epigenetic age than non-responders, providing the rationale for further study.Medicine, Department ofGraduat
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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