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MERKELCELCARCINOOM, IDENTIFICATIE VAN NIEUWE MOLECULAIRE TARGETS EN ONTWIKKELING VAN EEN DRIEDIMENSIONAAL CELCULTUURMODEL
No full textMerkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma. Nevertheless, its incidence has increased in the last few years and this trend is predicted to persist. Furthermore, with a general mortality rate of 33-46% and a high rate of recurrences, it is one of the most aggressive types of skin cancer. Currently, MCC has two recognized etiologies. Merkel cell polyomavirus (MCPyV) DNA is found clonally integrated in the genome of the tumor cells in around 80% of MCCs from the Northern hemisphere. MCPyV-positive (MCPyV+) MCCs constitutively express two viral oncoproteins, the small (sT) and the large (LT) tumor antigens (TAs), which contribute to the uncontrolled proliferation of the transformed cells. The MCPyV-negative (MCPyV-) subtype, which is predominant in Australia and New Zealand, is thought to arise upon accumulation of UV-light induced somatic mutations in proto-oncogenes and tumor suppressors. This is evidenced by a higher mutational burden than that of the MCPyV+ counterpart. In any case, the two subtypes of MCC share oncogenic mechanisms, such as inactivation of the retinoblastoma protein, which may explain the phenotypic similarities.
MCC is primarily a disease of the elderly, though its incidence is also higher among immunocompromised individuals. Until recently, the classic treatment modalities consisted of surgery and radiotherapy for local lesions or locally metastatic disease, and chemotherapy for metastasized MCC. Nevertheless, responses to chemotherapy were mostly short-lived and at expenses of an elevated general toxicity. Recently, the advent of immune checkpoint inhibitors, such as avelumab and pembrolizumab, has considerably improved the management of patients with metastasized disease. These immune checkpoint inhibitors induced durable responses with acceptable toxicity, highlighting the importance of tumor infiltrating lymphocytes and the role of the tumor microenvironment in the clinical outcome of patients with MCC. Hence, immunotherapies have become the standard of care for patients with metastatic MCC and their use at earlier stages or as adjuvant is currently under investigation in clinical trials. However, around half of the patients do not respond to the treatment or develop resistance after an initial response. Furthermore, a large proportion of patients, who are immunosuppressed or under immune suppressive treatment, are not eligible for immunotherapy. Similarly, elderly patients often suffer from other comorbidities or the tumors commonly appear at areas, such as the face, that are delicate for intervention with surgery or radiotherapy. Consequently, there is an unmet need for the development of alternative therapeutic approaches to treat patients with MCC that are not amenable to the current treatment possibilities.
MCPyV+ MCCs constitutively express both the viral sT and a truncated form of the LT. The truncated LT is unable to initiate viral replication due to lack of the carboxyl (C)-terminal sequence containing the DNA origin binding domain and the DNA helicase activity. These viral proteins have been shown to have important roles in tumorigenesis and in the maintenance of MCPyV+ MCC cells proliferation. Here, current evidence points towards a more predominant role of MCPyV sT in tumor initiation, whereas the LT has been suggested to be involved in maintenance of the oncogenic phenotype. Indeed, experiments with short hairpin RNAs (shRNAs) targeting the viral TAs showed that MCPyV+ MCC cells required the expression of these oncoproteins to maintain cell growth. Therefore, we hypothesized that the most advanced gene-editing tool currently available, CRISPR/Cas9, could be used to target the viral TAs and, consequently, hamper the growth of virus-positive MCCs. Thus, we applied this approach in two MCPyV+ MCC cell lines, MS-1 and WAGA, by designing single-guide RNAs (sgRNAs) targeting only the LT or both, sT and LT. Our results showed that this CRISPR/Cas9-based strategy caused a significant decrease of LT protein levels, impaired cell proliferation and reduced cell cycle progression. Importantly, cells transfected with a non-targeting sgRNA and HEK293T cells, which are MCPyV-, remained unaffected. Hence, our results further validated previous reports regarding the role of MCPyV TAs in the maintenance of the growth of MCC cells. Furthermore, these findings provided strong evidence for the development of a CRISPR/Cas9-based therapeutic approach against MCPyV+ MCC.
Targeted therapy presents as an attractive approach against the two subtypes of MCC. Several reports have shown that the PI3K/mTOR pathway is frequently activated in MCC, irrespective of the viral state. Consequently, MCC cell lines and xenografts were shown to be sensitive to MLN0128, a dual mTOR1/2 inhibitor. This drug is currently under clinical investigation for its use in the treatment of MCC and other solid tumors. Although the MAPK/ERK pathway has not been reported to be aberrantly activated in MCC, there is a crosstalk with the PI3K/mTOR pathway and compensatory activation has been largely proved in other malignancies. Therefore, these observations prompted us to test the effects of combining MLN0128 with trametinib, a MEK1/2 inhibitor that is currently used for the treatment of metastatic melanoma with the V600E or V600K changes in the BRAF protein. A panel of three virus-positive and three virus-negative MCC cell lines were treated with combinations of these two compounds. As determined by the Chou-Talalay method, the combined targeting was synergistic in the assayed MCC cells. Thus, a reduced dose of each inhibitor was necessary to reach the same efficacy in inhibiting cell growth than when used as single agents. Although the precise mechanism involved in this synergistic activity has not been completely elucidated, the combined targeting of the PI3K/mTOR and MAPK/ERK pathways appears to be a promising approach to treat MCC and to overcome the limitations of single drug regimens related to general toxicity and drug resistance.
One of the main hurdles for the development of new treatments against MCC is the difficulty to perform prospective clinical trials, due to the rarity and rapid evolution of the disease. Furthermore, there is lack of a model able to mimic the process of MCC tumorigenesis in vitro. Although cell lines and patient derived xenografts of MCC have been established, they present certain limitations, owing the physiological differences between mice and humans. Moreover, transgenic animals expressing the viral oncoproteins have not been able to fully resemble virus-positive MCCs thus far. Here, we developed organotypic epithelial raft cultures (OERCs) of MCC by using primary human keratinocytes (PHKs) and both MCPyV+ and MCPyV- MCC cell lines grown on top of an artificial dermal equivalent. The differentiation of the rafts and the growth of MCC cell lines was confirmed through histology and immunohistochemistry. Normal PHKs grown on top of an artificial dermal equivalent at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Important differences were noted in the behaviour of the different MCC cell lines tested, such as growth pattern, invasiveness, and requirement of keratinocytes to proliferate. Virus-positive MCC cell lines generally grew exclusively on top of the differentiated epithelium, forming cell clusters. Among the virus-negative MCC cell lines, only MCC14/2 cells showed robust proliferation, even in the absence of keratinocytes and when embedded into the artificial dermis. Gene expression analysis of OERCs of co-cultures of MCC cell lines and PHKs revealed that some genes, such as those encoding distinct types of collagens, were differently expressed in rafts from MCPyV+ cells and rafts from MCC14/2 cells. Furthermore, several molecules were found highly expressed in all the analysed OERCs (e.g., matrix metalloproteinases that are involved in the degradation of the extracellular matrix, thus contributing to cell invasion and migration) and they might serve as potential targets for novel MCC treatments or as diagnostic biomarkers. Despite the need to standardize and adapt this model to high throughput analysis, OERCs represent a suitable tool for evaluating the efficacy and selectivity of new drug candidates against MCC. Furthermore, they can potentially be employed to study the molecular events driving MCC.
In summary, the work presented in the current thesis provides new insights into the understanding of MCC and the bases for the investigation of new therapeutic approaches to treat patients suffering from this malignancy, either alone or in combination with the current treatment modalities. Moreover, advances in the development of OERCs will provide clinicians and researchers with new means to test novel drug candidates against MCC and to shed light into the molecular mechanisms involved in tumorigenesis.status: Publishe
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
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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