1,721,021 research outputs found
Electron Cryo-Tomography of Vitreous Cryo-Sections: Towards Imaging Biological Nanomachines in their Cellular Environment
No full textThe field of biological electron microcopy (EM) has evolved into a reliable imaging technique for examining the ultrastructure of cells and their constituents at molecular, and, in some cases, atomic1, 2 resolution. Conventional EM techniques are being overshadowed by cryo-techniques, which ensure a more native cellular preparation method. In addition, electron tomography eliminates the projection problem of single EM images and adds a third dimension to the analysis. The cryo-preparation technique combined with tomography is currently the most suitable technique to enable a direct three-dimensional observation of cells and their constituents in a close-to-native environment. Although several aspects of cell biology have profited from information obtained by cryo-ET of plunge frozen whole cells preparations or isolated organelles or molecular complexes studies, the majority of cells are too large to freeze using conventional plunge freezing techniques. Therefore, the technique of vitreous cryo-sectioning was introduced to incorporate large cells and pieces of tissue that are too large for conventional freezing and imaging techniques. As we have seen throughout this thesis, the technique of vitreous cryo-sectioning for electron cryo-tomography is a developing method that was difficult to implement in many EM labs. It requires a high level of dexterity for manipulating the thin ribbon of vitreous cryo sections and transferring it to an EM. In addition, the contamination levels within the microtome coat the equipment, EM grid, and sections with water contamination in the form of ice crystals. In Chapter 3 we addressed these limitations; section attachment to the support film and contamination within the cryo-ultramicrotome. By altering the output of the electrode, normally used to ionize the surface of the diamond for better surface gliding properties of the vitreous cryo-sections, we developed a method to electrostatically attach sections to the EM support film. We illustrated the advantage of these novel developments by exploring the ultrastructure of the 80S Saccharomyces cerevisiae ribosome in a close-to-native, cellular environment. As a result of the novel methods developed, the technique of electron cryo-tomography of vitreous cryo-sectioning is becoming a more widely and reliable method for ultra structural cellular studies. At the current resolution limit of 4-5 nm, large macromolecular complexes like 80S ribosomes are easy to recognize. However as the resolution increases by an order of magnitude, the near future should allow a full comprehensive description of supramolecular architecture within cells. Although the technique of vitreous cryo-sectioning is gaining importance it’s still plagued by section-induced cutting artifacts, as we have seen in Chapter 4. It was thought that vitreous cryo-section induced compression affected the specimen in a homogeneous manner. In other words, if the cell would be compressed by a given value then all its intracellular components would be compressed by the same value. What we observed, however, was at the current resolution limit of 4-5 nm, large macromolecular complexes like the 80S ribosomes remained unaltered by section-induced compression. We concluded that cells and their constituents are not affected by the forces of section-induced compression in a uniform manner. Therefore, the technique of vitreous cryo-sectioning will remain valuable for exploring macromolecular landscapes for large cells and tissues that cannot be imaged in toto. In Chapter 5 we applied the novel techniques from the previous studies to Escherichia coli cells that have been infected with bacteriophage T7. The main goal of this study was to visualize the structural components that are assembled by the phage in order to transfer its genome into the host cell, a feat that had never been achieved. In our resulting observations we found three distinct phenotypes of the capsid interacting with the cell. In the study we focused on empty capsids because they have assembled the genome transfer complex and expelled the majority of their genome into the host cell. We observe that at the infection site, the outer membrane of the bacterial envelope is pinched towards the inner membrane. We also observed proteins associated with the outer membrane and also within the envelope of the bacteria. Unfortunately the technique of vitreous cryo-sectioning lacks a protein localization method and therefore we cannot identify the proteins expelled by the bacteriophage. We conclude, however, that the phage must construct a complex of proteins that work together to reduce the distance of the cell membrane in order to transfer its genome.Kavli Insititute of NanoscienceApplied Science
Subcellular trafficking of mycobacteria: Implications for virulence and immunogenicity
No full textThe aim of this thesis is to determine the properties of the compartment where mycobacteria end up after phagocytosis and which mycobacterial genes play a role in this process. In most cases, bacterial pathogens are taken up by the cell, processed in the endocytic pathway and eventually bacterial derived peptides are presented on MHC class II molecules to CD4+ T-cells. Proteins from viral pathogens in contrast, are degraded in the cytosol and transported into the ER for presentation on MHC class I molecules to CD8+ T-cells. Sometimes, peptides from bacterial pathogens are also presented by MHC class I molecules, a process called cross-presentation. The exact mechanism is still unknown but essential in eliciting a good immune response. Several theories are mentioned; one of these theories is that the phagosome can fuse with the ER, allowing the pathogens and derived peptides to get in contact with MHC class I molecules. This theory raised a lot of questions, and was therefore examined carefully by us and others in chapter 1. Biochemical approaches, together with fluorescence and electron microscopy were used to quantitatively and dynamically assess the contribution of the ER to phagosome formation and maturation. We could not detect continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to the formation or maturation of phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main component of phagosomes. During maturation, the phagosomes fuse with endosomal and eventually lysosomal vesicles, to become acidic and degradative organelles. One of the bacterial pathogens whose peptides can be presented via the MHC class I pathway is Mycobacterium tuberculosis. For a very long time, it was not understood how this process takes place, as mycobacteria were reported to replicate in early phagosomes. In chapter 2 we show that after 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in non-apoptotic cells. Also M. leprae is able to translocate, while the vaccine strain M. bovis BCG or heat-killed mycobacteria remain phagosomal. The translocation process is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6, as mutants that do not secrete these proteins stay in the phagosome. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This cytosolic localization of virulent mycobacteria may also provide an explanation for the observed MHC class I based antigen presentation which is lacking in current vaccine strains. That it took a long time for this pathway to be discovered can be explained, as the set-up of the experiments is crucial to detect translocation. Translocation takes place later during infection and only with pathogenic species. Furthermore, not every technique is suitable to detect cytosolic mycobacteria. In chapter 3 we discussed why mycobacterial translocation to the cytosol has often been overlooked or has not been detected, but also addressed its importance for pathogenicity and future studies. The mycobacterial translocation pathway was further characterized in chapter 4, where we demonstrate the causality between translocation and pathogenicity. We show that various pathogenic, patient-derived mycobacteria are able to translocate to the cytosol, while non-pathogenic species or mutants can not. We further establish in this chapter that the mechanism of translocation is dependent on the ESX-1 (type VII) secretion system, as only strains that have a functional secretion system translocate, and reintroduction of the ESX-1 gene cluster into the non-virulent vaccine strain M. bovis BCG is sufficient to induce translocation. Several proteins are secreted by this secretion complex. We show that ESAT-6, and specifically the C-terminal region of the secreted ESAT-6 protein, is crucial for translocation. These data establish that the ESX-1 secretion system causes translocation and thereby determining mycobacterial pathogenicity. The compartment the mycobacteria reside in, determines which immune response will be triggered. The translocation to the cytosol gives more clarity on how antigen presentation via MHC class I molecules takes place. But there are also still question marks on the CD4+ T-cell activation, either via the MHC class II or CD1 presentation pathways. MHC class II molecules normally acquire peptide antigens and rapidly traffic from the phagolysosome to the plasma membrane, whereas CD1 molecules instead load lipid antigens, and continually recycle between the phagolysosome and the plasma membrane. In chapter 5 we show that both MHC class II and CD1b localize to bacterial phagosomes early after phagocytosis, but MHC class II traffics from the phagosome to the plasma membrane before the antigens are availabile and loading can take place. In contrast, CD1 antigen presentation is not hampered during M. tuberculosis infection, as a pool of CD1 molecules remains available on the phagolysosomal membrane that is able to acquire lipid antigens and deliver them to the plasma membrane. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow M. tuberculosis to use DC maturation as a mechanism of immune evasion. An important and powerful technique, which is used often in this thesis to answer biological questions, is the transmission electron microscope. This microscope can go to much higher resolutions than light microscopy. Although special sample preparation is necessary, there is no need for (antibody) markers to visualize cellular structures or compartments. By morphological distinctive features, organelles can be distinguished, although protein A gold markers can be used as well to detect specific proteins. The advantage of this is that you can visualize in addition the structures in the cell that you were not primarily looking at. In contrast to for example fluorescence microscopy, whereby only the labeled proteins are visible while the rest of the cell remains invisible. Without using the electron microscope, many results described in this thesis would not have been found. I would like to draw special attention to the findings of chapter 2. By examining human dendritic cells and macrophages, which were infected with M. tuberculosis, we unexpectedly found mycobacteria that were not surrounded by a phagosomal membrane. This was a serendipitous finding, as we were not looking for them and were not even aware of the possibility of M. tuberculosis to become cytosolic. If another technique had been used, this important discovery would not have been made. By exploring the cellular localization of mycobacteria in greater detail, also in relation to antigen presenting molecules, we now have more insight in this important mechanism. Not only do we have a better understanding why M. bovis BCG is not very effective as vaccine against tuberculosis, we also know the requirements on how to improve a future vaccine: getting it into the cytosol.Technische natuurwetenschappenApplied Science
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
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