1,721,053 research outputs found
Computational analysis of quantitative “omics” data
No full textOver the past decades, the rise of "omics" approaches has allowed for systematic, in-depth investigation of each aspect of molecular biology. It has contributed to our changed view on the on the linearity and the regulation of the informational flow of the central dogma. Different regulatory mechanisms have been identified, describing interaction and variety not only on the genetic level but also on the transcript and protein level. The development and integration of multi-omics have allowed for the uncovering of intricate molecular mechanisms underlying different phenotypic manifestations of traits at a high accuracy, in a systematic manner. With this, multi-omics is essentially the basis of network or systems biology.
In this thesis, I have utilized "omics" technologies, specifically proteomics, and the subsequent computational data analysis and integration to investigate the systematic DNA damage response in Tetrahymena thermophila and identify DNA damage proteins across the Tree of Life. Additionally, I co-developed a user-friendly computational pipeline for evolutionary positive selection analysis, which relies on comparative genomics and either large-scale genome sequencing or proteotranscriptomics data.
In Chapter 2, we mapped the system's response to DNA damage over time in Tetrahymena thermophila (Nischwitz, Schoonenberg et al., in preparation). To date, limited studies have combined the strength of proteomics and transcriptomics to investigate DNA damage kinetics in response to various DNA-damage treatments. Our study investigated DNA damage response (DDR) dynamics over eight hours after or during exposure to six different mutagens. We observed upregulation of previously identified DNA damage repair pathways and found novel crosstalk between DDR pathways. All treatments induced a dynamic response at both the transcript and protein levels. Using unsupervised self-organizing maps, we examined the clustering of expression profile trends to better understand the DDR. Many of the quantified proteins and transcripts exhibited damage-specific responses. We are currently employing a novel knockdown system to target a subset of PARP-related proteins to characterize their specific roles in Tetrahymena further.
In Chapter 3, we studied the interactome of specific DNA damage lesions across the Tree of Life, exploring the conservation of pathways responsible for repair and recognition of DNA damage lesions (Nischwitz, Schoonenberg et al., iScience, 2023). Due to the need for precise genome maintenance, DNA repair has been highly conserved across all domains of life. To study the shared and unique elements of the DNA damage response, we performed a phylointeractomic study to identify enriched DNA damage binders in 11 different species at the 8-oxoG and abasic lesions and at a uracil base incorporated into DNA. Our approach identified several known DNA damage factors as binders to the afore-mentioned lesions. Additionally, through orthology, network, and domain analysis, we linked 44 previously unassociated proteins to DNA repair.
Finally, in Chapter 4, we developed a computational pipeline to make positive selection analysis user-friendly (Ceron-Noriega et al., Genome Biology and Evolution, 2023). AlexandrusPS generates orthology relationships, sequence alignments, and phylogenetic trees with its automated process. It then performs site-specific (SSM), branch (BM), and branch-site (BSM) positive selection analyses and produces four main output files, including orthology relationships, positive selection results, and all intermediate files (sequence alignments, phylogenetic trees).xiv, 137 Seiten ; Illustrationen, Diagramm
Investigations of genome instability utilizing quantitative proteomics
Full text linkThe field of mass spectrometry-based proteomics has had a profound impact on discoveries in almost every field of science. Specifically, bottom-up proteomics enables the exploration of scientific questions at a high-throughput and proteome-wide level. In this thesis, mass spectrometry-based proteomics was employed to understand aspects of genome instability related to: 1) telomere biology in Caenorhabditis elegans , 2) phylogenetic diversity of recognition and repair of in vitro DNA damage lesions, and 3) DNA damage kinetics in Tetrahymena thermophila.
Article I (Dietz et al. 2021) describes the extensive characterization of the first novel double-stranded telomere binders in C. elegans , TEBP-1 and TEBP-2. These proteins were discovered using in vitro telomere pulldown assays coupled with label-free and dimethyl quantitative mass spectrometry. TEBP-1 and TEBP-2 bind directly and specifically to double-stranded telomeric DNA. Both proteins are critical to the negative and positive regulation of telomere homeostasis. The double knockout strain of tebp-1;tebp-2 exhibits severe germline arm atrophy and synthetic sterility, suggesting their critical role in fertility. TEBP-1 and TEBP-2dimerize and directly interact with the known single-stranded binder POT-1, thereby connecting them to the known telomere complex in C. elegans.
Article II (Nischwitz and Schoonenberg et al., 2023) explores the conservation of recognition and repair of DNA damage lesions. Due to the imperative need for accurate maintenance of the genome, DNA repair has been highly conserved across all domains of life. To study both the shared and unique elements of the DNA damage response, we conducted a phylointeractomic study to identify enriched binders in 11 different species at the 8-oxoG and abasic lesions, as well as a uracil base incorporated into DNA. While numerous binders were canonical DNA damage factors, we also observed enrichment of proteins not previously associated with DNA repair. Through orthology, network, and domain analysis, we linked 44 proteins that were previously unassociated to DNA repair.
Article III (unpublished, Nischwitz and Schoonenberg et al., xxxx) delves into the kinetics of the DNA damage response (DDR) in the ciliate Tetrahymena thermophila (Tetrahymena) . To date, there have been limited studies that combine the power of proteomics and transcriptomics to investigate DNA damage kinetics across various treatments. Our screen monitored the dynamic DNA damage response over eight hours after exposure to six different mutagens. We observed upregulation of previously associated DNA damage repair pathways, as well as unexpected DDR crosstalk. All treatments elicited a dynamic response at both the transcript and protein level. Through unsupervised machine learning clustering, we examined expression profile trends to gain a more comprehensive understanding of the DDR, as many of these proteins exhibited damage-specific responses. Currently, we are employing a knockdown system to target a subset of these PARP-related proteins to further characterize their specific roles in Tetrahymena.Artikel I (Dietz et al. 2021) beschreibt die umfassende Charakterisierung der ersten bekannten doppelsträngigen Telomerbinder in C. elegans, TEBP-1 und TEBP-2. Diese Proteine wurden mit Hilfe von in vitro Telomer-Pulldown-Assays in Verbindung mit markierungsfreier und quantitativer Dimethyl-Massenspektrometrie entdeckt. TEBP-1 und TEBP-2 binden direkt und spezifisch an doppelsträngige telomere DNA. Beide Proteine sind entscheidend für die negative und positive Regulierung der Telomer-Homöostase. Der Doppel-Knockout-Stamm vontebp-1;tebp-2 weist eine schwere Keimbahnarmatrophie und synthetische Sterilität auf, was darauf hindeutet, dass beide Proteine eine entscheidende Rolle für die Fruchtbarkeit spielen.TEBP-1 und TEBP-2 dimerisieren und interagieren direkt mit dem bekannten EinzelstrangbinderPOT-1, wodurch sie mit dem bekannten Telomerkomplex in C. elegans verbunden sind.
Artikel II (Nischwitz und Schoonenberg et al., 2023) befasst sich mit der Erhaltung der Erkennung und Reparatur von DNA-Schäden. Aufgrund der zwingenden Notwendigkeit, das Genom akkurat zu erhalten, ist die DNA-Reparatur in allen Bereichen des Lebens in hohem Maße konserviert. Um sowohl die gemeinsamen als auch die einzigartigen Elemente derDNA-Schadensreaktion zu untersuchen, haben wir eine phylointeraktomische Studie durchgeführt, um in 11 verschiedenen Arten angereicherte Proteine, die an den 8-oxoG- und abasischen Läsionen sowie an einer in die DNA eingebauten Uracilbase binden, zu identifizieren. Bei zahlreichen Bindungsstellen handelte es sich um kanonische DNA-Schadensfaktoren, aber wir beobachteten auch eine Anreicherung von Proteinen, die bisher nicht mit der DNA-Reparatur in Verbindung gebracht wurden. Durch Orthologie-, Netzwerk und Domänenanalysen konnten wir 44 Proteine identifizieren, die zuvor nicht mit der DNA-Reparatur assoziiert wurden.
Artikel III (unveröffentlicht, Nischwitz und Schoonenberg et al., xxxx) befasst sich mit der Kinetik der DNA-Schadensreaktion (DDR) in dem Ciliaten Tetrahymena thermophila( Tetrahymena ). Bislang gibt es nur wenige Studien, die die Leistungsfähigkeit von Proteomik und Transkriptomik kombinieren, um die Kinetik von DNA-Schäden bei verschiedenen Behandlungen zu untersuchen. In unserem Screening wurde die dynamische DNA-Schadensreaktion über acht Stunden nach der Exposition gegenüber sechs verschiedenen Mutagenen überwacht. Wir beobachteten die Hochregulierung von zuvor assoziierten DNA-Schadensreparaturwegen sowie unerwartete DDR-Crosstalk. Alle Behandlungen lösten eine dynamische Reaktion sowohl auf der Transkriptions- als auch auf der Proteinebene aus. Mit Hilfe von unüberwachten maschinellen Lernens untersuchten wir die Trends der Expressionsprofile, um ein umfassenderes Verständnis der DDR zu gewinnen, da viele dieser Proteine schadensspezifische Reaktionen zeigten. Gegenwärtig setzen wir ein Knockdown-System ein, um eine Untergruppe dieser PARP-verwandten Proteine anzugreifen und ihre spezifische Rolle in Tetrahymena weiter zu charakterisieren.142 Seiten ; Illustrationen, Diagramm
Proteome-wide positive selection analysis on improved nematode gene annotation by machine learning assisted proteotranscriptomics
No full textZahlreiche Studien an der Nematode und Modellspezies Caenorhabditis elegans haben zu bedeutenden Entdeckungen in den Bereichen Biologie und Biomedizin geführt. Diese sind im Kontext der Evolution schwer zu extrapolieren, da das Nematoden-Phylum eine beträchtlich große phylogenetische Vielfalt aufweist. Als Beispiel für dieses Problem haben wir versucht, die Evolutionsgeschichte der tebp-1- und tebp-2 Gene zu rekapitulieren, von denen wir gezeigt haben, dass sie eine bedeutende Rolle in der Telomerbiologie in C. elegans spielen (Artikel I). Wir konnten zeigen, dass Caenorhabditis briggsae Homologe dieser Proteine ebenfalls Telomere binden. Indem wir die evolutionäre Analyse durch Untersuchung der Phylogenie und Syntenie auf acht weitere Caenorhabditis-Nematoden ausweiteten, zeigten wir, dass diese Proteine möglicherweise eine konservierte Rolle in der gesamten Caenorhabditis-Gattung spielen. Mit dem Ziel, Anzeichen einer positiven Selektion zu erkennen, stellten wir bei vielen der Zielnematoden fehlende oder unzureichende Genannotationen fest. Die Genauigkeit der positiven Selektionsanalyse wird durch die Qualität der in der WormBase Nematoden-Informationsressource verfügbaren Genannotation beeinträchtigt, die in hohem Maße von automatisierten Annotationsworkflows unter Verwendung verfügbarer sequenzierter Genome abhängt.
Zur Schließung dieser Lücke setzten wir eine Proteotranskriptomik-Technik zusammen mit einer durch maschinelles Lernen unterstützten Qualitätskontrolle ein, um die Genannotationen für 12 Nematodenarten zu verbessern, was eine Systemanalyse und neue Einblicke in evolutionäre Prozesse ermöglicht (Artikel II). Durch den Vergleich unserer Annotation mit der sehr guten Annotation von C. elegans demonstrierten wir die Leistungsfähigkeit unserer Methode und identifizierten 2 zuvor nicht identifizierte Gene in dieser Spezies (autorisiert von WormBase Kuratoren), was nach mehr als 20 Jahren sorgfältiger manueller Annotation bemerkenswert ist . Mit unserer Technik konnten wir qualitativ hochwertige Annotationen für 9 genomsequenzierte Arten erstellen und neue proteinkodierende Genannotationen für 3 weitere Arten ohne sequenzierte Genome (C. droshophilae, R. regina und R. axei) in der gleichen Qualität wie die von C. elegans bereitstellen.
Um die Annotationen zu benchmarken und die evolutionäre Analyse zu erleichtern, haben wir eine Pipeline erstellt, die Orthologievorhersagen und positive Selektionsanalysen ermöglicht. Die Implementierung der Pipeline ermöglichte die Bestimmung von 23.090 orthologen Gruppen, die die proteotranskriptomische Annotation der protein-kodierenden Gene der 12 Nematodenarten umfassen. Unter Verwendung der Pipeline für umfassende positive Selektionsanalysen haben wir Orthologiegruppen unter positiver Selektion entdeckt. Ermutigt durch diese Ergebnisse haben wir den Nutzen der Pipeline für die wissenschaftliche Gemeinschaft erkannt und werden sie der Allgemeinheit unter dem Namen AlexandrusPS als Docker-Image zur Verfügung stellen. AlexandrusPS ermöglicht es Benutzern, CodeML-Protokolle automatisiert parallel auf einem Desktop-Computer auszuführen, was Analysen mit hohem Durchsatz ermöglicht, ohne dass Hochleistungs-Computersysteme erforderlich sind. Die Pipeline wird der Community über einen Application Note-Artikel in einer der größeren Bioinformatik-Fachzeitschriften vorgestellt werden (Artikel III).Getrennte Zählung ; Illustrationen, Diagramm
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Mass spectrometry-based quantitative proteomics to investigate RNA-protein interactions
Full text linkMass spectrometry-based proteomics is a versatile tool, offering a global and unbiased
approach to analyse proteins and their interacting partners. Within the realm of molecular
biology, RNA-protein interactions stand as fundamental and intricate components that oversee
vital processes in the cell. These interactions, often mediated by specific RNA-binding proteins
(RBPs), orchestrate a wide array of cellular functions. From the regulation of gene expression to
the maintenance of genomic stability, the post-transcriptional processing of RNA molecules, and
even the spatial organisation of the cell nucleus, RNA-protein interactions play a central role in
shaping the intricate web of cellular activities. In this thesis, the interaction between RNA and
proteins is investigated using state-of-the-art MS.
Article Ⅰ delved into the characterization of Telomeric repeat-containing RNA (TERRA)
molecules in M. musculus, examining their genomic origins and comparing their interactomes to
their H. sapiens counterparts. RNA-FISH analysis revealed disparities in behaviour, with M.
musculus TERRA foci primarily located outside of telomeres, in contrast to H. sapiens TERRA
foci, which recurrently resided at telomeres. As a result, a distinct genomic origin for M.
musculus TERRA molecules outside telomeres was hypothesised. Through a comprehensive
genomic analysis, four major chromosomal regions, including known Telo 18q, PAR-Xq/Yq, and
ChrX Tsix locus regions, and a novel Chr2 region, were identified as potential sources of
TERRA molecules. Conservation of TERRA-associated functions was evaluated with an affinity
purification-mass spectrometry (AP-MS) approach. A comparison of the enriched proteins with
publicly available H. sapiens TERRA-interacting protein datasets revealed that, despite having a
distinct genomic origin, functions are conserved between M. musculus and H. sapiens.
Article Ⅱ centred on the functional assignment of RBPs in S. cerevisiae. An AP-MS
screen was designed to elucidate the interaction partners of 40 selected RBPs, which were
chosen based on their involvement in various stages of mRNA processing. Functional analysis
of the collected data highlighted the overrepresentation of canonical RNA-binding domains
(RBDs) and RNA binding-related GO molecular function terms among the RBPs' interaction
partners. KEGG pathway analysis demonstrated the enrichment of RNA pathways, consistent
with the RBP selection criteria, as well as involvement in metabolic and synthesis pathways.
Finally, network-based function assignment of RBPs was facilitated by concurrent binding
patterns within the network.ix, 108 Seiten ; Illustrationen, Diagramm
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe 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
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