1,720,966 research outputs found
Monitoring cellular Guanosine triphosphate (GTP) and GTP associated proteins
Get PDFGuanosine-5'-triphosphate (GTP) is an essential molecule for cell survival and function. Although free GTP plays a crucial role in several cellular processes, most studies focus solely on the protein bound GTP and its association with cancer. Efficient methods for determining cellular GTP concentration are currently lacking. Chromatography or capillary electrophoresis are commonly utilized for separation and mass spectrometry (MS) for quantification of GTP in biological samples. For monitoring GTPases and their interactions with ligands, differential scanning fluorimetry, differential scanning calorimetry, or differential static light scattering are typically utilized. However, these methods either lack sensitivity or throughput.
In this PhD project, the Protein-Probe method, based on a Eu3+-chelate peptide probe that interacts with the hydrophobic core of the protein, has been developed and applied for studying different factors that affect the thermal stability of small GTPases, such as inhibitors, buffer ions and nucleotides. Furthermore, the method was explored for a better understanding of the binding specificity of the recent Food and Drug Administration approved KRASG12C covalent inhibitors to RAS GTPases.
Additionally, a novel dual-labeled Förster Resonance Energy Transfer (FRET)-based peptide probe was introduced and compared to the Protein-Probe method in the thermal stability assay. The FRET-Probe technique was also applied to determine the chemical stability of proteins with different chemical denaturants.
For measuring cellular GTP level, a homogenous high throughput assay was developed to measure the amount of GTP in cells utilizing a highly GTP-specific antibody. The assay yielded comparable results to those obtained with CE/MS, demonstrating a similar level of accuracy, while also exhibiting a substantial enhancement in throughput.
In conclusion, this thesis work aimed at developing novel and robust methods to address the current limitations in monitoring cellular GTP concentration and to study small GTPases. The focus of the study was free GTP, recognizing its essential role in various cellular processes.------
Guanosiini-5'-trifosfaatti (GTP) on välttämätön molekyyli solujen selviytymiselle ja toiminnalle. Vaikka vapaalla GTP:llä on ratkaiseva rooli useissa soluprosesseissa, suurin osa tutkimuksista keskittyy vain GTPaaseihin sitoutuneeseen GTP:hen ja sen linkittymisessä syövän kehittymiseen. Tällä hetkellä solun GTP-konsentraation määrittämiseksi ei ole tehokkaita menetelmiä. Käyttämällä kromatografiaa tai kapillaarielektroforeesia (CE) erotukseen ja massaspektrometriaa (MS) kvantifiointiin voidaan biologisten näytteiden GTP-pitoisuutta seurata, mutta se on melko työlästä. GTPaasien ja niiden vuorovaikutusten seurantaan käytetään tyypillisesti differentiaalista pyyhkäisyfluorimetriaa, differentiaalista pyyhkäisykalorimetriaa tai differentiaalista staattista valonsirontaa. Kaikilta näistä menetelmistä puuttuu kuitenkin herkkyys tai suorituskyky. Tästä syystä tämän opinnäytetyön tavoitteena oli kehittää uusia herkkiä ja toimintavarmoja menetelmiä GTP-pitoisuuden seurantaan ja GTPaasien tutkimiseksi uudesta näkökulmasta.
Solujen GTP-tasojen mittaamiseksi kehitettiin homogeeninen tehoseulontaan yhteensopiva määritys käyttämällä GTP-spesifistä vasta-ainetta. Määritys tuotti samanlaisia tuloksia kuin vertailumenetelmänä käytetty CE/MS, mutta huomattavasti nopeammin ja helpommin. Tässä väitöskirjatyössä esiteltiin myös uusi kaksoisleimattu FRET-pohjainen peptidikoetin, jota käytettiin yhdessä aiemmin kehitetyn ”Protein-Probe” menetelmän kanssa. FRET-Probe tarjoaa saman korkean herkkyyden kuin Protein-Probe tekniikka, mutta se mahdollistaa proteiinin stabiilisuuden mittaamisen neutraalissa pH:ssa ja yhdessä vaiheessa. Protein-Probe menetelmää käytettiin pienten GTPaasien ja niiden lämpöstabiilisuuteen vaikuttavien tekijöiden tutkimiseen. Lisäksi kehitettyjä menetelmiä käytettiin FDA:n äskettäin hyväksymien kovalenttisten KRAS(G12C) inhibiittorien sitoutumisspesifisyyden, toiminnan ja resistenttiyden muodostumismekanismien tutkimiseen. FRET-Probe-tekniikkaa ei sovellettu ainoastaan proteiinien lämpöstabiilisuuden seurantaan, vaan sen osoitettiin soveltuvan myös proteiinien isotermaalisen kemiallisen stabiilisuuden seurantaan.ei tietoa saavutettavuudest
Droplet digital PCR for detection of mutations in high-grade serous ovarian cancer
No full textOvarian cancer (OC) is one of the most fatal cancers that affect female reproductive system. It is frequently discovered at advanced stage therefore, the 5-year survival rate is typically low. High grade serous ovarian cancer (HGSOC) is the most aggressive and has the worst prognosis of all types of OC. It is characterized by copy number variation (CNV) and genetic instability.
Circulating free DNA (cfDNA) is a potential non-invasive alternative to tissue biopsy that can be used to detect mutations for early screening and targeted treatment of HGSOC. Nevertheless, methods with higher sensitivity than real-time PCR (qPCR) are required for amplification and quantification of plasma cfDNA. Droplet digital PCR (ddPCR) is a very sensitive tool in rare mutations detection. It produces absolute quantification without the need for a standard curve. Additionally, it divides the sample into droplets where each droplet acts as an individual PCR reaction. This leads to high sensitivity in identification rare targets. The aim of this study was to evaluate the sensitivity of ddPCR in detection of TP53 mutation and three novel unpublished fusion genes in HGSOC.
For TP53 detection, serial dilutions of Mutant (MT) in genomic DNA (gDNA) was tested to identify limit of detection (LOD). Next, different concentrations of plasma cfDNA (5, 10, 20 and 40 ng per reaction) from HGSOC patient were analyzed by both qPCR and the optimized ddPCR assay. DdPCR showed 1 % LOD and recorded 3720 MT copies/ μl when 40 ng cfDNA per reaction was used. On the other hand, qPCR LOD was 5 % and it failed to detect cfDNA. Based on these results, we suggest that ddPCR is more sensitive than qPCR in detection of rare mutations. As for the fusion genes, ddPCR was more challenging and required further optimization
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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