1,720,968 research outputs found
Interpreting the biosensor data of biomolecular interactions
No full textThe literature part of this thesis reviewed the process of obtaining affinity information with quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensors. Basic principles of these biosensors were also evaluated, along with the principles of data acquisition and finally the data processing. The raw data produced by QCM or SPR can be used to study biomolecular interactions qualitatively and quantitatively. These techniques are also powerful in obtaining kinetic and thermodynamic information of the biomolecular interactions. SPR and QCM can produce data easily, but data interpretation can be sometimes problematic. This is partly due to misconceptions on how the sensograms should be interpreted. Many of the interpretational problems can and should be avoided long before the modeling of the data takes place to obtain reliable affinity data.
The literature part of this thesis also presents tools for developing good experimental design. Well-designed experimental set-up is the most important element for producing good biosensor data. One should also estimate from the sensogram shapes what kind of analysis is needed. This was explained in detail in the literature part, pointing out the key elements how sensograms with certain shape should be interpreted and further analyzed to obtain affinity constants. Data analysis part of the literature review provides also information how to use appropriate models (e.g. fitting equilibrium, kinetic or complex data) with extensive examples. Surface site distribution model will be also covered as the tool to analyze complex biomolecular interactions by QCM and SPR.
In the experimental part, affinity of anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) towards different lipoproteins was studied with partially filling affinity capillary (PF-ACE) electrophoresis and QCM. PF-ACE with adsorption energy distribution (AED) calculations provided information on the heterogeneity of the interactions. For the first time, a modified surface site distribution model called Interaction map was utilized to model QCM data of lipoprotein interactions with anti-apoB-100 Mab. With the Interaction maps, it was possible to distinguish different kinetics of low-density lipoprotein (LDL) and anti-apoB-100 Mab interactions. Affinity constants obtained were used to evaluate thermodynamics of these interactions. Both methods were also used to evaluate interactions with other apoB-100 containing lipoproteins: intermediate-density lipoprotein (IDL) and very lowdensity lipoprotein (VLDL). It was found that the Interaction maps could distinguish two different kinetics from the mixture of IDL-VLDL with distinct affinity constants. Both methods agreed well with the affinity constants. It was found that the anti-apoB-100 Mab used in this study, had a high affinity towards apoB-100 containing lipoproteins.
In the second part of the experimental, a convective interaction media (CIM) based LDL isolation platform was developed. In these studies, anti-apoB-100 Mab was immobilized on the CIM-disk and was used to isolate LDL from human plasma and serum samples. It was found that apolipoprotein based separation of LDL from plasma was possible, although not without difficulties, since apoB-100 is not only present in LDL, but also in VLDL and IDL. To circumvent this problem different antibodies (anti-apoE and anti-apoAI) were utilized to capture VLDL and IDL from the plasma before the interaction of LDL with the anti-apoB-100 CIM-disk. LDL was successfully isolated with this approach in a significantly reduced time compared to conventional ultracentrifugation method used for LDL isolation
Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules
Get PDFThis doctoral thesis describes the development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules. The focus of the study was on subpopulations of lipoproteins and extracellular vesicles (EVs) that are important in the detection of different diseases, such as atherosclerotic cardiovascular diseases and cancer, and may even possess therapeutic potential. In the thesis, immunoaffinity chromatography (IAC) with selective antibodies immobilized on the monolithic disk columns were utilized for the selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) was able to fractionate relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Continuous flow quartz crystal microbalance (QCM) and partial filling affinity capillary electrophoresis (PF-ACE) were employed to study the affinity of the interactions between the antibody and lipoproteins.
The first step was to develop a method to study interactions between antibody and lipoproteins to select a high affinity antibody useful for the isolation of lipoprotein subpopulations by IAC. The interaction data obtained with PF-ACE was analyzed to determine the heterogeneity of the interactions with adsorption energy distribution calculations, while the QCM data was processed with interaction maps. The affinity constants obtained with QCM and PF-ACE agreed well with each other.
Next, the IAC methods were developed to capture EVs of different cellular origins from human plasma using anti-CD9 monoclonal antibody (mAb), while anti-CD61 mAb was exploited to capture platelet-derived EVs. The anti-apolipoprotein B-100 (anti-apoB-100) mAb was exploited to immunocapture apoB-100 containing lipoproteins. The anti-apoB-100 mAb was also characterized by the PF-ACE and QCM studies. Appropriate elution conditions were found for the IAC methods, which has often been an issue with magnetic beads-based immunoaffinity methods.
Since IAC allowed selective isolation of EVs and lipoproteins, a size-based separation to their subpopulations with AsFlFFF was introduced as a successive step. This enabled additional characterization of subpopulations by nanoparticle tracking analysis, western blotting, electron microscopy, capillary electrophoresis coupled with laser-induced fluorescent detection, zeta potential measurements, as well as free amino acids and glucose analysis with hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Finally, IAC was successfully on-line coupled to AsFlFFF, resulting in quick and automated isolation and fractionation of the subpopulations of EVs and lipoproteins. The constructed IAC-AsFlFFF system was able to process reliably 18–38 samples in 24 h with only minor operator involvement, resulting in highly reproducible and gentle fractionation of EV subpopulations in the size range of exomeres and exosomes.
Polymeric monolithic disk columns were utilized for the first time for the IAC-based isolation of EVs and their subpopulations from human plasma, and for the detection of exomeres in CD9+ EVs and CD61+ platelet-derived EVs from human plasma samples. The results demonstrated that CD61+ EVs are potentially taking part in gluconeogenesis based on free amino acids and glucose present as cargo.Väitöskirjassa kehitettiin nopeita, luotettavia ja automatisoituja eristämis- ja fraktiointimenetelmiä ihmisen biomakromolekyylien nanokokoisille alaryhmille. Tutkimuksen painopiste oli lipoproteiinien ja solunulkoisten vesikkelien (EV) alaryhmissä. Näiden biomakromolekyylien alaryhmien tutkiminen on tärkeää eri sairauksien, kuten ateroskleroottisten sydän- ja verisuonitautien ja syöpien havaitsemisessa.
Biomakromolekyylien eristämiseen ihmisen veriplasmasta käytettiin ensimmäistä kertaa immunoaffiniteettikromatografiaa, jossa oli selektiivisiä monoklonaalisia vasta-aineita immobilisoituna polymeerisille monoliittisille kiekkopylväille. Eristettyjen biomakromolekyylien jatkokäsittely epäsymmetrisen virtauskenttävirtausfraktioinnin (AsFlFFF tai AF4) avulla erotteli niiden alaryhmät toisistaan koon perusteella (esimerkiksi eksomerit ja eksosomit). Kvartsikidemikrovaakaa (QCM) ja kapillaarielektroforeesia (PF-ACE) käytettiin monoklonaalisen vasta-aineen ja lipoproteiinien välisen vuorovaikutuksen affiniteetin tutkimiseksi.
Lopuksi immunoaffiniteettikromatografia liitettiin yhteen epäsymmetriseen virtauskenttävirtausfraktiointiin automatisoiduksi järjestelmäksi. Näin mahdollistettiin lipoproteiinien ja solunulkoisten vesikkelien alaryhmien nopea, toistettava ja automaattinen eristäminen veriplasmasta. Järjestelmä kykeni käsittelemään 18–38 näytettä 24 tunnissa vähäisellä operaattorin osallistumisella. Ensimmäistä kertaa oli mahdollista eristää ja fraktioida veriplasmanäytteistä erittäin toistettavasti ja hellävaraisesti solunulkoisten vesikkelien alaryhmiä eksomerien ja eksosomien kokoluokassa. Alaryhmien jatkotutkimuksissa paljastui myös, että verihiutaleiden solunulkoisten vesikkelien alaryhmät osallistuvat mahdollisesti glukoneogeneesiin.ei saavutettav
Potential and utilization of water extracts from spruce bark
Get PDFThe aim of this thesis was to investigate potential ways to utilize water extracts from spruce bark. Spruce bark has been found to contain three main stilbene glucosides (astringin, isorhapontin, polydatin), which have potential in the treatment of skin aging and cosmetics applications. Since stilbene glucosides are water soluble, they are dissolved into the debarking process waters of the pulp and paper industry. These waters are not fully utilized at the moment, and being considered as a waste stream, which adds to the costs of the mill by increasing COD levels of the waste water. This waste stream could potentially be transformed to a source of stilbene glucosides, to provide additional revenue to the mill.
In the experimental part, temperature effect on hot water extraction yields of industrial spruce bark was studied. Mechanical pressing of heated spruce bark was performed with a laboratory scale mechanical press to study the effect on the COD levels. Finally, bark press waters obtained from the mill were characterized, with partial purification of stilbene glucosides by ultrafiltration with 2 and 5 kDa filters. The yields were calculated by gravimetric and spectroscopic analysis. Two dimensional (2D) solution-state 1H–13C correlation NMR spectroscopy provided the structural verification of the stilbene glucosides and other compounds present in the samples.
Extraction yield of industrial spruce bark was increasing 1.4 times every 20 ◦C, on average, reaching yield of 5.0% at 80 ◦C. The mechanical pressing was able to press out 26±5% of the total mass of the sample. COD level was 75,100 mg/L for 80 ◦C sample. This was two times higher compared to bark press sample of taken from a paper mill, which had COD level of 37,500 mg/L. Ultrafiltration could remove 90% of polyphenols and permeated 14% of the stilbenes of the feed. With 2D-HSQC NMR, all three major stilbene glucosides were identified from the
filtrate and the feed
Modern isolation and separation techniques for extracellular vesicles
Get PDFExtracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation techniques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share similar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent development using an automated on-line system including selective affinity-based trapping unit and asymmetrical flow field-flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma. (C) 2020 The Author(s). Published by Elsevier B.V.Peer reviewe
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
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