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1
Chan, Hong-Lin. "A 2D-difference gel electrophoresis strategy for redox proteomics." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444604/.
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Post-genomic biomedical science requires quantitative proteomics. In most cases this involves differential protein expression analysis using matched pairs of simultaneously detectable labelling reagents for specific protein amino acids. In this thesis the development and optimisation of a novel cysteine labelling strategy, that is based on the use of iodoacetyl derivatives of Cy3 and Cy5 (ICy3/5) and 2D-difference gel electrophoresis (2D-DIGE) is described. The differentially labelled samples are separated on a single 2D gel and detected by multi-wavelength fluorescence scanning. The method is used to analyse standard proteins and then cell lysates to define the stoichiometry, sensitivity and specificity of this labelling technique. A comparative study of this new proteomic ICy dye protocol with the current NHS-Cy dye labelling system and methods that employ commonly used protein staining methods is described. The method is then used for cysteine labelling of proteins in non-reduced, denatured biological samples allowing accurate monitoring and sensitive detection of redox-dependent thiol modifications and expression level changes. The method is shown to be compatible with the use of MALDI mass spectrometry to identify proteins by analysis of trypsinised ICy labelled peptide digests. Using parallel sample analysis within single gels, the ICy-dye reagents were used to detect redox-, ErbB-2- and growth factor-dependent changes in a human mammary luminal epithelial cell system which was exposed to hydrogen peroxide or to growth factor stimulation. The conventional lysine labelling 2D-DIGE technique was also used in parallel to assess the new ICy labelling strategy for determination of the effects of oxidative stress on protein isoform levels. This study has revealed the identity of proteins involved in the response to oxidative stress and growth factor stimulation in the context of ErbB-2 growth factor receptor over-expression. In addition, this labelling strategy was also used to detect changes in thiol reactivity that follow the UV irradiation of plasma proteins as part of a study designed to evaluate the effect of UV disinfection on plasma product safety for clinical use.
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Brown, Andrew S. "Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) Characterization of Decorin." Youngstown State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1311873768.
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Pandey, Archana. "Proteome analysis of Pseudomonas putida KT2440 using 2D gel electrophoresis and LC/ESI-Q-TOF mass spectrometry /." Online version of thesis, 2007. https://ritdml.rit.edu/dspace/handle/1850/3848.
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Šopíková, Martina. "Změny proteinového profilu v průběhu sladování ječmene." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2008. http://www.nusl.cz/ntk/nusl-216437.
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This diploma thesis is focused on studies of changing of protein profile during barley malting. Substantial part of this work is devoted to the proteomics identification of barley proteins which change during malting and so become more stationary and they influence quality of beer (haze and foam in beer). For this experiment was used barley variety Jersey. In the theoretical part of this thesis there is information about beer, manufacturing of beer with description of important commodities for manufacturing of beer and information about barley malting and information about malting process. Next there is description of methods for separation of proteins (1D gel electrophoresis and 2D gel electrophoresis), MALDI TOF/TOF mass spectrometry and this use for the analysis and identification of proteins, the use of matrices and ways of the sample preparation. In the experimental part of this thesis there was carried out the optimisation of the dosage of sample for 1D gel electrophoresis and the optimisation of staining. The 15 % TRIS-HCl gel was the best, this gel was stained by Commassie Brilliant Blue G-250. For illustration of changes was made 2D gel electrophoresis. With help of method peptide mass fingerprinting and MS/MS protein of barley – protein Z, -amylase subtilisin inhibitor, -amylase a peroxidase were identificated. The analysis of barley extract intact proteins was carried out, this analysis was focused on changes of important barley protein LTP 1.
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Bartee, Eric Carter. "Discovery and characterization of a novel family of human ubiquitin ligases termed Membrane Associated RING-CH (MARCH) proteins." Oregon Health & Science University, 2007. http://content.ohsu.edu/u?/etd,629.
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Ph.D.Molecular Microbiology and Immunology
Both poxviruses and γ2-herpesviruses share the K3-family of viral immune evasion proteins. These proteins are characterized by an amino-terminal RING-CH domain followed by two transmembrane domains. We analyzed several human homologues of the K3-family termed membrane-associated RING-CH (MARCH) proteins. All MARCH proteins localized to subcellular membranes while several reduced surface levels of known K3-family substrates. Thus, MARCH proteins appear to be structurally and functionally homologous to viral K3 proteins. One of the major challenges in determining the function of this family is the identification of their physiological substrates. To overcome this we created a quantitative proteomics approach which can be used to identify novel substrates for both the K3- and MARCH-families. Using stable isotope labeling by amino acids in cell culture, we compared the proteome of plasma membrane, golgi, and endoplasmic reticulum membranes in the presence and absence of K5 and MARCH-VIII. Quantitative mass spectrometric protein identification from these fractions revealed that CD316 (bone marrow stromal antigen 2), CD166 (activated leukocyte cell adhesion molecule) and syntaxin-4 were consistently underrepresented in the plasma membrane of K5 expressing cells, while CD44, CD81 (TAPA-1) and B-cell receptor-associated protein 31kDa (Bap31) were consistently underrepresented in the plasma membrane of MARCH-VIII expressing cells. Furthermore, downregulation of each of these proteins was independently confirmed. Our results both identify and characterize a novel family of human ubiquitin ligase enzymes and elucidate a novel technique which can analyze this family and be easily adapted to the analysis of other cellular enzymes viral immune modulators.
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Guterres, Sheila Barreto. "Busca de biomarcadores para esquizofrenia em plaquetas utilizando eletroforese diferencial em gel bidimensional (2D-DIGE) e espectrometria de massas." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/75/75132/tde-16112011-150931/.
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A esquizofrenia é uma doença crônica, grave e incapacitante que afeta cerca 24 milhões de pessoas em âmbito mundial. É caracterizada por uma desorganização no pensamento que prejudica a funcionalidade do indivíduo. Existem intervenções que são efetivas e contribuem para a diminuição da prevalência do transtorno, pois ajudam o portador a levar uma vida produtiva e integrada à sociedade, porém devem ser ministradas nos estágios iniciais da doença. No entanto, existe uma grande dificuldade em se diagnosticar a esquizofrenia precocemente devido a sua complexidade e às sutilezas dos seus sintomas apresentados antes do surgimento da psicose. O cérebro não é acessível a exames invasivos in vivo e por esse motivo a exploração de fluidos periféricos é de grande importância. As plaquetas e neurônios serotonérgicos possuem características bioquímicas e morfológicas em comum que possibilitam a comparação entre a estrutura e a função de ambos e, por causa dessa similaridade, muitos trabalhos utilizam plaquetas como modelo para o estudo de doenças neuropsiquiátricas, inclusive a esquizofrenia. A detecção precoce da esquizofrenia é um objeto de investigação atual e relevante não somente para revolucionar os meios atuais de diagnóstico, mas também para desenvolver novos tratamentos aplicados aos estágios iniciais da doença, diferenciar os subgrupos de doentes e monitorar as intervenções preventivas. A proposta do presente trabalho é fazer o estudo da expressão de proteínas em plaquetas de pacientes esquizofrênicos e controles com o objetivo de identificar proteínas candidatas a biomarcadores utilizando técnicas proteômicas quantitativas e confiáveis, como 2D-DIGE e a espectrometria de massas.Schizophrenia is a disabling, serious, and chronic illness, which affects about 24 million people worldwide. It is characterized by a severe disorganization of the thoughts that harms the social life of patients becoming them dependent of the family and/or government. There are effective treatments that contribute to decrease the prevalence of the disorder because they improve the life and social conditions of the patients, but they are only advantageous if the intervention is made in the early stages of the disease. It is difficult to obtain early diagnosis due to the complexity of the disease and its insidious symptoms before the beginning of the psychosis. The brain is not easily accessed in vivo and, because of this, it is very important to study the peripheral tissues like blood, which makes the use of the platelets very interesting. Furthermore, platelets and serotonergic neurons share biochemical and morphological characteristics that allows the comparison between structure and function of both. From these similarities many authors has used platelets as a neuron model to study many neurodegenerative diseases including schizophrenia. The early detection of schizophrenia is a current and suitable goal, not only to improve the early diagnosis but also to develop new treatments, differentiate the subtypes, and monitor the preventive interventions. The purpose of this project is to do a comparative screening of expressed proteins in platelets from schizophrenics and controls with the objective of finding differently expressed proteins that could be candidates to biomarkers using 2D-DIGE and mass spectrometry.
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Woolard, Christopher Lee. "Identification of Potential Protein Biomarkers of Low Level Kidney Degradation." Wright State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=wright1247498817.
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Leung, Man Ching. "Identification of human hair follicle antigens targeted in the presumptive autoimmune hair follicle disorder Alopecia Areata and their potential functional relevance In Vitro. Methods development for isolation and identification of Alopecia Areata-relevant human hair follicle antigens using a proteomics approach and their functional assessment using an Ex Vivo hair follicle organ culture model." Thesis, University of Bradford, 2008. http://hdl.handle.net/10454/4330.
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Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.
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Muchindu, Munkombwe. "Electrochemical ochratoxin a immunosensors based on polyaniline nanocomposites templated with amine- and sulphate-functionalised polystyrene latex beads." Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3815_1306752491.
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Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3 &minus
) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate (PSNP-OSO3 &minus
) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 &minus
) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10&minus
8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were <
100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 &mu
A/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 &mu
M NO2 &minus
, were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 &minus
, respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors.
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Kierul, Kinga. "Comprehensive proteomic study of Bacillus amyloliquefaciens strain FZB42 and its response to plant root exudates." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16805.
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Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde.Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.
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Zabel, Claus. "Veränderungen im Proteom von Maus und Mensch durch Huntington's Chorea." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2003. http://dx.doi.org/10.18452/14825.
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Die Erkrankung Huntington s Chorea ist eine autosomal dominant vererbte Erkrankung, die gewöhnlich im mittleren Lebensabschnitt beginnt und unausweichlich zum Tode führt. In unserem Bestreben, Proteine zu identifizieren, welche an Prozessen "Upstream" oder "Downstream" des krankheitsverursachenden Proteins Huntingtin beteiligt sind, wurde das Proteom eines sehr gut etablierten Mausmodells mit Hilfe der Großgel 2D-Elektrophorese untersucht. Es konnte zum ersten Mal auf Proteinebene nachweisen werden, dass die Expression von zwei Serinproteasehemmern, alpha1-Antitrypsin und Contraspin und darüber hinaus eines Chaperons, alphaB-Kristallin, im Verlauf der Erkrankung abnimmt. Reduzierte Expression von alpha1-Antitrypsin und Contraspin konnte in Gehirn, Leber, Herz und Testes nahe dem Endstadium der Erkrankung nachgewiesen werden. Hier ist es wichtig festzustellen, dass die Expressionsabnahme von alpha1-Antitrypsin im Gehirn der Abnahme in der Leber im Herzen und in den Testes vorangeht. Eine verminderte Expression des Chaperons alphaB-Kristallin wurde nur im Gehirn gefunden. Für ein weiteres Protein, das Major Urinary Protein, wurde eine verminderte Expression in der Leber und im Urin von betroffenen Mäusen festgestellt. Damit konnte demonstriert werden, dass die Erkrankung auf Proteinebene auch ein Protein, das im Gehirn von transgenen Mäusen nicht vorkommt, beeinflusst. Bei Untersuchungen am Menschen wurde in drei Gehirnregionen von Postmortem-Gehirnen von Huntington s Chorea Patienten eine veränderte Expression von alpha1-Antitrypsin festgestellt. Wenn gewährleistet werden kann, dass die Konzentration von alpha1-Antitrypsin und alphaB-Kristallin während Huntington s Chorea im Gewebe nicht absinkt, könnte dies vielleicht neuronalen Zelltod verhindern und somit bei der Verzögerung des Krankheitsverlaufs nutzbringend eingesetzt werden.Huntington disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In an effort to identify proteins involved in processes upstream or downstream of the disease causing huntingtin, the proteome of a well-established mouse model was studied by large-gel 2D electrophoresis. It could be demonstrated for the first time at the protein level that two serin protease inhibitors, alpha1-antitrypsin and contraspin and the chaperone alphaB-crystallin decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver, heart and testes in mice. Reduced expression of alpha1-antitrypsin and contraspin could be detected in the brain, liver heart and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. Reduced expression of the liver specific major urinary proteins not found in the brain, was seen in affected mice, demonstrating that the disease exerts its influence on a protein not present in the brain of transgenic mice at the protein level. When investigating three human brain regions obtained post-mortem from Huntington s disease patients, alpha1-antitrypsin expression was also altered. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington s disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.
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Fulton, Benjamin L. "2D-PAGE Analysis of Myocardial Collagen in Male and Female Spontaneously Hypertensive Rats." Connect to resource online, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1219668882.
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Noma, Alexandre. "Duas abordagens para casamento de padrões de pontos usando relações espaciais e casamento entre grafos." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/45/45134/tde-15072010-104140/.
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Casamento de padrões de pontos é um problema fundamental em reconhecimento de padrões. O objetivo é encontrar uma correspondência entre dois conjuntos de pontos, associados a características relevantes de objetos ou entidades, mapeando os pontos de um conjunto no outro. Este problema está associado a muitas aplicações, como por exemplo, reconhecimento de objetos baseado em modelos, imagens estéreo, registro de imagens, biometria, entre outros. Para encontrar um mapeamento, os objetos são codificados por representações abstratas, codificando as características relevantes consideradas na comparação entre pares de objetos. Neste trabalho, objetos são representados por grafos, codificando tanto as características `locais\' quanto as relações espaciais entre estas características. A comparação entre objetos é guiada por uma formulação de atribuição quadrática, que é um problema NP-difícil. Para estimar uma solução, duas técnicas de casamento entre grafos são propostas: uma baseada em grafos auxiliares, chamados de grafos deformados; e outra baseada em representações `esparsas\', campos aleatórios de Markov e propagação de crenças. Devido as suas respectivas limitações, as abordagens são adequadas para situações específicas, conforme mostrado neste documento. Resultados envolvendo as duas abordagens são ilustrados em quatro importantes aplicações: casamento de imagens de gel eletroforese 2D, segmentação interativa de imagens naturais, casamento de formas, e colorização assistida por computador.Point set matching is a fundamental problem in pattern recognition. The goal is to match two sets of points, associated to relevant features of objects or entities, by finding a mapping, or a correspondence, from one set to another set of points. This issue arises in many applications, e.g. model-based object recognition, stereo matching, image registration, biometrics, among others. In order to find a mapping, the objects can be encoded by abstract representations, carrying relevant features which are taken into account to compare pairs of objects. In this work, graphs are adopted to represent the objects, encoding their `local\' features and the spatial relations between these features. The comparison of two given objects is guided by a quadratic assignment formulation, which is NP-hard. In order to estimate the optimal solution, two approximations techniques, via graph matching, are proposed: one is based on auxiliary graphs, called deformed graphs; the other is based on `sparse\' representations, Markov random fields and belief propagation. Due to their respective limitations, each approach is more suitable to each specific situation, as shown in this document. The quality of the two approaches is illustrated on four important applications: 2D electrophoresis gel matching, interactive natural image segmentation, shape matching, and computer-assisted colorization.
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Kultima, Kim. "Transcriptomics and Proteomics Applied to Developmental Toxicology." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7921.
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Yang, Shun-Chieh, and 楊順傑. "Registration of Protein Spots in 2D Gel Electrophoresis Images." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/59262899855964638252.
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碩士淡江大學
資訊工程學系碩士班
95
In proteomics, 2D gel electrophoresis plays a very important role. We need some processes on these 2D gel electrophoresis images to get information we want. These processes include detection and registration of protein spots. Traditionally, researchers can pick protein spots in the gel images manually. As a result, they spent much time but still made mistakes. For this reason, we proposed a system to assist researchers in dealing with this problem and analyzing protein characteristics. For instance, we got two 2D gel images. One is protein with germs infective, the other is protein with germs anti-infective. In two images, protein spots are different from each other. We take results of detection of protein spots to determine if protein spots change in two images. These changes like getting bigger or smaller, darker or lighter, even disappearing. And, these protein spots are what we are interested. Therefore, we design a system in accordance with demands of researchers. In this system, we mainly take results of detection of protein spots in 2D gel images and develop follow-up capability of matching protein spots. We use methods on mathematics, that is, to select several pairs of spots in two images as landmarks, and then we can find an equation that could transform the source image into the target image. Thus, all spots in images will satisfy this equation and our aim to match these protein spots will be achieved. We show our results of matching depending on demands of users to let them get results efficiently.
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Tsai, Ming-Hung, and 蔡明宏. "Detection and Analysis of Protein Spots in 2D Gel Electrophoresis Image." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/36156173056614970670.
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碩士淡江大學
資訊工程學系碩士班
94
2D gel electrophoresis plays an important role in proteomics. It can not only separate protein spots effectively but also identify and quantify the function of proteins. Traditionally, researchers can merely pick protein spots in the gel images with manpower. As a result, the efficiency of the research is critically reduced, and lots of mistakes take place at that time. Therefore, we proposed a system to deal with 2d gel images and to solve this problem. In this system, we mainly use some methods in morphlolgy, for example, we hope to retrieve protein spots from complicated gel images within watershed images segmentation algorithm. When it comes to images segmentation, watershed is the commonest approach, for its ability to segment boundary of spot objects. On the contrary, problems of over segmentation usually occur due to the production of too many local minimums, and this will cause fracture division. As a result, a few pre-processing procedures which can be easily achieved with existing filters should be utilized to improve the effect. By the way, we can also modify local minimums on gradient images before doing images segmentation with images reconstruction technique, that is, local minimums on the protein spots are kept while others are repaired. Therefore, problems of over segmentation can be avoided, and boundary of spots produced are quite complete, too.Using these method, we can detect all of the protein spots in 2D gel images. The system is also able to assign ID numbers to the protein spots and retrieve some annotated information of the spots such as area or location, which can be further analyzed for more researches. Finally, the comparison between the proposed system and another commercial software called Image Master is proceeded, which stands for the reliability of our system.
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Chang, Chung-Min, and 張景閔. "A JPEG-LS Based New Lossless Compression Method of 2D Gel Electrophoresis HDR Images." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/j774kt.
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碩士臺中技術學院
資訊科技與應用研究所
98
Proteomics, the study on proteins, was first introduced in 1995 by V. Wasinger. Techniques developed, thereafter, for the study of proteins can effectively analyze and identify the various types of protein in very short time. These resulted in increase protein researches using these techniques to study normal and diseased tissue organization; pathogenic and non-pathogenic bacteria; before and after treatment; and protein changes within cells. Proteomics is gradually becoming widely applied in bioinformatics, disease diagnosis and drug therapy design. Currently, the 2DGE image is a popularly used method in the medical community for studying proteins via the electrophoresis separation of proteins. After electrophoresis the protein spots are distributed on a gel. The amount of proteins on the gel is different and their color gradient dependent on the staining. The photographed proteins of the 2DGE images are also dependent on image resolution and brightness contrast settings (controlled by shutter exposures). An inaccurate image could sometimes cause wrong disease diagnostic and also wrong administration of drugs to a patient. viii The High Dynamic Range (HDR) is a technique that renders images photographed in different exposures. This technique ensures the detailed information of an image by rendering images taken at different exposures so that details from different contrastive can preserved in the resulting image. In the proposed research, this concept is applied to the 2DGE images photographed in different brightness at different exposures so that all details on the protein information are retained. Therefore the need to store the different resolution images is important and essential. Currently, the two classes of commonly used lossless compression methods can be distinguished as compression software tools and image compression methods. Compression software tools include WINRAR, WINZIP, 7-ZIP and more. On the other hand, image compression methods include JPEG2000, JPEG-LS, GIF and more. In the paper, we proposed using the Jpeg-LS compression method with its low complexity and high compression efficiency and integrated it with HDR; the improved propose method is called the HDR JPEG-LS compression method. With HDR JPEG-LS, we can effectively increased compression rate and to retain more protein information that is essential for accurate medical diagnostics. Furthermore, the technique can be used to project images for a different exposure based on existent images to study for possible missing protein details to ensure accurate diagnostics.
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Bazra, Souad. "Untersuchung der Proteinmusterveränderungen renaler Fibroblasten nach TGFß-1-Behandlung." Doctoral thesis, 2014. http://hdl.handle.net/11858/00-1735-0000-0022-5E5B-5.
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Rinke, Kathinka. "Analyse prognostischer Faktoren für die TNFα Antagonisten-Therapie bei Rheumatoider Arthritis." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-B1F7-3.
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Schwartz, Logan. "Impact d'une mitochondrie exogène sur le protéome du cybride Chrosomus eos." Thèse, 2014. http://hdl.handle.net/1866/11530.
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On retrouve dans le complexe Chrosomus eos-neogaeus une forme cybride ayant le génome nucléaire de C. eos et le génome mitochondrial de C. neogaeus. Ce modèle particulier fournit une occasion unique d’étudier l’influence d’une mitochondrie exogène sur le métabolisme et la physiologie d'organismes vivant en milieu naturel, et s'étant donc adaptés à cette situation cellulaire atypique. La mitochondrie jouant un rôle fondamental vital, nous nous attendons à ce que la présence d’une mitochondrie exogène chez la forme cybride ait un impact sur l’expression de son génome et du protéome qui en découle.
L’objectif de ce projet est d’étudier les différences au niveau protéomique entre des individus C. eos purs (forme sauvage) et des cybrides provenant d'habitats similaires afin de faire ressortir au maximum les différences dues à la présence de mitochondries C. neogaeus chez la forme cybride. Pour ce faire, nous avons comparé les protéomes des formes cybride et sauvage en utilisant l'électrophorèse en deux dimensions. Un sous-groupe de protéines produisant un signal spécifique révélé par l’analyse comparative a été identifié et analysé par spectrométrie de masse (LC/MS).
Les résultats indiquent que la présence de mitochondries C. neogaeus chez le cybride influence fortement la régulation génique chez ce dernier. De plus, les protéines identifiées apportent des pistes intéressantes supportant l'hypothèse que la présence de mitochondries C. neogaeus chez le cybride rendrait ce biotype plus résistant au froid que la forme sauvage.The Chrosomus eos-neogaeus genetic complex regroups different forms of hybrids of these two species, among which a cybrid form, that harbours the nuclear genome of C. eos and the mitochondria of C. neogaeus. This peculiar model is thus a unique opportunity to study the influence of an exogenous mitochondria on the metabolism and cellular physiology in a living animal in the wild, and thus perfectly adapted to this atypical cellular environment. Mitochondria being at the core of fundamental biological processes, we expect that the presence of foreign mitochondria will modify gene expression and the resulting proteome of these fishes. The overall goal of this master thesis is thus to compare the proteome of pure (wild type) C. eos with the cybrid form sampled in similar lakes from the same geographical area so that most differences could be attributed to the different mitochondrial genomes. To achieve this goal, we used two dimensional electrophoresis. We selected a sub-group of proteins that showed the most extreme expression differences and identified these spots by mass spectrometric analyses (LC/MS). Results demonstrate that C. neogaeus mitochondria has a strong influence on gene expression in cybrid. Proteins identified bring new clues supporting the hypothesis that cybrid are more cold tolerant than the wild type biotype.
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Dieks, Jana-Katharina. "Liquorproteomveränderungen bei Patienten mit Lewy-Körperchen Demenz." Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0001-BB2D-0.
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Die Demenz mit Lewy-Körperchen (DLB) ist eine progrediente neurodegenerative Erkrankung und stellt nach der Alzheimer-Erkrankung eine der häufigsten Ursachen einer Demenz dar. Betroffene leiden neben dem zentralen Merkmal Demenz an Fluktuationen der Kognition, Parkinsonismus und visuellen Halluzinationen. Charakteristische neuropatholgische Kennzeichen der DLB sind α-Synuklein-enthaltende Lewy-Körperchen und -Neuriten, die sich in kortikalen und subkortikalen Hirnregionen finden. Bei der klinischen Diagnostik dieser Erkrankung sind neben der Beurteilung klinischer Befunde laborchemische, psychometrische, apparative und bildgebende Verfahren von Bedeutung, jedoch ist eine sichere Diagnose nur bioptisch zu stellen.
Gegenstand dieser Arbeit war die Untersuchung des Liquorproteomprofils von DLB-Patienten im Vergleich zu neurologisch gesunden Kontrollen und die Identifikation von regulierten Proteinen im Liquor bei der DLB durch Verwendung klassischer Methoden der Proteomik. Nach initialer Depletion von zwölf häufigen Proteinen wurden die Liquorproben mittels zweidimensionaler Gelektrophorese aufgetrennt, die Proteinexpressionsmuster quantitativ verglichen und anschließend insgesamt 23 verschiedene Proteine aus 44 regulierten Gelspots massenspektrometrisch identifiziert. Es fanden sich Proteine involviert in die Immunantwort, den Lipidstoffwechsel, den Glukosestoffwechsel, die Signaltransduktion und die Zellstruktur sowie einige, die sich keiner dieser funktionellen Gruppen zuordnen ließen. Von vier ausgewählten Proteinen - Complement C4a, Transthyretin, Contactin-1 und Chromogranin A - wurden Western Blots angefertigt, wofür Liquor sowohl von DLB-Patienten und gesunden Kontrollen als auch zum weiterführenden Vergleich von Parkinson- und Alzheimer-Patienten verwendet wurde.
Die Ergebnisse dieser Arbeit zeigen auf Proteinebene die Vielfalt der biologischen Prozesse, die bei der DLB gestört ist. Zum Teil lassen sich Parallelen zu anderen neurogenerativen Erkrankungen erkennen, einige Proteine konnten jedoch erstmalig und einzig als bei der DLB reguliert nachgewiesen werden.
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Koehler, Gage. "Overwintering Survival of Strawberry (Fragaria x ananassa): Proteins Associated with Low Temperature Stress Tolerance during Cold Acclimation in Cultivars." 2012. http://hdl.handle.net/1805/2925.
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Indiana University-Purdue University Indianapolis (IUPUI)Winter survival is variable among commercially grown strawberry (Fragaria x ananassa) cultivars. The main objectives of this study were to evaluate the molecular basis that contribute to this difference in strawberry cultivars and to identify potential biomarkers that can be used to facilitate the development of new strawberry cultivars with improved overwintering hardiness. With these goals in mind, the freezing tolerance was examined for four cultivars, ‘Jonsok’, ‘Senga Sengana’, ‘Elsanta’, and ‘Frida’ (listed from most to least freezing tolerant based on survival from physiological freezing experiments) and the protein expression was investigated in the overwintering relevant crown structure of strawberry. Biomarker selection was based on comparing the protein profiles from the most cold-tolerant cultivar, ‘Jonsok’ with the least cold-tolerant cultivar ‘Frida’ in a comprehensive investigation using two label-free global proteomic methods, shotgun and two dimensional electrophoresis, with support from univariate and multivariate analysis. A total of 143 proteins from shotgun and 64 proteins from 2DE analysis were identified as significantly differentially expressed between ‘Jonsok’ and ‘Frida’ at one or more time points during the cold treatment (0, 2, and 42 days at 2 ºC). These proteins included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis related proteins and flavonoid pathway proteins. The proteins that contributed to the greatest differences between ‘Jonsok’ and ‘Frida’ are candidates for biomarker development. The novel and significant aspects of this work include the first crown proteome 2DE map with general characteristics of the strawberry crown proteome, a list of potential biomarkers to facilitate the development of new strawberry cultivars with improved cold stress tolerance.
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Seibert, C., B. R. Davidson, B. J. Fuller, Laurence H. Patterson, W. J. Griffiths, and Y. Wang. "Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry." 2009. http://hdl.handle.net/10454/6179.
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Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample.