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Dissertations / Theses on the topic 'Proteomic'
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1
Lang, Alastair Michael. "Developing tissue proteomics : differential in gel electrophoresis in biomarker discovery and proteomic degradation." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4642/.
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The field of proteomics and functional genomics has developed steadily since the completion of the human genome project. The wealth of genomic information and the pace at which it was compiled was astounding. Proteomics, despite considerable effort, on the other hand has not seen quite the same pace of development. The progress being considerably hindered by the lack of an amplification process and the relative complexity of the proteome in comparison to the genome. These intrinsic difficulties have led to the sensitivity of proteomic techniques being pushed closer to physical limits. There is therefore a further need to re-evaluated techniques such as sample preparation and integrity, analytical methods and collaborative strategies to maximise the effectiveness and quality of data collected. The importance of tissue in scientific and clinical research is unequivocal. However, tissue is difficult to collect, store and work with due to issues with proteomic degradation and storage. Good lab practices can minimise the effect of degradation but degradation of proteins can be rapid. Strategies to minimise degradation include freezing, formalin fixing and microwave treatment which all have their relative advantages and disadvantages. The importance of sample preparation as being the top of the workflow is often acknowledged but improvements are not well described in the literature. The main aim of this thesis is to present investigative studies into the mitigation of some of the limitations in tissue sample degradation, analytical approaches in differential in gel electrophoresis and accessing DiGE spot and tissue profile data. Presented is the evaluation of the effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and to aid in the cessation of proteomic degradation. A multifaceted analytical approach of differential in Gel electrophoresis spot data is assessed, giving proteomic profiles of mouse brain tissue. Preliminary data is presented showing that the process of heat-treatment has had a predominantly beneficial effect on mouse brain tissue, with a higher percentage of spots stabilised in heat-treated samples compared to snap-frozen samples. However, stabilisation did occur in snap-frozen samples for different protein spot so the appropriateness of using heat-treatment is as yet not fully determined and requires further analysis. In addition, the variation in tissue profiles of WKY, SP.WKYGla.2a and SHRSP rat model for hypertension is investigated with the future prospect of providing that vital connection between genomic and proteomic data and link phenotype and genotype preliminary investigated. A number of putative markers were identified and quantified using DiGE analysis. In order for these markers to be accepted as biomarkers, more downstream validation is required, however this study provides a good spring board as a proof of concept in using DiGE as an global putative biomarker discovery platform.
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Tabb, David L. "Bioinformatics of proteomic tandem mass spectra : selection, characterization, and identification /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/10847.
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Vogel, Martin Joseph. "Proteomic profiling following cryopreservation." Diss., Online access via UMI:, 2004. http://wwwlib.umi.com/dissertations/fullcit/1424168.
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Jufvas, Åsa. "Human Adipocytes : Proteomic Approaches." Doctoral thesis, Linköpings universitet, Avdelningen för cellbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-125907.
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Type 2 diabetes is characterized by increased levels of glucose in the blood originating from insulin resistance in insulin sensitive tissues and from reduced pancreatic insulin production. Around 400 million people in the world are diagnosed with type 2 diabetes and the correlation with obesity is strong. In addition to life style induction of obesity and type 2 diabetes, there are indications of genetic and epigenetic influences. This thesis has focused on the characterization of primary human adipocytes, who play a crucial role in the development of type 2 diabetes. Histones are important proteins in chromatin dynamics and may be one of the factors behind epigenetic inheritance. In paper I, we characterized histone variants and posttranslational modifications in human adipocytes. Several of the specific posttranslational histone modifications we identified have been characterized in other cell types, but the majority was not previously known. Moreover, we identified a variant of histone H4 on protein level for the first time. In paper II, we studied specific histone H3 methylations in the adipocytes. We found that overweight is correlated with a reduction of H3K4me2 while type 2 diabetes is associated with an increase of H3K4me3. This shows a genome-wide difference in important chromatin modifications that could help explain the epidemiologically shown association between epigenetics and metabolic health. Caveolae is a plasma membrane structure involved in the initial and important steps of insulin signaling. In paper III we characterized the IQGAP1 interactome in human adipocytes and suggest that IQGAP1 is a link between caveolae and the cytoskeleton. Moreover, the amount of IQGAP1 is drastically lower in adipocytes from type 2 diabetic subjects compared with controls implying a potential role for IQGAP1 in insulin resistance. In conclusion, this thesis provides new insights into the insulin signaling frameworks and the histone variants and modifications of human adipocytes.
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Le, Thi Tam. "Proteomic signatures of Bacillus subtilis." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=984429247.
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Schiess, Ralph. "Proteomic strategy for biomarker discovery /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=18097.
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Mujahid, Sana. "PROTEOMIC ANALYSIS OF LISTERIA MONOCYTOGENES." MSSTATE, 2008. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11012007-174823/.
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Listeria monocytogenes is a deadly, Gram-positive foodborne pathogen that is ubiquitous in the environment. The bacterium expresses a number of virulence and stress adaptation proteins that support its pathogenic capabilities. Two-dimensional gel electrophoresis (2-DE) was used to map L. monocytogenes surface proteins, which play a central role in virulence, and to examine protein expression by L. monocytogenes grown on ready-to-eat meat, an important source of Listeria infections. A novel method for solubilization of surface proteins from L. monocytogenes for 2-DE was developed. Additionally, the unique proteome expressed by L. monocytogenes grown on a meat matrix was uncovered. The developed solubilization method will facilitate efforts to identify and routinely compare surface proteins of Listeria by 2-DE. Furthermore, the 2-DE database of proteins expressed by L. monocytogenes grown on a meat matrix will allow further understanding of the interactions of Listeria with its food environment that influence its ability to cause disease.
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Mi, Jia. "Proteomic Analysis of Peroxisomal Proteins." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7943.
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Chen, R. "Proteomic study of mitochondrial proteins." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597549.
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Several proteins in bovine heart mitochondria with molecular masses of 29, 18 and 10 kDa have been demonstrated to be phosphorylated in a cAMP-dependent manner. The 18 kDa and 10 kDa proteins are subunits of complex I. In the presence of protein kinase A, subunits of purified complex I with the same molecular masses are phosphorylated in vitro. By Edman degradation and mass spectrometry of the radioactive protein bands from 32P-phosphorylated mitochondria and 32P-phosphorylated complex I, the 18kDa protein has been identified as subunit ESSS of complex I. It is phosphorylated at serine-20. In the 10 kDa band, subunit MWFE of complex I and subunit d of complex II are phosphorylated at serine-55 and serine-7, respectively. Five distinct 14-3-3 proteins have been detected in bovine heart mitochondria by immunological methods and by mass spectrometry. They are the b, g, e, h and ζ isoforms of the protein. Recombinant 14-3-3 b, g and ζ proteins have been over-expressed in Escherichia coli, and purified to homogeneity. The hydrophobic membrane protein, subunit c, has been isolated from bovine heart mitochondria, and from ovine and human lysosomal storage bodies associated with ceroid lipofuscinosis (Batten disease). By mass spectrometry, a post-translational modification with a mass of 42 Da is associated with a chymotryptic fragment of this protein in all three samples. By tandem MS sequencing, it has been shown that lysine-43 is trimethylated, and not acetylated, at the e-N-position of the residue is subunit c from all three samples. Therefore, trimethylation of subunit c is not, as has been suggested, a cause of abnormal accumulation of the subunit in lysosomes. This modification is not found in the equivalent lysine-44 of subunit c from yeast mitochondria. Therefore, the role of the modified lysine-43 in the assembly and (or) the functioning of the mitochondrial ATP synthase complex appears to be confined to higher organisms.
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Zhang, Meng. "Proteomic analysis of streptococcus pyogenes." Thesis, Northumbria University, 2007. http://nrl.northumbria.ac.uk/842/.
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Streptococcus pyogenes (group A streptococcus, GAS) is a major human Gram-positive pathogen that causes infections that normally occur in the respiratory tract, the skin, the wound, the lung, the bloodstream and/or muscle tissues and result in millions of deaths every year. To cause such infections, S. pyogenes produces a wide range of virulence factors. The destruction of connective tissue and the hyaluronic acid therein plays an important role in pathogenesis. S. pyogenes was propagated in hyaluronic acid rich growth media in an attempt to create a simple biological system that could reflect some elements of the pathogenesis. The growth of bacteria was analyzed in the hyaluronic acid rich media and control media and a proteomic approach was applied to identify those proteins that were differentially expressed by the streptococcal pathogens growing in the different media. The techniques of two dimensional gel electrophoresis and static nanospray mass spectrometry were optimized and proteome maps for S. pyogenes grown in both media were constructed. The differentially expressed proteins by S. pyogenes were identified and analyzed using bioinformatics. Our results showed that several recognized virulence factors of S. pyogenes were upregulated in hyaluronic acid rich media, including the Ml protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in GAS pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential streptococcal pathogens virulence factors.
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Vitorino, Rui Miguel Pinheiro. "Dental caries: a proteomic approach." Doctoral thesis, Universidade de Aveiro, 2004. http://hdl.handle.net/10773/17671.
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Doutoramento em BioQuímicaA cárie dentária é uma doença complexa que afecta uma grande parte da população mundial independentemente do sexo, idade ou etnia. Este processo é dependente de factores biológicos que se encontram presentes na saliva e placa dentária. Em seguimento do referido, amostras de saliva foram colectadas de indivíduos caracterizados em função dos índices DMFT e DMFS. A avaliação dos convencionais parâmetros clínicos como por exemplo fluxo salivar, capacidade tampão, pH usados na avaliação do risco para a cárie dentária em combinação com dieta, hábitos de higiene e tabagismo foram realizados para todos os indivíduos participantes do qual se observou a ausência de uma positiva correlação com o índice DMFT. Uma vez que os factores biológicos presentes na saliva influenciam o processo da cárie dentária, o objectivo deste trabalho consistiu na investigação de uma possível correlação entre as proteínas e peptídeos da saliva e o processo da cárie dentária. A caracterização das proteínas e peptídeos da saliva foi alcançada utilizando electroforese bidimensional (2-DE), cromatografia líquida de alta resolução (HPLC) combinada com a espectrometria de massa (MS), do qual resultou a identificação de 38 proteínas das quais 12 foram identificadas pela primeira vez por 2-DE e 22 peptídeos por HPLC-MS também identificados pela primeira vez. Ensaios realizados para o estudo da composição da película dentária seguiram a mesma metodologia descrita para a caracterização das proteínas e peptídeos da saliva sendo realizados inicialmente in vitro e confi rmados posteriormente por ensaios in vivo. A adsorção dos componentes salivares à hidroxiapatite é um processo selectivo com predominância de componentes salivares de baixo peso molecular. Contudo, amilase, lactoferrina, IgA salivar e anidrase carbónica VI foram também identificadas. A extracção sequencial usando guanidina e ácido trifluoroacético das proteínas/peptídeos adsorvidas à hidroxiapatite permitiu uma avaliação da força das ligações estabelecidas. Destes ensaios verificou-se que proteínas ricas em prolina (PRP-1/3), cistatina S, statherina e histatina 1 estabeleciam interacções fortes com a hidroxiapatite permanecendo adsorvidas após extracção com guanidina. As proteínas caracterizadas da saliva e da película dentária foram correlacionadas com o índice DMFT apresentando uma predominância de elevadas quantidades de cistatinas, PRP -1/3, statherina e histatina 1 no grupo de indivíduos sem cárie. O reduzido número de fragmentos em associação com as elevadas quantidades de cistatinas podem sugerir um controle mais eficiente da actividade proteólitica evitando desta maneira a degradação de importantes proteínas salivares no grupo de indivíduos sem cárie. A composição da película dentária é afectada pela composição proteica da saliva encontrando-se as referidas proteínas em maior quantidade. Os dados obtidos sugerem uma eficiente protecção por parte das proteínas da saliva contra a cárie dentária em particular a PRP-1/3, statherina e histatina 1, provavelmente devido à sua participação nos processos de remineralização na superfície do dente, e das cistatinas na diminuição da actividade proteólitica.
Dental caries is a complex disease process that affects a large proportion of the world population, regardless of gender, age and ethnicity. This process is dependent upon biological factors that are present within saliva and dental plaque. Following this, whole saliva was collected from selected individuals characterised according its DMFT and DMFS scores. Evaluation of the conventional clinical parameters such as flow rate, buffering capacity, pH used for caries risk assessment in combination with diet, hygiene and smoke habits was performed for all participating subjects showing absence of a statistic positive correlation with DMFT index. Since biological factors present on saliva influence dental caries process, the aim of this study was to investigate how salivary proteins and peptides are correlated with this pathology. Characterisation of salivary proteins and peptides was achieved using twodimensional gel electrophoresis (2-DE) and high performance liquid chromatography (HPLC) in combination with mass spectrometry (MS) resulting in the identification of 38 proteins, being 12 proteins identified by 2-DE and 22 peptides by HPLC-MS were identified for the first time. Experiments to study enamel pellicle composition were performed following the same methodology described for salivary proteins and peptides, initially in vitro being supported with in vivo assays. Adsorption of salivary components to hydroxyapatite showed to be a selective process with a predominance of low molecular weight salivary components. However, amylase, lactoferrin, S-IgA, carbonic anhydrase VI were also identified. A sequential extraction, using of guanidine and trifluoroacetic acid, of the adsorbed proteins/peptides to hydroxyapatite allowed to evaluate the strength of the establish interactions. From this experiments, proline-rich proteins (PRP -1/3), cystatin S, statherin, histatin 1 exhibited a strong interaction with hydroxyapatite remaining adsorbed after guanidine extraction. Characterised salivary proteins from whole saliva and enamel pellicle were correlated with DMFT index showing a predominance of higher amounts of cystatins, PRP-1/3, statherin and histatin 1 in caries free group. Decreased number of fragments in association with higher amounts of cystatins may suggest a more effective control in proteolytic activity which avoid the degradation of important salivary proteins from caries free group. Acquired pellicle composition is affected by whole saliva protein composition being the above referred proteins present in higher amounts. Obtained data suggest an effective protective role of several salivary proteins to dental caries in particular of PRP-1/3, statherin and histatin 1, possibly due to their participation on remineralization processes at the tooth surface, and of cystatins probably by decreasing proteolytic activity.
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Granlund, Irene. "Proteomic analysis of Arabidopsis thaliana." Doctoral thesis, Umeå : Department of Chemistry, Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1820.
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Hassan, Hanna. "Proteomic profiling of vesicular organelles." Thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215028.
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Alvarez, de Eulate Diaz de San Martin Eva Maria. "Electrochemical studies toward proteomic analysis." Thesis, Curtin University, 2014. http://hdl.handle.net/20.500.11937/702.
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This thesis provides the basis for a label-free bioanalytical platform using electrochemical analysis at liquid –liquid interfaces. The possibility to detect biomolecules such as proteins in a label-free manner via adsorption and ion-transfer was achieved. Several pre-treatment steps used in proteome analysis, such as protein pre-concentration and digestion, were studied. The results demonstrate the promise of this strategy for the detection and identification of proteins.
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Samji, S. M. "Proteomic pattern in pre - eclampsia." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2017. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/5891.
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Velázquez, Sánchez Diego. "Exploring the regulatory mechanisms of the S. cerevisiae Ppz1 protein phosphatase and the molecular basis for its toxicity." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667958.
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Uno de los más notables componentes en la homeostasis de cationes en levaduras es la proteína fosfatasa Ppz1. El papel de Ppz1 es doble: inhibe la entrada de potasio mediante el control de la actividad de los transportadores Trk1 y Trk2 y atenúa la expresión del gen de la Na+-ATPasa ENA1. Ppz1, está controlada negativamente por dos subunidades reguladoras estructuralmente relacionadas, Hal3 y Vhs3, siendo la primera la más relevante funcionalmente. A parte de esto se ha demostrado que niveles altos de Ppz1 son extremadamente perjudiciales para la célula.
El primer objetivo de este trabajo ha sido estudiar la interacción entre Ppz1 y Hal3. A pesar de las numerosas evidencias de que ambas proteínas interaccionan físicamente, se desconoce que regiones de cada proteína están involucradas en dicha interacción y hasta qué punto es un proceso dinámico. Para ello se han diseñado dos aproximaciones experimentales, por un lado se han creado una serie de cepas con etiquetas fluorescentes en Ppz1 y Hal3, para seguir su interacción in vivo gracias al fenómeno de la transferencia de energía por resonancia de fluorescencia (FRET) mediante citometría de flujo. Estas fusiones de Hal3 y Ppz1 con proteínas fluorescentes, además de ser completamente funcionales, han demostrado la interacción in vivo de ambas proteínas y permiten estudiar posibles variaciones en la interacción. Por otro lado, mediante experimentos de crosslinking in vitro de ambas proteínas y un posterior análisis por LC-MS/MS se han mostrado dos regiones del dominio C-terminal de Ppz1 y tres regiones de Hal3, que podrían ser las responsables de llevar a cabo esta interacción.
A pesar de ser regulada negativamente por Hal3 y Vhs3, la región más N-terminal de Ppz1 es muy rica en residuos fosforilables. De hecho, el 30% de este dominio son serinas o treoninas, y algunas de ellas han sido identificadas como fosforiladas. Sin embargo, apenas ha sido estudiado que efectos funcionales pueden dar lugar cambios en el estado de fosforilación de Ppz1. En este trabajo hemos conseguido demostrar que la MAPK Hog1 fosforila a Ppz1 en la posición S265, pero este hecho no es suficiente para variar el comportamiento de la fosfatasa. Además, hemos conseguido demostrar que el estado de fosforilación de Ppz1 puede variar dependiendo de factores externos como alta concentración de sal o deficiencia de potasio.
Recientemente se ha demostrado que Ppz1 es la proteína más tóxica de levadura cuando se sobredosifica. Recientemente, en nuestro laboratorio se ha verificado que la actividad fosfatasa de Ppz1 es necesaria para causar dicha toxicidad, ya que la sobreexpresión de una versión catalíticamente inactiva no causa problemas de crecimiento en las células. Sin embargo, las bases moleculares de esta toxicidad son desconocidas. En nuestro caso hemos realizado experimentos para conocer el estado del proteoma y del fosfoproteoma de S. cerevisiae cuando se sobreexpresa Ppz1, y hemos descrito una variación en el estado de fosforilación en numerosas proteínas de la levadura. De hecho se han identificado cambios importantes en proteínas relacionadas con la traducción, polarización celular, la transición G1/S del ciclo celular o con el metabolismo de carbohidratos. Además, algunos experimentos paralelos han reforzado los resultados obtenidos mediante espectrometría de masas, como la defosforilación de Mig1 y Rps6, o una hiperfosforilación de eIF2α en la Ser-51, indicando una posible inhibición en la iniciación de la traducción.
En resumen los resultados obtenidos en esta tesis han puesto a punto herramientas para conocer más sobre el dinamismo de la interacción Ppz1-Hal3, han aportado datos sobre la fosforilación de Ppz1, y han generado una gran cantidad de datos de fosfoproteómica cuyo estudio detallado podría llevar a esclarecer bases moleculares de la toxicidad de Ppz1 en S. cerevisiae.The protein phosphatase Ppz1 is one of the most relevant components in cation homeostasis in yeast. It has two major roles: on one hand, it inhibits the potassium influx by controlling the activity of the Trk1 and Trk2 potassium transporters and, on the other hand, it represses the expression of the ENA1 gene, coding for a Na+-ATPase. Ppz1 is negatively regulated by two structurally related subunits, Hal3 and Vhs3, being Hal3 the most functionally relevant. Previous studies have shown that high levels of Ppz1 are extremely detrimental for yeast cell growth. The first objective of this work has been to study the interaction between Ppz1 and Hal3. Although there is evidence of both proteins interacting physically, the regions of each protein involved in the interaction, as well as its dynamism of the interaction, are still unknown. Therefore, two different experimental approaches have been designed. The first approach is based on the detection of fluorescence resonance energy transfer (FRET) by flow-cytometry. Several strains have been constructed with fluorescent tags on Ppz1 and Hal3, to monitor the interaction in vivo. These tagged versions of Ppz1 and Hal3, in addition to be fully functional, have been shown suitable to demonstrate the interaction in vivo between both proteins and to allow the study of possible variations on the interaction. The second approach, based on in vitro crosslinking of both proteins, followed by LC-MS/MS analysis, has shown two regions of Ppz1 C-terminal and three from Hal3 that could be involved in the interaction. Even though Ppz1 is negatively regulated by Hal3 and Vhs3, the most N-terminal region of Ppz1 is very rich in phosphorylable residues (in fact, 30 % of the N-terminal residues are serine or threonine). Some of them have been identified as phosphorylated in several studies. However, how phosphorylation could affect Ppz1 function remains unknown. In this work we show that the MAPK1 Hog1 phosphorylates Ppz1 at S265, but this is not enough to alter the behaviour of the phosphatase. Moreover, we demonstrated that the phosphorylation state of Ppz1 can change depending on external factors, such as high salt or low potassium concentrations. It has been recently demonstrated that Ppz1, when overexpressed, is the most toxic protein for yeast cells. We described in our laboratory that the phosphatase activity of Ppz1 it is crucial for this toxicity, since overexpression of a catalytically inactive version does not negatively affect cell growth. However, the molecular basis of this toxicity is still unknown. To address this question our approach was to characterize the proteomic and phosphoproteomic landscape of S. cerevisiae cells overexpressing Ppz1. We have found changes in the phosphorylation state of numerous proteins, including proteins related to translation, polarized cell growth, G1/S transition and carbon metabolism. Moreover, we provide additional evidence that reinforce the results obtained by mass-spectrometry, such in the case of Mig1 and Rps6 dephosphorylation, or hyperphosphorylation of eiF2α at Ser-51, pointing to a possible inhibition of translation initiation. Overall, the results obtained in this thesis have set tools to dynamically study in vivo the Ppz1-Hal3 interaction, have provide information on the phosphorylation of Ppz1, and have generated a large database of proteomic and phosphoproteomic data whose future analysis might provide the clues about the toxicity of Ppz1 in S. cerevisiae.
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Geletu, Heye Mulu. "Proteomic analysis of acute promyelocytic leukemia." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-63200.
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Alm, Henrik. "Proteomic Characterization of Induced Developmental Neurotoxicity." Doctoral thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-99652.
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The developing brain goes through a number of developmental periods during which it displays an increased sensitivity to exogenous disturbances. On such period is the so called “Brain growth spurt” (BGS) which in humans takes place starting from the third trimester of pregnancy and throughout the first few years of life. The corresponding period in rats and mice is the first postnatal weeks. Exposure to relatively modest concentrations of the brominated flame retardant PBDE-99 during the second week of life in mice causes a more or less permanent impairment in the ability of the animals to adjust properly to environmental changes at adulthood. This “late response on early exposure” reflects the long-term consequences of disrupting the developing brain during a sensitive time period. The cellular mechanisms underlying the behavioral effects are far from clear. To address the initial damage occurring around the time of exposure, the approach used in this thesis is to use proteomics to analyze the effects of PBDE-99 on protein expression soon (24 hours) after exposure of the neonatal mouse on postnatal day (PND) 10.The thesis comprises the effects on the proteome in three distinct brain parts: cerebral cortex, striatum and the hippocampus. In addition, an in vitro model was developed and used to evaluate the PBDE-99 effects on cultured cerebral cortex cells from embryonic rat brains. Gel-based proteomics (2D-DIGE) coupled to MALDI- or ESI-MS has been used throughout for the proteomics experiments, but other techniques aimed at analyzing both proteins and mRNA have also been used to better characterize the effects. Even if the protein complements expressed by the different brain parts and separated with 2D-DIGE are seemingly similar, the effects are apparently specific for the different brain regions. In hippocampus, PBDE induces effects on proteins involved in metabolism and energy production, while the effects in striatum point towards effects on neuroplasticity. PBDE-99 changes the expression of cytoskeletal proteins in the cerebral cortex 24 hours after exposure. Interestingly, in vitro exposure of cerebral cortex cells to a PBDE-99 concentration in the same order of magnitude as in the in vivo neonatal brain also induces cytoskeletal effects, in the absence of cytotoxicity. This may suggest effects on regulatory aspects of cytoskeletal dynamics such as those involved in neurite sprouting. This thesis also addresses the problems involved in presenting proteomics data. Many of the available methods and approaches for presenting transcriptomics data are not suitable for isoform rich protein data. Modifications of existing methods and the development of a new approach (DEPPS) is also presented. Most importantly, the thesis presents the application and usefulness of proteomics as hypothesis generating techniques in neurotoxicology.
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Karsani, Saiful Anuar. "Proteomic analysis of Leishmania mexicana differentation." Thesis, Imperial College London, 2006. http://hdl.handle.net/10044/1/11356.
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Raine, John Dale. "Proteomic analysis of the malarial parasite." Thesis, Imperial College London, 2005. http://hdl.handle.net/10044/1/11358.
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Chilton, Caroline Hazel. "Comparative proteomic analysis of Clostridium difficile." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5960.
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The recent increase in availability of next generation sequencing methodologies has led to extensive analysis of the genome of Clostridium difficile. In contrast, protein expression analysis, crucial to the elucidation of mechanisms of disease, has severely lagged behind. In this study, in-depth proteomic analysis of three strains of varying virulence, demonstrated previously in an animal model, has been undertaken against a background of the sequenced genomes. Strain B-1 is a historic, virulent, ribotype 005 clone, strain A represents the emerging hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low virulence. To undertake a comprehensive overview of the expressed proteome, both 1D and 2D gel electrophoresis were used to separate and display the protein content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass spectrometry for protein identification. A total of 888 different proteins were characterised by comparative analysis of isolates grown in parallel for 64 hours on blood agar. Of these, only 38% were shared between all isolates. An additional 350, 243 and 398 proteins were detected from broth cultures, and the use of a hexapeptide bead library, designed to capture low abundance proteins, led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and Tra5/5 respectively. Relative differential expression was investigated using Differential In Gel Electrophoresis (DIGE), and five proteins were shown to have a statistically higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5. A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in both A and B-1. Furthermore, differential post-translation modification events were seen in flagellar and S-layer proteins. In-vivo expression of these proteins was mapped using Western blotting. Immunodetection of the majority of these, including FliC and the high molecular weight S-layer protein, were conserved between the three strains, but a notable series of immunoreactive protein spots were present in strains A and Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein present in the genomes these strains, but not that of B-1. Protein expression differences for a number of previously proposed virulence proteins were evident between strains, including toxin B, sporulation, flagella and the S-layer proteins, metabolic enzymes, stress response proteins and ABC transporters. This study strongly supports the view that the virulence of Clostridium difficile is multifactorial, and that a number of related factors, although not directly required for pathogenicity, may serve to modulate the virulence of individual strains.
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Westbrook, James Arthur. "Proteomic Analysis of Heart Transplant Rejection." Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498689.
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Sharif, Amar. "Metabolic and proteomic profiling in cholangiocarcinoma." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486601.
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Background: Cholangiocarcinoma (CCA) has a very poor prognosis and the aetiology is not clearly understood. Analytical biochemical techniques may provide insights into pathogenesis and cellular signalling pathways as well as identifying novel diagnostic and prognostic biomarkers of disease. Particularly relevant are mass spectrometry (MS)-based quantitative proteomics of tissue and magnetic resonance spectroscopy (MRS) of bile. Methods: MS: 28-80 /-lg of microsomal protein from paired CCA and normal adjacent tissue from two patients were reduced, alkylated, trypsin digested and subsequently labelled with isobaric tag reagents, combined and separated by two-dimensional high-performance liquid chromatography and analysed by tandem mass spectrometry. MRS: In vitro proton MR data were acquired from bile from five CCA cases and compared to disease control bile from benign biliary disease (seven gallstones, eight sphincter of Oddi dysfunction, and five primary sclerosing cholangitis [PSC]). Results: MS: Proteins over-expressed in both CCA tissues included galectin-3-binding protein precursor, proteasome subunit beta type 1 precursor and type 6 precursor. Proteins involved in bile acid synthesis (sterol 12-hydroxylase, CYP8B1) conjugation (bile acyl CoA synthetase, BACS) and glucuronidation (UDP-glucuronsyltransferases precursor proteins, UGT2B4, UGT2827) were downregulated in CCA tissue, while the bile acid transporter, bile salt export pump (BSEP), was over-expressed. MRS: Taurine-and glycine-conjugated bile acids were elevated in bile in patients with CCA when compared to bile from patients with PSG (p=O.016, p=0.008). Biliary phosphatidylcholine levels were reduced in patients with CGA when compared to those with gallstones (p=0.030). Conclusions: The proteomic and MRS data suggest altered bile acid metabolism plays an important role in CGA aetiopathogenesis.
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Pleat, Jonathon Michael. "A proteomic analysis of human scarring." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534204.
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Somasundaram, Brinda. "Proteomic Characterization of Hemogen in Erythropoiesis." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23474.
Full textAbstract:
Hemogen (Hemgn) is reported as a tissue specific transcriptional regulator in testis as well as hematopoietic tissues. It is known that Hemgn positively regulates erythroid differentiation; however,the underlying molecular mechanism is not well understood. In the current study, using proteomic approach in combination with other molecular biology tools,we have attempted to decipher the role of Hemgn in differentiating Murine erythroblast leukemia (MEL) cells as a model system. Our study reveals that Hemgn predominantly interacts with transcriptional regulators, chromatin modifiers and histones. Furthermore, using Chromatin Immunoprecipitation and knockdown approach, we have demonstrated that Hemgn is recruited to the b-globin locus, which is known to be activated during erythroid differentiation. Based on the results,we speculate that Hemgn acts as a tissue specific histone chaperone that regulates transcription during erythroid differentiation.
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Wilkins, Joanna Claire. "Proteomic analysis of aciduric oral streptococci." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252409.
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Fowsantear, Winita. "Comparative proteomic analyses of Helicobacter species." Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=201662.
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Helicobacter pylori infects and colonises in the human gastric where the infection can lead to gastric diseases. There are also a number of other Helicobacter species referred to as non-pylori Helicobacters that colonise other parts of the gastrointestinal tract. To understand those characteristics of the non-pylori Helicobacters that allow them to colonise different sites in the body resulting in different patterns of infection, the analysis of Helicobacter pylori and non-pylori Helicobacter proteomes was initiated. Total cellular proteins were extracted and compared using 2-DE. The Helicobacter proteome showed a high level of variability between different H. pylori strains as well as between the eight Helicobacter species analysed. It was proposed that some of the proteomic variation related to the pathogenic potential of the bacteria. Differential syntheses of specific proteins were found to be associated with H. pylori isolates associated with two different disease outcomes. Differential patterns of protein synthesis were also observed to discriminate between the Helicobacter isolates according to their site of colonisation defined as gastric and enterohepatic Helicobacter groups. Significant protein spots were identified by peptide fragment fingerprinting and LC-MS/MS. To provide additional functional information on the bacterial proteins, the Helicobacter hydrophobic proteins, which are typically located in the bacterial membrane and may be involved in bacterial host interactions, were analysed. Triton X-114 was used to enrich the bacterial hydrophilic and hydrophobic proteins which were analysed by 2-DE. There was significant enrichment of specific proteins to both the hydrophobic or hydrophilic fractions. Some of the enriched hydrophobic proteins which discriminated the Helicobacter species were identified. The Helicobacter species that colonise at different sites in the gastrointestinal tract are exposed to different acidic environments. The ability of the bacteria to survive the different levels of acid stress may contribute towards the observed proteomic variation. Quantitative differences in protein abundance were demonstrated between bacteria grown under neutral and acidic conditions. The differential patterns of protein synthesis were most clearly detected when the sub-cellular hydrophobic and hydrophilic fractions were analysed by 2-DE.
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Bozanic, Vesna. "Protein stability in a proteomic perspective." Doctoral thesis, Universidade Nova de Lisboa.Instituto de Tecnologia Química e Biológica, 2013. http://hdl.handle.net/10362/10531.
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Dissertation presented to obtain the Ph.D degree in BiochemistryThis work involved the identification and analysis of the properties of the most stable proteins present within proteomes, aiming at obtaining a general perspective of the factors that determine protein stability. As models we have focused on ensembles of proteins with high intrinsic stability, and for this purpose we have studied proteomes from the hyperthermophilic archaeon Sulfolobus solfataricus and Sulfurisphaera sp., whose properties were compared to those of the mesophilic bacterium Escherichia coli.(...)
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Mallawaaratchy, Duthika Marian. "Proteomic insights into gliobastoma tumour invasion." Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14731.
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Glioblastoma (GBM) is the most common, primary malignant brain tumours in adults. A better understanding of GBM biology is required to identify targets and develop new therapeutics. Tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that degrade the ECM and thereby actively re-modelling the surrounding tumour microenvironment. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia to some degree, with U87MG the most and LN229 the least invasive cells. Overall, 1,141 proteins were identified and the abundance levels of 49 proteins correlated with invasiveness, many of which were previously linked to invadopodia formation, epithelial-mesenchymal transition and GBM cell invasion. Invadopodia act as multi-vesicular endosome docking sites and have been shown to be a site of exosome release. Overall, 844 proteins were identified in the extracellular vesicles (EVs) protein profiles, where approximately 50% were also identified in the membrane dataset. The abundance levels of 14 proteins correlated with the invasiveness. GBM-derived EVs can cross the blood brain barrier and are detectable in peripheral blood, the profiles and significant markers presented here could be of interest to diagnostics. Bioinformatics, Western blot analysis, co-localisation immunofluorescence and a siRNA knockdown substantiated some of interesting membrane and EV proteomics findings. In silico analysis of publically available gene databanks demonstrated the clinical prognostic significant of several invasion-related targets identified in these analyses. GBM invasion is regulated by a dynamic cross-talk between tumour cells and the brain microenvironment. After confirming the uptake of fluorescently labelled GBM-EVs, astrocytes were observed to assume a more invasive phenotype, using the same invadopodia assay. Whole cell proteome analysis of astrocyte before and after exposure to GBM EVs provides insight into the intercellular communication between GBM cells and surrounding normal astrocytes. This study highlights the signalling pathways that contribute to GBM invasion and may help to understand the aggressiveness of this disease.
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Branson, Owen E. "Improved tag-count approaches for label-free quantitation of proteome differences in bottom-up proteomic experiments." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1471553685.
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Hou, Weimin. "Developing Mass Spectrometry-Based Analytical Methodologies for Analyzing Complex Protein and Lipid Samples." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26134.
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Mass spectrometry has increasingly become the method of choice for the analysis of
complex biological samples, including proteins and lipids. This thesis describes the
development of MS-based analytical methodologies for the analysis of complex proteomic
and lipidomic samples.
Chapter 3 describes the development of microfluidic proteomic reactors, in the
formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic
digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These
microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex
proteomic samples by combining multiple processing steps, such as rapid extraction and
enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all
performed in a few nanoliters effective volume, resulting in an increased protein digestion
efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that
were compatible with HPLC-MS/MS analysis.
In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of
PAF and LPC lipid species is described. In this method, lipid extracts from biological
samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000
mass spectrometer, where the lipid species of interest were detected using a precursor ion
scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with
standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0,
C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of
identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative
comparisons of other LPC lipid species of interest between different samples. The developed
methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated
PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8
transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42
treatment, respectively, in comparison to controls.
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Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.
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Crawford, Scott Daniel. "Sources of Variability in a Proteomic Experiment." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1534.pdf.
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Grönwall, Caroline. "Affibody molecules for proteomic and therapeutic applications." Doctoral thesis, KTH, Molekylär Bioteknologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4674.
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This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells.QC 20100729
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Alruwaili, Jamal A. "Serum proteomic analysis of prostate cancer progression." Thesis, University of Portsmouth, 2011. https://researchportal.port.ac.uk/portal/en/theses/serum-proteomic-analysis-of-prostate-cancer-progression(03249d0a-9e7e-4d61-8b75-7e5f94693b68).html.
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Background: The reported incidence of prostate cancer (PCa) has increased in recent years due to the aging of the population and increased testing; however mortality rates have remained largely unchanged. Studies have shown deficiencies in predicting patient outcome for both of the major PCa diagnostic tools, namely prostate specific antigen (PSA) and trans rectal ultrasound ‐guided biopsy (TRUS). Therefore, serum biomarkers are needed that accurately predict prognosis of PCa (indolent vs. aggressive) and can thus inform clinical management. Aim: This study uses surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI‐TOF‐MS) analysis to identify differential serum protein expression between PCa patients with indolent vs. aggressive disease categorised by Gleason grade and biochemical recurrence. Materials and Methods: A total of 99 serum samples were selected for analysis. According to Gleason score, indolent (45 samples) and aggressive (54) forms of PCa were compared using univariate analysis. The same samples were then separated into groups of different recurrence status (10 metastatic, 15 biochemical recurrence and 70 nonrecurrences) and subjected to univariate analysis in the same way. The data from Gleason score and recurrence groups were then analysed using multivariate statistical analysis to improve PCa biomarker classification. Using gel‐electrophoresis technique, candidate biomarkers were separated and identified by LC‐MS/MS and validated using optimised Western blot (WB) immunoassay against 100 PCa serum samples from the Wales Cancer Bank (50 as indolent group & 50 as aggressive group). Results: The comparison between serum protein spectra from indolent and aggressive samples resulted in the identification of twenty‐six differentially expressed protein peaks (p<0.05), of which twenty proteins were found with 99% confidence. A total of 18 differentially expressed proteins (p<0.05) were found to distinguish between recurrence groups; three of these were robust with P<0.01. Sensitivity and specificity within the Gleason score group was 73.3% and 60% respectively and for the recurrence group 70% and 62.5%. Four candidate biomarkers (categorised by Gleason score) were identified using a novel 1 D LC‐MS/MS technique. The candidate biomarker with m/z of 9.3 kDa was found to be upregulated in aggressive PCa patients, and was identified as Apolipoprotein C‐I (ApoC‐I). Another three candidate biomarkers (22.2, 44.5 and 79.1 kDa) were found downregulated in the aggressive group and up‐ regulated in the indolent group and identified as apolipoprotein D (ApoD), putative uncharacterised protein (PUP) and Transferrin (TF), respectively. The utility of the putative biomarkers was examined by Western blot (WB) analysis of 100 blinded PCa serum samples. None of the three SELDI identified biomarkers were able to statistically identify PCa patients’ progression. Conclusion: The use of SELDI to identify potential PCa progression biomarkers has been confirmed in PCa patients. However, immunovalidation of prospective biomarkers in blinded PCa serum samples was unsuccessful. This study demonstrates the importance of validation in ascertaining the true clinical applicability of a cancer biomarker.
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Waterbury, Raymond. "The electron microscopy proteomic organellar preparation robot /." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102768.
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An Electron Microscopy Proteomic Organellar Preparation (EMPOP) robot was developed as a tool for high-throughput preparation of subcellular fraction samples for electron microscopic identification. It will provide a means for validation of subcellular sample purity and confirmation of protein localization needed for organellar proteomics.The device automates all chemical and mechanical manipulations required to prepare organelles for electron microscopic examination. It has a modular, integrated design that supports automated filtration, chemical processing, delivery and embedding of up to 96 subcellular fraction samples in parallel. Subcellular fraction specimens are extremely fragile. Consequently, the system was designed as a single unit to minimize mechanical stress on the samples by integrating a core mechanism, composed of four modular plates, and seven support subsystems for: (1) cooling, (2-3) fluid handling, (4-7) positioning. Furthermore, control software was developed specifically for the system to provide standardized, reproducible sample processing while maintaining flexibility for adjustment and recall of operational parameters.
Development of the automated process progressed from initial validation experiments and process screening to define operational parameters for preservation of sample integrity and establish a basic starting point for successful sample preparation. A series of successive modifications to seal the local environment of the samples and minimize the effect of fluidic perturbations further increased process performance. Subsequent testing of the robot's full sample preparation capacity used these refinements to generate 96 samples in approximately 16 hours; reducing the time and labor requirement of equivalent manual preparation by up to 1,000 fold.
These results provide a basis for a structured approach toward process optimization and subsequent utilization the device for massive, parallel preparation of subcellular fraction samples for electron microscopic screening and quantitative analysis of subcellular and protein targets necessary for high-throughput proteomics.
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Allingham, Heather. "Development of proteomic techniques for biomarker discovery." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3145/.
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The main aim of my research, presented here, is to develop proteomic research techniques, for their use in biomarker discovery and identification. This is broken down into three main chapters: • Biomarker Identification in Stroke Brain guided by MALDI-imaging. • Evaluation of the Effectiveness of Heat Treatment for Prevention of Proteomic Sample Degradation using Label Free Relative Quantitation. • Discovery and Identification of Biomarkers for Hypertension Within these chapters special attention is paid to sample preparation, development and assessment of new methods for biomarkers discovery and identification, the importance of experimental design and the application of relevant and useful statistical methods to enable the mining of useful information from rich datasets. The biological changes in stroke induced mouse brain tissue are studied, the prevention of degradation to tissue samples by a novel heat treatment method and changes to plasma samples from hypertensive, wild type and a congenic strain of rat are also studied. The outcomes of this work are multiple, namely: • The identification of a possible marker for stroke from mouse brain tissue • The effect of a new heat treatment device on proteomic data obtained from mouse brain tissue • The novel application of a statistical analysis to a new type of dataset (LC-MS and label free quantitation of biological samples) • The identification of possible biomarkers for hypertension in rat plasma using this method
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Yang, Qian. "Proteomic investigation of the group B streptococcus." Thesis, Northumbria University, 2011. http://nrl.northumbria.ac.uk/2119/.
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The Group B Streptococcus (GBS) is a Gram-positive opportunistic pathogen which is a leading cause of neonatal disease globally. In 2000-2001, the general incidence of neonatal GBS infection was 0.72 per 1000 live births in U.K. and the mortality rate is about 10%, because of which neonatal GBS disease is a significant burden on society. GBS is part of the commensal flora, colonising the vagina and gastrointestinal tract of women. Vertical transmission is the main cause of early onset GBS disease. During the process of GBS neonatal disease, GBS must be able to survive in several very different host environments, including the vagina, amniotic fluid, the neonate's lung and blood. The vagina is normally acidic, low oxygen and with limited nutrients while the neonate's lung and blood are neutral, high oxygen and with abundant nutrient. Proteomic investigations of GBS protein expression under conditions representing those associated with benign maternal colonisation and foetal exposure may help us understand the molecular basis of GBS virulence. GBS growth characteristics, long term survival, acid adaptation, viable but non-culturable state and biofilm formation were investigated to help us understand how GBS survives in different environments and also help us to develop an in vitro model to reflect in vivo conditions during GBS disease development. An in vitro model of GBS growth under conditions reflecting maternal vaginal carriage (low pH, low oxygen, nutrient stress) and exposure to body fluids during invasive disease (neutral pH, aeration, nutrient sufficient) was established. Proteins expressed under each growth conditions were separated by two dimensional electrophoresis. Individual proteins were subjected to in-gel trypsin digestion and identified using liquid chromatography-mass spectrometry with peptide mass fingerprinting followed with bioinformatic research. A total of 76 proteins were identified and 16 of these were expressed differentially. The putative virulence factor C protein β antigen and proteins involved in responses to oxidative stress were up-regulated under the conditions reflecting neonatal exposure. Another in vitro model of GBS growth on Todd Hewitt agar in the presence or absence of 10% human serum was established and followed by proteomic investigation of proteins differentially expressed under these two conditions, as this model reflects GBS neonatal septicaemia (exposure to serum). A total of 84 proteins were identified and 11 of which were expressed differentially. The putative virulence factor C protein β antigen, arginine deiminase, an ABC transporter substrate-binding protein and glyceraldehyde-3-phosphate dehydrogenase were up-regulated in the presence of human serum.
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Chan, C. W. "A proteomic approach to studying oligodendrocyte signalling." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597430.
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In this study, my aim was to investigate oligodendrocyte signalling using a non-candidate, unbiased, proteomic approach. First, in collaboration with my industrial sponsors Merck Sharpe and Dohme, the potential of using 2-dimensional differential in gel electrophoresis (2D DIGE) to study oligodendrocyte signalling was evaluated. Although differentially expressed proteins between the 2 proteomes under analysis could be identified, it was concluded that 2D DIGE would be an unsuitable method to study oligodendrocyte biology. An alternative approach was therefore pursued in which a specific subset of the proteome was analysed. Previously, α6β1 integrin has been shown to be involved in oligodendrocyte survival and morphological differentiation. A protocol was developed whereby protein complexes co-immunoprecipitated with α6β1 integrin could be identified by mass spectroscopy analysis. Multiple proteins were identified including β1 integrin, contactin, plectin and heterogeneous ribonuclear protein K (hnRNP K). Characterisation and function of contactin in oligodendrocyte α6β1 integrin signalling was then investigated. Contactin expression throughout oligodendrocyte in vitro differentiation was confirmed by both Western blotting and Immunocytochemistry. The interaction of contactin and α6β1 was also verified by traditional co-immunoprecipitation. siRNA knockdown of contactin had no effect on in vitro oligodendrocyte morphological differentiation. However, contactin siRNA knockdown did block oligodendrocyte enhanced survival of the α6β1 integrin substrate, laminin-2, in response to platelet derived growth factor (PDGF). Therefore, by taking a proteomic approach, I have identified a novel interaction between α6β1 integrin and contactin, and a potential role of contactin in α6β1 integrin mediated survival signalling.
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Clarke, Fiona Margaret. "Genomic and proteomic markers of aggressive periodontitis." Thesis, Queen Mary, University of London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497877.
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Paton, Louise Nancy. "Intermediate filament protein assembly : a proteomic approach." Thesis, University of Canterbury. Biological Science, 2005. http://hdl.handle.net/10092/7989.
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Intermediate filament proteins (IFPs) form the main structural elements of a wool fibre. The IFPs of wool are comprised of two families; the acidic type I family and the neutral-basic type II family. During follicle development, one type I and one type II IFP develop into an obligate heteropolymer, which, through a series of associations with other heteropolymers, forms an intermediate filament.
Two-dimensional polyacrylamide gel electrophoresis (20-PAGE) methods have been used to provide high-resolution separation of wool IFPs. Improvements in the method for maintaining reducing conditions and chaotrope constitution, combined with low % T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern, with numerous discrete minor spots not previously observed.
Genomic studies have reported that there are eight genes which produce eight abundant IFPs in wool. It was hypothesised that the large number of additional spots seen on a 20-PAGE gel was due to post-translational modification (PTM) of the protein. Several common PTMs of proteins produce charge heterogeneity, including phosphorylation and glycosylation. However, analysis of wool IFPs by 20- PAGE techniques and mass spectrometry revealed no evidence of phosphorylation or glycosylation modifications.
Conformational equilibria as a cause of protein charge heterogeneity has recently been reported. Investigations with both the type I and type II IFPs have shown that when single protein spots from a 20-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. The cause of this heterogeneity is thought to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension. This technique allowed the accurate assignment of IFPs resolved by 20-PAGE to protein families.
Fractionation methods to separate the IFPs and intermediate filament associated proteins (IFAPs) were successfully developed. Further fractionation into the type I and type II IFPs was achieved along with partial success at isolating individual spots. In vitro assembly experiments with the different IFP families gives important information about the strength of different protein pairings. To date there are no reproducible, efficient, in vitro assembly conditions for keratinised wool IFPs. A comprehensive study to investigate assembly conditions for keratinised wool IFPs was undertaken.
Assembly of filaments from IFPs was achieved after a partial digestion with chymotrypsin. Filaments were formed that varied in diameter from 10 to 40 nm, showing that higher ordered structures were being formed. This demonstrates that IFPs can be successfully assembled in vitro to form filamentous structures that may be able to be manipulated for biomaterial uses.
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Handley, Kelly. "Statistical analysis of proteomic mass spectrometry data." Thesis, University of Nottingham, 2007. http://eprints.nottingham.ac.uk/10287/.
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This thesis considers the statistical modelling and analysis of proteomic mass spectrometry data. Proteomics is a relatively new field of study and tried and tested methods of analysis do not yet exist. Mass spectrometry output is high-dimensional and so we firstly develop an algorithm to identify peaks in the spectra in order to reduce the dimensionality of the datasets. We use the results along with a variety of classification methods to examine the classification of new spectra based on a training set. Another method to reduce the complexity of the problem is to fit a parametric model to the data. We model the data as a mixture of Gaussian peaks with parameters representing the peak locations, heights and variances, and apply a Bayesian Markov chain Monte Carlo (MCMC) algorithm to obtain their estimates. These resulting estimates are used to identify m/z values where differences are apparent between groups, where the m/z value of an ion is its mass divided by its charge. A multilevel modelling framework is also considered to incorporate the structure in the data and locations exhibiting differences are again obtained. We consider two mass spectrometry datasets in detail. The first consists of mass spectra from breast cancer cells which either have or have not been treated with the chemotherapeutic agent Taxol. The second consists of mass spectra from melanoma cells classified as stage I or stage IV using the TNM system. Using the MCMC and multilevel techniques described above we show that, in both datasets, small subsets of the available m/z values can be identified which exhibit significant differences in protein expression between groups. Also we see that good classification of new data can also be achieved using a small number of m/z values and that the classification rate does not fall greatly when compared with results from the complete spectra. For both datasets we compare our results with those in the literature which use other techniques on the same data. We conclude by discussing potential areas for further research.
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Henrich, Sebastian [Verfasser], and Bernd [Akademischer Betreuer] Fakler. "Proteomic analysis of the metabotropic glutamate receptors." Freiburg : Universität, 2017. http://d-nb.info/1191095053/34.
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Davies, Claire Rebecca. "Proteomic analysis of the mouse mammary gland." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426507.
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Suggett, Nigel Ross :. "A proteomic analysis of colorectal cancer progression." Thesis, Birmingham City University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479124.
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Spandidos, Athanasia. "Proteomic methods applied to renal cell carcinomas." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620561.
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Tan, Kit-Yee. "The characterisation of RKIP using proteomic approaches." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2569/.
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Proteomics have come a long way since the mid 1990s, with many life-scientists adopting proteomic approaches to understand gene function. Whilst genomic information itself provides an excellent foundation for biomedical research, the complexity of a human cannot be explained by the genomics approach alone. Proteins are the ultimate effector of cell function, thus, studying the dynamic proteome complement of a cell at any given time is a better reflection of the immediate cell environment and its subsequent disease states. Proteins exert their roles in a living organism by interacting with other proteins, so it is believed that a protein's interactions and its functions are related. The study of protein interactions for biological discovery is now routinely carried out in high-throughput formats. Protein microarrays, in particular protein-protein interaction arrays, are valuable tools in protein functional studies, facilitating the unbiased systematic screening for potential interactors. This hypothesis- generating approach is essential in this post-genomic era to bridge the gap between genomics and proteomics, increasing the efficiency of biological research. This PhD describes the functional characterisation of Raf Kinase Inhibitor Protein (RKIP) using protein microarrays for the first time. Whilst the exact physiological role of RKIP remains unclear, RKIP has been associated with an increasing number of diseases, particularly in cancer. This microarray study provided an insight into RKIP’s role in the cell, with the identification of a broad spectrum of interactors not previously associated with its current known function. Findings from this study supported RKIP’s role as a metastatic suppressor, and its function as a scaffold protein. Whilst the advantages of protein microarrays for functional studies are clear, a number of limitations remain. The limitations associated with the current detection strategies in protein microarrays were addressed, with the application of a novel quantum dot-based detection strategy for the detection of protein interactions on microarrays. The performance of quantum dots was comparable to current Alexa-based detection strategies. Biological validation of the novel interactors are routinely carried out by immunoprecipitations (IP) and western blots, which introduces a bottle neck in the high throughput workflow. This thesis reports the development of an integrated co-IP / SILAC (stable isotope labelling by amino acids in culture) approach to compliment the rate of discovery made possible by microarrays.
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Liu, Yiding. "Technologies for Proteomic and Genomic Biomarker Analysis." Cleveland State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1229461302.
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Giles, Robert J. "Proteomic Analysis of Myogenesis: Defining the Cytoskeletome." Youngstown State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1378915844.
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