Relevant bibliographies by topics / Proteomics research / Dissertations / Theses
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Dissertations / Theses on the topic 'Proteomics research'

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Published: 4 June 2021
Last updated: 30 January 2023

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1

Eriksson, Cecilia. "Affinity based proteomics research tools /." Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11184.

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2

Falk, Ronny. "Systems enabling antibody-mediated proteomics research." Doctoral thesis, Stockholm, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4025.

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3

Hoffman, Melissa. "Quantitative Proteomics to Support Translational Cancer Research." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7303.

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Altered signaling pathways, which are mediated by post-translational modifications and changes in protein expression levels, are key regulators of cancer initiation, progression, and therapeutic escape. Many aspects of cancer progression, including early carcinogenesis and immediate response to drug treatment, are beyond the scope of genetic profiling and non-invasive monitoring techniques. Global protein profiling of cancer cell line models, tumor tissues, and biofluids (e.g. serum or urine) using mass spectrometry-based proteomics produces novel biological insights, which support improved patient outcomes. Recent technological advances resulting in next-generation mass spectrometry instrumentation and improved bioinformatics workflows have led to unprecedented measurement reproducibility as well as increased depth and coverage of the human proteome. It is now possible to interrogate the cancer proteome with quantitative proteomics to identify prognostic cancer biomarkers, stratify patients for treatment, identify new therapeutic targets, and elucidate drug resistance mechanisms. There are, however, numerous challenges associated with protein measurements. Biological samples have a high level of complexity and wide dynamic range, which is even more pronounced in samples used for non-invasive disease monitoring, such as serum. Cancer biomarkers are generally found in low abundance compared to other serum proteins, particularly at early stages of disease where cancer detection would make the biggest impact on improving patient survival. Additionally, the large-scale datasets generally require bioinformatics expertise to produce useful biological insights. These difficulties converge to create obstacles for down-stream clinical translation. This dissertation research demonstrates how proteomics is applied to develop new resources and generate novel workflows to improve protein quantification in complex biosamples, which could improve translation of cancer research to benefit patient care. The studies described in this dissertation move from assessment of quantitative mass spectrometry platforms, to analytical assay development and validation, and ending with personalized biomarker development applied to patient samples. As an example, four different quantitative mass spectrometry acquisition platforms are explored and comparisons of their ability to quantify low abundance peptides in a complex background are explored. Lung cancers frequently have aberrant signaling resulting in increased kinase activity and targetable signaling hubs; kinase inhibitors have been successfully developed and implemented clinically. Therefore, changes in amounts of kinase peptides in the complex background of peptides from all ATP-utilizing enzymes in a lung cancer cell line model after kinase inhibitor treatment was selected as a model system. Traditional mass spectrometry platforms, data dependent acquisition and multiple reaction monitoring, are compared to the two newer methods, data independent acquisition and parallel reaction monitoring. Relative quantification is performed across the four methods and analytical performance as well as downstream applications, including drug target identification and elucidation of signaling changes. Liquid chromatography – multiple reaction monitoring (LC-MRM) was selected for development of multiplexed quantitative assays based on superior sensitivity and fast analysis times, allowing for larger peptide panels. Method comparison results also provide guidelines for quantitative proteomics platform selection for translational cancer researchers. Next, a multiplexed quantitative LC-MRM assay targeting a panel of 30 RAS signaling proteins was developed and described. Over 30% of all human cancers have a RAS mutation and these cancers are generally aggressive and limited treatment options, leading to poor patient prognosis. Many targeted inhibitors have successfully shut down RAS signaling, leading to tumor regression, however, acquired drug resistance is common. The multiplexed LC-MRM assays characterized and validated are a publically available resource for cancer researchers to interrogate the RAS signal transduction network. Feasibility has been demonstrated in cell line models in order to identify signaling changes that confer BRAF inhibitor resistance and biomarkers of sensitivity to treatment. This analytical LC-MRM panel could support meaningful development of new therapeutic options and identification of companion biomarkers, with the end goal of improving patient outcomes. Multiplexed LC-MRM assays developed for personalized disease biomarkers using an integrated multi-omics approach are described for Multiple Myeloma, an incurable malignancy with poor patient outcomes. This disease is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin, or M-protein. Clinical treatment decisions are based on multiple semi-quantitative assays that require manual evaluation. In the clinic, minimal residual disease quantification methods, including multi-parameter flow cytometry and immunohistochemistry, are applied to bone marrow aspirates, which is a highly invasive technique that does not provide a systemic evaluation of the disease. To address these issues, we hypothesized that unique variable region peptides could be identified and LC-MRM assays developed specific to each patient’s M-protein to improve specificity and sensitivity in non-invasive disease monitoring. A proteogenomics approach was used to design personalized assays for each patient to monitor their disease progression, which demonstrate improved specificity and up to a 500-fold increase in sensitivity compared to current clinical methods. Assays can be developed from marrow aspirates collected when the patient was at residual disease stage, which is useful if no sample with high disease burden is available. The patient-specific tests are also multiplexed with constant region peptide assays that monitor all immunoglobulin heavy and light chain classes, which could reduce analysis to a single test. In conclusion, highly sensitive patient-specific assays have been developed that could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy in the hopes of achieving a cure, or to intervene at an earlier time point in disease recurrence. This study also provides a blueprint for future development of personalized diagnostics, which could be applied to biomarkers of other cancer types. Overall, these studies demonstrate how quantitative proteomics can be used to support translational cancer research, from the impact of different mass spectrometry platforms on elucidating signaling changes and drug targets to the characterization of multiplexed LC-MRM assays applied to cell line models for translational research purposes and in patient serum samples optimized for clinical translation. We believe that mass spectrometry-based proteomics is poised to play a pivotal role in personalized diagnostics to support implementation of precision medicine, an effort that will improve the quality and efficiency of patient care.
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4

Gibson, Frank. "The development of standards for proteomics research and a proteomics investigation of diabetic adipocyte models." Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445541.

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5

Larsson, Karin. "Generation and characterization of antibodies for proteomics research." Doctoral thesis, Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11425.

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6

Pujari, Goutam. "Current and future trends in proteomics (SELDI-TOF) in clinical diagnosis and clinical research." Thesis, Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31972111.

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7

Muir, Matthew Stewart. "Proteomics of the ovine cataract." Diss., Lincoln University, 2008. http://hdl.handle.net/10182/792.

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The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.
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8

Spicka, Kevin James. "Design and synthesis of fluorescent dyes for use in proteomic research." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/spicka/SpickaK0808.pdf.

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Proteomics is a rapidly developing field requiring powerful new technology in order to be able to detect proteins at increasingly lower concentrations. To aid in the detection of proteins at lower concentrations, DIGE dyes, a family of spectrally resolved fluorescent dyes, are currently available to proteomic researchers for 2D gel analysis. However, the demands of protein detection dictate that dyes that are even more sensitive and versatile be created. The syntheses of highly sensitive, water soluble BODIPY fluorophore dyes are described. These dyes are proposed to have the necessary sensitivity to allow for detection of proteins in much lower concentrations, providing an improvement over current protein detection limits. The BODIPY dyes that have been synthesized are available in a variety of absorbances and emissions. While fluorescent dyes that are amine-reactive are the most popular covalently binding protein labeling markers being used in today's proteomic research, thiol-reactive fluorescent markers are gaining importance in proteomic research. Since thiol residues are less common in proteins compared to their amine counterparts, saturation labeling and quantification are more easily achieved. The syntheses of sensitive thiol- reactive fluorescent dyes are described. These syntheses allow for quick generation of thiol-reactive fluorescent markers to be used in proteomic research.
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9

Balluff, Benjamin. "MALDI imaging mass spectrometry in clinical proteomics research of gastric cancer tissues." Diss., lmu, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-155986.

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10

Zhang, Songya [Verfasser], Andreas [Akademischer Betreuer] Bechthold, and Irmgard [Akademischer Betreuer] Merfort. "Genomics, proteomics and secondary metabolites biosynthesis research on Streptomyces asterosporus DSM 41452." Freiburg : Universität, 2018. http://d-nb.info/115950511X/34.

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11

Pueblo, Hanna Elizabeth. "APPLICATIONS OF DYNAMIC ISOELECTRIC/ANISOTROPY BINDING LIGAND ASSAY FOR PROTEOMIC RESEARCH." OpenSIUC, 2012. https://opensiuc.lib.siu.edu/dissertations/494.

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The work presented in this dissertation centers around the development of analytical tools for the study of advanced proteomics. Section 1 of this work reviews the need for high efficiency protein separation techniques. Dynamic isoelectric focusing (DIEF) is new technique similar to capillary isoelectric focusing (CIEF) invented by Dr. Luke Tolley at Southern Illinois University Carbondale. Using DIEF, the electric field inside the separation capillary can be modified using high voltage electrodes, additional to the anode and cathode, to control the depth and shape of the resulting pH gradient. By changing the pH gradient, the location and width of focused protein bands can be controlled. As a new analytical technique, the development of DIEF required the design and fabrication of special holders which allow for electrical connections to be made at lengths along the separation capillary. These holders were also designed to have a removable section of capillary to extract very specific pH range proteins from high-resolution separations. Higher throughput DIEF systems were investigated, as well as multiplexed DIEF systems. Section 2 covers the topic of dynamic isoelectric/anisotropy ligand binding assay (DIABLA). DIABLA is a new method used to identify proteins in a complex sample that bind to a known molecule. DIABLA has the potential to be used in two complimentary ways, discovery mode and scanning mode. Both modes are accomplished by using DIEF, followed by fluorescence anisotropy as a sensitive detection method. This allows the entire length of capillary to be scanned to identify areas of non-zero anisotropy, which indicate binding interactions between the protein and target molecule. The binding protein(s) can then be extracted using the removable section of capillary from the DIEF holder, and can be identified by using a second dimension analysis, such as LC/MS/MS. DIABLA was verified in a series of proof-of-concept experiments in both discovery and scanning modes. These experiments involved fluorescently tagging proteins that were focused in the presence of a ligand tagged with a different fluorophore. The usefulness of DIABLA as a separation technique was demonstrated in four specific analyses of complex protein samples in Chapter 10.
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Williams, Taufika Ialam. "Methods development in biological mass spectrometry applications in small molecule research and proteomics /." Lexington, Ky. : [University of Kentucky Libraries], 2005. http://lib.uky.edu/ETD/ukychem2005d00368/tiwdis.pdf.

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Thesis (Ph. D.)--University of Kentucky, 2005.
Title from document title page (viewed on January 19, 2006). Document formatted into pages; contains: xiv, 247 p. :ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 224-244).
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Williams, Taufika Islam. "METHODS DEVELOPMENT IN BIOLOGICAL MASS SPECTROMETRY: APPLICATIONS IN SMALL MOLECULE RESEARCH AND PROTEOMICS." UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/288.

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Technological developments have enabled mass spectrometry (MS) to evolve asone of the most versatile, sensitive and widely used analytical methods. Key areas ofresearch in biological MS include the development of analyte-selective MSmethodologies, along with the design of MS compatible separation technology. Analytesof interest range from small, biologically active molecules in disease progressionresearch, to macromolecules such as proteins, in proteomics investigations. Advances inthese areas are vital to maintaining the level of sophistication that has become thebenchmark for MS analyses.Mass spectrometry has found a permanent station in disease progression studies,particularly in biomarker discovery. This is especially true for Alzheimer's disease (AD),a condition marked by widespread lipid peroxidation (LPO) in the brain. The mainhypothesis of the first part of this dissertation is that LPO produces aldehydes that canpotentially be exploited as AD biomarkers. Design of novel LC-MS/MS methods forbrain aldehyde analysis is described. The methods were applied towards aldehydequantification in the hippocampus, superior and middle temporal gyrus and cerebellum ofsubjects with early AD (EAD), mild cognitive impairment (MCI) and age-matchedcontrols. Results obtained indicated elevation of neurotoxic aldehydes in MCI and EADbrain and suggested that LPO occurred early in AD. Understanding AD progression hasbecome important for developing diagnostic methods and treatments.Mass spectrometry is also the major analytical tool in proteomics, where gelelectrophoresis is dominant in pre-MS separations. The main hypothesis of the latter partof this dissertation is that exposure of microbe fermenters including Clostridiumthermocellum to an external stimulus, such as ethanol, can alter the membrane proteome.Design of novel doubled-SDS-PAGE (dSDS-PAGE) methods for membrane proteinanalysis is described, as these proteins are under-represented in standard 2D-PAGE. Thenewly developed Bicine-dSDS-PAGE offered superior separation over other methods andwas applied towards analysis of wild type and ethanol-adapted C. thermocellum cellmembranes. Significant differences in protein expression were observed. Anunderstanding of ethanol adaptation will promote the design of more ethanol-tolerantstrains. Such an outcome can have dramatic effects in the fuel industry as the trendtowards more efficient fuel development gathers momentum.
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Bateson, Hannah. "Detection and enrichment of cytochrome P450s using bespoke affinity chromatography and proteomic techniques : development of chemical immobilisation and novel affinity chromatography methods, with subsequent proteomic analysis, for the characterisation of cytochrome P450s important in cancer research." Thesis, University of Bradford, 2012. http://hdl.handle.net/10454/5712.

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Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.
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Amelina, Hanna. "Proteomics in biomarker research : Insights into the effects of aging and environment on biological systems." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-56483.

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Proteomics is the global analysis of proteins that covers a broad range of technologies aimed at determining the identity and quantity of proteins expressed in the cell, their three-dimensional structure and interaction partners. In contrast to genome, proteome reflects more accurately on the dynamic state of the cell, tissue, or an organism. Therefore much is expected from proteomics to yield better disease markers for early diagnosis and therapy monitoring, as well as biomarkers that would indicate environmental exposure or provide prediction of biological age. In this thesis, I have developed and applied robust and sensitive subproteomic approaches to study the effect of aging as well as and environmental pollution using different animal models. In the first part, a high-throughput proteomic method based on liquid chromatography coupled to 2-dimensional gel electrophoresis (LC/2-DE) was developed. The usefulness of this method has been demonstrated by applying it to the assessment of marine pollution in a field experiment. Next, I have utilized this subproteomic approach to study the effect of aging in mouse kidney of both genders. As a result, a protein expression signature of aging kidney was obtained, revealing gender-dependent alterations in proteome profiles of aging mouse kidney. In order to further reduce the dynamic range of protein expression and increase the sensitivity of proteomic analysis, I have applied a shotgun mass spectrometry-based proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to study age-related differences in peroxisome-enriched fractions from mouse liver. Only eight proteins showed statistically significant difference in expression (p<0.05) with moderate folds. This study indicates that age-depended changes in the liver proteome are minimal, suggesting that its proteome is efficiently maintained until certain age. Finally, in the context of aging studies and the role of peroxisomes in aging, I have tested the utility of cell-penetrating peptides (CPPs) as agents for protein delivery into acatalasemic peroxisomes using yeast as a model. The results obtained suggest that CPPs may be suitable for the delivery of antioxidants to peroxisomes and in future could provide a tool for the protein therapy of age-related diseases.
At the time of the doctoral defense, the following publications were unpublished and had a status as follows: Paper 3: Submitted, Paper 4: Submitted.
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16

Zhou, Cong. "Machine Learning Based Protein Identitification and Partial Granger Causality : Novel Bioinformatics Approaches for Proteomics Research." Thesis, University of Sussex, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505888.

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17

Potier, David N. "The development of mass spectrometry based approaches to monitor protease activity in biological fluids." Thesis, University of Manchester, 2012. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:185527.

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When treating patients with cancer, the ability to predict a patient’s response to treatment is an important tool to allow therapy to be tailored for best outcome. Therefore, the need exists for a test to forecast a patient’s response using a sample that is readily accessible, and provides an accurate reflection of a patient’s response to a disease or treatment. Profiling biological fluids, such as plasma or urine, has gained considerable interest in recent years. This is because these fluids are readily available and are expected to provide an accurate representation of a patient’s response to treatment. As such, much effort has been put into finding biomarkers or prognostic indicators. Abnormal protease activity has been linked to the progression of cancer due to their involvement in several processes vital to the survival and proliferation of the disease. These include metastasis, resistance to apoptosis and angiogenesis, amongst others. In addition, dysregulated protease activity has been linked to poor response to chemotherapy as well as tumour regrowth following radiotherapy. Therefore, an increased understanding regarding the activity of a patient’s proteases may provide the clinician with more information as to how best to treat the patient. Therefore, monitoring protease activity has been suggested as a potential marker to predict a patient’s response to cancer treatment. Most enzyme activity assays are currently performed by fluorescence spectroscopy. However, these workflows suffer from limited sensitivity and linear range. Therefore, an alternative, more sensitive assay is required. Mass spectrometry (MS) is a highly sensitive analytical technique routinely used to quantify changes in biological systems. As such, MS has the potential to be used in enzyme activity assays. This study illustrates the development of a novel MS based method to monitor the activity of target enzymes in plasma; specifically asparaginyl endopeptidase (AEP) and caspase-3 using mass spectrometry. These enzymes have been linked to poor chemotherapeutic response in childhood leukaemia and tumour regrowth post-radiotherapy respectively. This project will describe the development and optimisation of each stage of a five step sample preparation and analysis method. This includes the design of enzyme substrates designed to be cleaved by the target enzyme, whilst reducing the effect of other enzymes acting on this substrate, how best to enrich samples for these target peptides, as well as determining the best MS technique to monitor these peptides. In addition, this project describes a comparison between this assay and an existing fluorescence assay when monitoring AEP activity in biological samples such as plasma and whole cell lysates. The application of this method in quantifying caspase-3 activity in plasma samples is also examined.
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Balluff, Benjamin [Verfasser], and Horst [Akademischer Betreuer] Zitzelsberger. "MALDI imaging mass spectrometry in clinical proteomics research of gastric cancer tissues / Benjamin Balluff. Betreuer: Horst Zitzelsberger." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/103375210X/34.

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19

Wang, Linan. "Proteomic Based Approaches for Differentiating Tumor Subtypes." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1482248318956052.

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20

Abbaoui, Besma. "BROCCOLI ISOTHIOCYANATES AS CHEMOPREVENTIVE AGENTS AND EPIGENETIC MODULATORS OF BLADDER CANCER." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1312835540.

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Hagerty, James Robert. "Developmental Regulation of Translation in Parasitic Flatworms." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1623424469091568.

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22

Speirs, Monique Merilyn. "Development and Use of Lipidomics and Proteomics Methods to Identify and Measure Pro-Survival Metabolic Pathways in Cancer." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7601.

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Throughout society’s continual war against cancer, we have attempted pharmacological intervention only to find that tumors develop modes of resistance. It is well known that genetics play an integral role in cancer. Technological advances have greatly improved our ability to study cancer biochemistry beyond the genome by measuring changes in the expression and activity of RNA, proteins, and lipids in experimental models and human patients. As our techniques and technology to perform cancer research progresses, it is becoming more evident that cancer cells develop stress tolerance mechanisms at multiple levels within the central dogma, including altering mRNA expression, enzyme concentrations, and functional activity of cellular proteins and lipids. In the first chapter, I review previous discoveries demonstrating the importance of metabolic reprogramming in cancer cells and how shifts in metabolic pathways contribute to cancer progression and therapeutic challenges. I discuss how mass spectrometry is a multifunctional research tool that can be used to identify global shifts in gene expression, identify oncogenic roles of specific metabolites and corresponding metabolic pathways, conduct enzyme activity assays, and understand the effects of drugs on cell signaling and metabolic flux through specific pathways. While metabolic reprogramming is a complex and multifaceted concept, the following chapters focus on two specific stress tolerance pathways of lipid and protein metabolism we have shown to significantly promote cancer cell evolution, proliferation, and drug resistance in models of human pancreatic and colon cancer. I describe novel mass spectrometry-based lipidomics and proteomics methods we developed to measure and determine the biological impact of these pathways in each model. I discuss the contributions we have made toward increasing general knowledge of metabolic reprogramming networks in cancer and how they may be targeted in more specific and effective manners to sensitize cancers to therapeutic drugs. Specifically, the second chapter entails our study of a pro-survival lipid metabolic pathway driven by the sphingolipid modifying enzyme sphingosine kinase in a panel of differentially reprogrammed pancreatic cancer subclones. The third chapter describes our novel kinetic proteomics approach to identify how the cellular degradation system autophagy is used to selectively remodel the proteome of colon tumor cells in a xenograft mouse model of colon cancer. Lastly, I discuss how these and other projects completed during my graduate work lay a foundation for ongoing research to further our fundamental understanding of cancer metabolism and treatment development.
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Maltman, Daniel James. "Characterization of endoplasmic reticulum from castor bean and the cloning of a plant phosphatase : a basis for comprehensive plant organelle proteomics research." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4958/.

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The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum.
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Harshman, Sean William. "Characterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385544818.

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Swensen, Adam Clayton. "Investigation of Dynamic Biological Systems Using Direct Injection and Liquid Chromatography Mass Spectrometry." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6574.

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In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.
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26

Nibbe, Rod K. "Systems Biology of Human Colorectal Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1264179836.

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Komar, Hannah Marie Komar. "Identifying pathogenic stromal and acinar signaling for improved diagnosis and treatment of chronic pancreatitis." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1509529255511222.

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28

Wagner, Gregory Randall. "Identification and characterization of altered mitochondrial protein acetylation in Friedreich's ataxia cardiomyopathy." Thesis, Hindawi Publishing Corporation and Oxford Journals and SAGE Journals, 2011. http://hdl.handle.net/1805/4209.

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Indiana University-Purdue University Indianapolis (IUPUI)
Friedreich’s Ataxia (FRDA) is a rare and poorly understood autosomal recessive disease caused by a pathological deficiency of the mitochondrial protein frataxin. Patients suffer neurodegeneration, ataxia, diabetes, and heart failure. In an effort to understand the mechanisms of heart failure in FRDA, we investigated the role of the protein modification acetylation, which is highly abundant on mitochondrial proteins and has been implicated in regulating intermediary metabolism. Using mouse models of FRDA, we found that cardiac frataxin deficiency causes progressive hyperacetylation of mitochondrial proteins which is correlated with loss of respiratory chain subunits and an altered mitochondrial redox state. Mitochondrial protein hyperacetylation could be reversed by the mitochondria-localized deacetylase SIRT3 in vitro, suggesting a defect in endogenous SIRT3 activity. Consistently, frataxin-deficient cardiac mitochondria showed significantly decreased rates of fatty acid oxidation and complete oxidation to carbon dioxide. However, the degree of protein hyperacetylation in FRDA could not be fully explained by SIRT3 loss. Our data suggested that intermediary metabolites and perhaps acetyl-CoA, which is required for protein acetylation, are accumulating in frataxin-deficient mitochondria. Upon testing the hypothesis that mitochondrial protein acetylation is non-enzymatic, we found that the minimal chemical conditions of the mitochondrial matrix are sufficient to cause widespread non-enzymatic protein acetylation in vitro. These data suggest that mitochondrial protein hyperacetylation in FRDA cardiomyopathy mediates progressive post-translational suppression of mitochondrial oxidative pathways which is caused by a combination of SIRT3 deficiency and, likely, an accumulation of unoxidized acetyl-CoA capable of initiating non-enzymatic protein acetylation. These findings provide novel insight into the mechanisms underlying a poorly understood and fatal cardiomyopathy and highlight a fundamental biochemical mechanism that had been previously overlooked in biological systems.
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Leary, Dagmar Hajkova. "CIRCADIAN PROTEOME CHANGES IN PHOTORECEPTOR OUTER SEGMENTS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1264276011.

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30

Daubenspeck, April Arnold. "Proteomic Analysis of Ischemic Stroke Blood Biomarkers." Wright State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=wright1515748115902114.

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31

Head, Talia B. "Proteomic Analysis of the Crustacean Molting Gland (Y-Organ) Over the Course of the Molt Cycle." DigitalCommons@CalPoly, 2017. https://digitalcommons.calpoly.edu/theses/1830.

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Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of the molting hormones, ecdysteroids, by the YO. Analysis of gene expression in the XOs and YOs has led to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting in crustaceans. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle (intermolt, early premolt, mid premolt, and late premolt) induced by multiple leg autotomy (MLA) in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected in the molting gland using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle (one-way permutation ANOVA, p≤0.05). Changes in protein abundance were most notable between the intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch in the Y-organ. Several hemolymph species proteins, including hemocyanin, cryptocyanin, and transglutaminase, were identified which characterized physiological changes associated with molting beyond the Y-organ. An abundance of cytoskeletal proteins were identified which correspond with glandular hypertrophy and are indicative of vesicular-mediated exocytosis, possibly of ecdysteroids. Further, several proteins involved in the immune, proteostasis, and oxidative stress response are characteristic of supporting the dynamic and demanding cellular changes associated with ecdysteroidogenesis and the transition of the Y-organ from the basal to the highly active state. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one-carbon metabolism changed in abundance in response to both the higher energy demands and the requirement for precursors of macromolecular synthesis of the YO over the molt cycle. Taken together, these changes in diverse physiological pathways represent the complexity involved with regulation of the Y-organ, even with just the single proposed physiological purpose of ecdysteroidogenesis.
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32

Michaels, Todd Richard. "Advancements in hardware design for proteomic research /." Available to subscribers only, 2007. http://proquest.umi.com/pqdweb?did=1456289281&sid=12&Fmt=2&clientId=1509&RQT=309&VName=PQD.

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33

Hoover, Michael. "Proteomic Characterization of Linker Histone Variants in Breast Cancer." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1497604765734645.

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34

Cho, Chi Shing. "Proteomic and medicinal approaches to diabetes and its complications." HKBU Institutional Repository, 2006. http://repository.hkbu.edu.hk/etd_ra/663.

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35

Hitron, John Andrew. "AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY." UKnowledge, 2018. https://uknowledge.uky.edu/toxicology_etds/22.

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The reversible oxidation of cysteine by reactive oxygen species (ROS) is both a mechanism for cellular protein signaling as well as a cause of cellular injury and death through the generation of oxidative stress. The study of cysteine oxidation is complicated by the methodology currently available to isolate and enrich oxidized-cysteine containing proteins. We sought to simplify this process by reducing the time needed to process samples and reducing sample loss and contamination risk. We accomplished this by eliminating precipitation steps needed for the protocol by (a) introducing an in-solution NEM-quenching step prior to reduction and (b) replacing soluble dithiothreitol reductant with a series of newly-developed high-capacity polyacrylamide-based solid-phase reductants that could be easily separated from the lysate through centrifugation. These modifications, collectively called resin-assisted reduction and capture (RARC), reduced the time needed to perform the RAC method from 2-3 days to 4-5 hours, while the overall quality and quantity of previously-oxidized cysteines captured was increased. In order to demonstrate the RARC method’s utility in studying complex cellular oxidants, the optimized methodology was used to study cysteine oxidation caused by the redox-active metals arsenic, cadmium, and chromium. As(III), Cr(VI), and Cd(II) were all found to increase cysteine oxidation significantly, with As(III) and Cd(II) inducing more oxidation than Cr(VI) following a 24-hour exposure to cytotoxic concentrations. Label-free proteomic analysis and western blotting of RARC-isolated oxidized proteins found a high degree of commonality between the proteins oxidized by these metals, with cytoskeletal, translational, stress response, and metabolic proteins all being oxidized. Several previously-unreported redox-active cysteines were also identified. These results indicate that cysteine oxidation by As(III), Cr(VI), and Cd(II) may play a significant role in these metals’ cytotoxicity and demonstrates the utility of the RARC method as a strategy for studying reversible cysteine oxidation by oxidants in oxidative signaling and disease. The RARC method is a simplification and improvement upon the current state of the art which decreases the barrier of entry to studying cysteine oxidation, allowing more researchers to study this modification. We predict that the RARC methodology will be critical in expanding our understanding of reactive cysteines in cellular function and disease.
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36

Bigi, Mila <1985&gt. "Genomic and proteomic approaches in pig meat quality research field." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6606/.

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Pig meat and carcass quality is a complex concept determined by environmental and genetic factors concurring to the phenotypic variation in qualitative characteristics of meat (fat content, tenderness, juiciness, flavor,etc). This thesis shows the results of different investigations to study and to analyze pig meat and carcass quality focusing mainly on genomic; moreover proteomic approach has been also used. The aim was to analyze data from association studies between genes considered as candidate and meat and carcass quality in different pig breeds. The approach was used to detect new SNP in genes functionally associated to the studied traits and to confirm as candidate other genes already known. Five polymorphisms (one new SNP in Calponin 1 gene and four additional polymorphism already known in other genes) were considered on chromosome 2 (SSC2). Calponin 1 (CNN1) was associated to the studied traits and furthermore the results reported confirmed the data already known for Lactate dehydrogenase A (LDHA), Low density lipoprotein receptor (LDLR), Myogenic differentiation 1 (MYOD1) e Ubiquitin-like 5 (UBL5), in Italian Large White pigs. Using an in silico search it was possible to detect on SSC2 a new SNP of Deoxyhypusine synthase (DHPS) gene partially overlapping with WD repeat domain 83 (WDR83) gene and significant for the meat pH variation in Italian Large White (ILW) pigs. Perilipin 1 (PLIN1) mapping on chromosome 7 and Perilipin 2 (PLIN2) mapping on chromosome 1 were studied and the results obtained in Duroc breed have shown significant associations with carcass traits. Moreover a study of protein composition of porcine LD muscle, indicated an effect of temperature treatment of carcass, on proteins of the sarcoplasmic fraction and in particular on PGM1 phosphorylation. Future studies on pig meat quality should be based on the integration of different experimental approaches (genomics, proteomics, transcriptomics, etc).
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37

Jones, Andrew. "The development of data standards and a database to aid proteomic research." Thesis, University of Glasgow, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418904.

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38

Mu, Huawei. "Mechanisms of species invasion in apple snails: proteome of the egg perivitelline fluid, and proteomic responses of the adults to abiotic stressors." HKBU Institutional Repository, 2016. https://repository.hkbu.edu.hk/etd_oa/329.

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The Pomacea apple snails are amphibious operculate freshwater gastropods. They exhibit different stress tolerant abilities as some of them are invasive (e.g., Pomacea maculata and Pomacea canaliculata) and others are non-invasive species (e.g., Pomacea diffusa). By using a comparative approach, I have profiled the perivitelline fluid of P. maculata, compared the physiological and proteomic responses of P. canaliculata and P. diffusa to thermal and hypoxic stressors, and examined their sequence divergences. 74 proteins were identified from the PVF of P. maculata. Comparison of gene expression levels between albumen gland (the organ that secrets PVF) and other organs showed that 24 PVF genes were specifically expressed in albumen gland. Base substitution analysis of PVF and housekeeping orthologs between P. maculata and its congener P. canaliculata showed that these reproductive genes have higher evolutionary rate. The LT50 of P. canaliculata was significantly higher than that of P. diffusa. More than 3,350 proteins were identified from the hepatopancreas of the snails exposed to acute and chronic thermal stress using iTRAQ-coupled mass spectrometry. Chronic exposure caused differential expression of more proteins, with many of them related to restoration of damaged molecules, ubiquitinating dysfunctional molecules, and utilization of energy reserves in both species; but only in P. diffusa there was a shift from carbohydrate to lipid catabolism. Analysis of orthologous genes encoding the differentially expressed proteins revealed nine candidate genes may have undergone positive selection (Ka/Ks > 0.5). A 72 h mortality experiment showed that P. canaliculata is much more tolerant to hypoxia than P. diffusa. The two species were then exposed to four levels of dissolved oxygen (6.7, 4.1, 2.0 and 1.0 mg O2 L-1) for 8 h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. Compared with P. diffusa, P. canaliculata had more up-regulated signal transduction proteins and down-regulated proteins which are involved in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis indicated that selection has acted on some of the hypoxia responsive genes. The comparative method and results provide a framework for studying the genetic basis of species invasion and predicting their further expansion in a changing climate for non-model species.
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39

Basu, Amrita. "Spatial Learning and Memory, Transcriptional and Proteomic Analysis of Growth Hormone Action in the Brain of bGH and GHA Mice." Ohio University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1440073471.

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40

Rivera, Reyes Reginaldo [Verfasser], Andreas [Akademischer Betreuer] Kispert, Thomas [Akademischer Betreuer] Thum, and Ina [Akademischer Betreuer] Gruh. "Two complementary proteomic analyses uncover new regulators of TBX18 transcriptional function / Reginaldo Rivera Reyes ; Akademische Betreuer: Andreas Kispert, Thomas Thum, Ina Gruh ; Hannover Biomedical Research School, Institut für Molekularbiologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2020. http://d-nb.info/1212872134/34.

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41

Martín, Pérez Miguel. "Physiological changes in the muscle of gilthead sea bream induced by culture and feeding conditions: a stable isotopes (15(N) and 13(C)) and proteomic study Cambios fisiológicos en el músculo de dorada inducidos por las condiciones de cultivo y alimentación: Estudio con isótopos estables (15(N) y 13(C)) y proteómica." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/104148.

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Stable isotopes, as tracers of the food chain, have been used to characterize the relationship between consumers and their food since isotopic fractionation implies discrimination against certain isotopes. But stable isotope analyses (SIA) can also be carried out in fish reared with artificial diets, as gilthead sea bream (Sparus aurata), the species most cultivated in the Mediterranean. Changes in natural abundance of stable isotopes (13C and 15N) in tissues and its reserves may reflect changes in the use and turnover of nutrients as the catabolic enzymes involved in the processes of decarboxylation and deamination show a preference for the lighter isotopes. Therefore these analyses can provide us useful information about the nutritional and metabolic status of fish. The aim of this project was to determine the capacity of stable isotopes to be used as potential markers of growth capacity and rearing conditions in sea bream. In this sense, stable isotope analyses have been combined with other metabolic (cytochrome-c-oxidase, COX, and citrate synthase, CS, activities) and growth parameters (RNA, DNA). The set of results obtained from the different studies conducted in this project shows that SIA, in combination with other metabolic parameters, can serve as an effective tool in discriminating fish with better growth potential as well as sensitive marker of nutritional status and fattening. Moreover, combination of stable isotopes analysis with emerging tools such as proteomic techniques (2D-PAGE), provide new insights on the metabolic changes occurring in fish muscles during exercise induced growth.
Els isòtops estables com a traçadors de la cadena alimentària s'han utilitzat per caracteritzar la relació entre els consumidors i els seus aliments, ja que el fraccionament isotòpic implica una discriminació en contra de certs isòtops. Però les anàlisis d'isòtops estables (SIA), també es poden dur a terme en peixos cultivats amb dietes artificials, com la orada (Sparus aurata), la especie més cultivada a la Mediterrània. Canvis en l'abundància natural d'isòtops estables (13C i 15N) en els teixits i les seves reserves poden reflectir els canvis en l'ús i reciclatge dels nutrients ja que els enzims catabòlics implicats en els processos de descarboxilació i desaminació mostren una preferència pels isòtops més lleugers. Per tant, aquestes anàlisis ens poden proporcionar informació útil sobre l'estat nutricional i metabòlic dels peixos. L'objectiu d'aquest projecte va ser determinar la capacitat dels isòtops estables per ser utilitzats com a marcadors potencials de la capacitat de creixement i condicions de cria de l'orada. En aquest sentit, les anàlisis d'isòtops estables s'han combinat amb altres metabòlics (activitats citocrom-c-oxidasa, COX, i citrat sintasa, CS) i els paràmetres de creixement (ARN/ADN). El conjunt de resultats obtinguts en els diferents estudis realitzats en aquest projecte demostra que el SIA, en combinació amb altres paràmetres metabòlics, pot servir com una eina eficaç per discriminar els peixos amb millor potencial de creixement, així com a marcador sensible de l'estat nutricional i d'engreix. D'altra banda, la combinació de l'anàlisi d'isòtops estables amb les eines emergents, com ara tècniques de proteòmica (2D-PAGE), ens proporciona nous coneixements sobre els canvis metabòlics que ocorren en els músculs dels peixos durant l’increment del creixement muscular induït per l'exercici.
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42

You-Chia, Yeh, and 葉又嘉. "The Proteomics Research of Colorectal Cancer." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/22341619152128110348.

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碩士
國立臺灣大學
生化科學研究所
91
In attempt to have better understanding of possible mechanism causing colon cancer and identify disease-specific proteins for early screening, we adopted 2-D electrophoresis in conjunction with Mass spectrum techniques to approach this issue. We focused the research mainly on the characterization of different spot patterns between normal and cancerous tissue from patients with colon cancer in Taiwan. The results obtained from analyzing cancerous tissues of colon indicates that six proteins with increased expression level are Hsp60, S100A11, S109A9, tropomyosin, mutant β-globin, acyl-CoA binding protein and four proteins with declined level are fatty acid-binding protein, Hsp27, apolipoprotein A1,α1-antitrypsin. Among these significant spots, three proteins worthy of further investigation are: (1) acyl-CoA binding protein: related with energy consume and metabolism of colon cancerous cells (2) α1-antitrypsin: the different chemical properties among isoforms (3) S100A11: with post-translational modification.
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43

Atkinson, Kelly Rene LeFevre. "Proteomic biomarker discovery for preeclampsia." 2008. http://hdl.handle.net/2292/2565.

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Preeclampsia is a serious multisystem complication of late pregnancy with adverse effects for mothers and babies. Currently this disorder is diagnosed from clinical observations occurring late in the disease process. Unknown factors in the maternal circulation, possibly released by the preeclamptic placenta, have been linked to the pathophysiological changes characteristic of the disorder. The research in this thesis used proteomic techniques to identify putative preeclampsia biomarkers from two sources: secreted from a placental cell line undergoing differentiation, and directly sampled from the serum and plasma of women with late-onset preeclampsia. The first part of this research examined the secreted proteome of a placental choriocarcinoma cell line (BeWo) undergoing forskolin-mediated differentiation. Development of serum-free culture techniques enabled analysis of these secreted proteins by two-dimensional gel electrophoresis (2DE). Statistical testing revealed the significant involvement of seven spots during this differentiation model, with VE-cadherin and matrix metalloproteinase 2 among the proteins identified. In the second part of this research, maternal serum and plasma proteins were compared from women with preeclampsia and healthy pregnant women. Serum samples were analyzed using 2DE, and plasma was subjected to difference gel electrophoresis (DIGE). Bioinformatic analysis of both datasets identified multiple spot clusters able to classify samples according to disease state. Five of these serum proteins were differentially regulated in preeclampsia, including two isoforms of apolipoprotein E whose isoform-specific expression was confirmed using western blots. Analysis of plasma from preeclamptic women identified six proteins, again including apolipoprotein E. Proteins from both studies are linked to preeclampsia pathophysiology through lipid transport, complement, and retinol transport systems. The culture methods and secreted proteomic techniques developed in this work have uncovered proteins in a placental cell line and maternal serum and plasma that are associated with preeclampsia. These methods can be extended to any system where secreted proteins are of interest. The differentially regulated proteins found in this study provide an important first step towards developing effective biomarkers for diagnosing and/or predicting preeclampsia.
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44

Sahab, Ziad Joseph Sang Qing-Xiang Amy. "Batch anion exchange separation a prefractionation technique for proteome research and its applications on in vivo cancer samples /." Diss., 2005. http://etd.lib.fsu.edu/theses/available/etd-11102005-151301.

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Thesis (Ph. D.)--Florida State University, 2005.
Advisor: Qing-Xiang A. Sang, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed May 11, 2006). Document formatted into pages; contains xvii, 118 pages. Includes bibliographical references.
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45

Alakhras, Nada S. "A method to isolate the CTD of RNA Polymerase II for proteomics analysis." Thesis, 2014. http://hdl.handle.net/1805/6179.

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Indiana University-Purdue University Indianapolis (IUPUI)
In an effort to advance the methodology in analyzing RNAPII protein-protein interaction network and to determine the role of the CTD in controlling RNAPII transcription, we devised a method to specifically isolate the CTD-associated and CTD-less RNAPII to identify the proteins that interact with both the CTD and the globular core of RNAPII using novel purification scheme coupled to quantitative proteomics.
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46

(5930141), Minervo Perez. "HIGH-THROUGHPUT IDENTIFICATION OF ONCOGENIC TYROSINE KINASE SUBSTRATE PREFERENCES TO IMPROVE METHODS OF DETECTION." Thesis, 2021.

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The use of computational approaches to understand kinase substrate preference has been a powerful tool in the search to develop artificial peptide probes to monitor kinase activity, however, most of these efforts focus on a small portion of the human kinome. The use of high throughput techniques to identify known kinase substrates plays an important role in development of sensitive protein kinase activity assays.
The KINATEST-ID pipeline is an example of a computational tool that uses known kinase substrate sequence information to identify kinase substrate preference. This approach was used to design three artificial substrates for ABL, JAK2 and SRC family kinases. These biosensors were used to design ELISA and lanthanide-based assays to monitor in vitro kinase activity. The KINATEST-ID pipeline relies on a high number of reported kinase substrates to predict artificial substrate sequences, however, not all kinases have the sufficient number of known substrates to make an accurate prediction.
The adaptation of kinase assay linked with phosphoproteomics technique was used to increase the number of known FLT3 kinase variant substrate sequences. Subsequently, a set of data formatting tools were developed to curate the mass spectrometry data to become compatible with a command line version of the KINATEST-ID pipeline modules. This approach was used to design seven pan-FLT3 artificial substrate (FAStides) sequences. The pair of FAStides that were deemed the most sensitive toward FLT3 kinase phosphorylation were assayed in increasing concentrations of clinically relevant tyrosine kinase inhibitors.
To improve the automation of the mass spectrometry data analysis and formatting for use with the KINATEST-ID pipeline, a streamlined process was developed within a bioinformatic platform, GalaxyP. The data formatting tools used to process the FLT3 mass spectrometry data were converted into compatible versions to execute within the GalaxyP framework. This process was used to design four BTK artificial substrates (BAStide) to monitor kinase activity. Additionally, one of the BAStide sequences was designed in the lanthanide chelating motif to develop an antibody-free activity assay for BTK.
Lastly, a multicolored time resolved lanthanide assay was designed by labeling SYK artificial substrate and a SRC family artificial substrate to measure the activity of both kinases in the same kinase reaction. This highlighted the functionality of lanthanide-based time resolved assays for potential multiplexing assay development.

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47

Tsai, Chou-Huang, and 蔡宙晃. "Studies of antilithic effects of Wulingsan on calcium oxalate stones – an experimental induced-nephrocalcinosis rat model and proteomics research." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/94010800970695926609.

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博士
中國醫藥大學
中國醫學研究所博士班
97
Urolithiasis is a common disease in Taiwan, and the medical expenditure is quite huge. The purposes of this study are to explore the mechanism of nephrocalcinosis using molecular technologies and to evaluate the antilithic abilities of a Traditional Chinese herbal formula, Wulingsan (WLS), by an ethylene glycol-induced nephrocalcinosis rat model. Forty one male Sprague-Dawley rats were divided into four groups. Eight rats were fed with normal chaw and water servered as the normal control, and the others received 0.75% ethylene glycol-added water as a stone inducer. The placebo group (11 rats) received a daily gastric gavage of starch, the low-dose group (11 rats) received WLS (375mg/kg), and the high-dose group (11 rats) received WLS (1125mg/kg) in stead. Four weeks later, animals were killed. The kidneys had harvested and the changes of body weight were measured. The biochemical data of urine and serum were obtained and analyzed. The kidney specimens were examined by a polarized light microscopy and the pictures were taken by a digital camera. The severity of crystal deposits in rat kidneys were evaluated by a semi-quantitative scoring method assisted with a self-composed picture browsing software named ImageScoring. Using this software, pictures were randomly and anonymously displayed allowing the investigators makeing scores. Six investigators had employed to accomplish their scoring works independently; finally those data were collected and analyzed. Representative samples of rat kidneys were selected from each group. Isoelectric focusing gel electrophorisis (IEF) and Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to separate the protein extracts. The PDQuest software was applied for gel comparisons. The interest proteins were digested with trypsin and sent to mass spectrometry (MALDI-TOF) for peptide analysis. The peptid fragments were macthed by MASCOT web interface from several sequence databases. Our results revealed that the rats of placebo group gained the least body weights; on the contrary, the rats of WLS-fed groups could normally grow just the same as control group did. The placebo group exhibited lower levels of urine-free calcium (p=0.059) and significantly lower serum phosphorus (p=0.015) than WLS-fed rats. Histological findings of kidneys revealed tubular destruction, damage and inflammatory reactions in the EG-water rats. The crystal deposit scores dropped significantly in the WLS groups, from 1.40 to 0.46 in the low-dose group and from 1.40 to 0.45 in the high-dose group. Overall, WLS effectively inhibited the deposition of calcium oxalate crystal and lowered the incidence of stones (p=0.034, Kruskal-Wallis test). The serum levels of calclium were no difference in four groups. Bu the free calcium level in the WLS-fed groups(group 3 and 4) were significantly lower than the placedo group, the low-dose group is 0.91±0.07 mmol/L and the high-dose group is 0.94±0.17 mmol/L, compared with the placebo group is 1.10 ± 0.06 mmol/L (p=0.001). The serum levels of phosphorus of placebo group is 9.45 ± 2.26 mg/dl, this is much lower than the control group(12.54±1.25 mg/dl) (p=0.015)。The serum pH levels of WLS-fed rats were significantly lowerthan the placebo rats, the low-dose group is 6.96±0.07, the high-dose group is 6.96±0.17, and the placebo group is 7.13 ± 0.09 (p=0.006).
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48

Djukic, Michael. "Proteomic investigations and biomarker discovery in transient ischaemic attack." Thesis, 2017. http://hdl.handle.net/2440/112817.

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Between 15-26% of ischaemic strokes are preceded by transient ischaemic attack (TIA) making accurate and timely diagnosis of TIA important for stroke prevention. However, TIA diagnoses are highly reliant on subjective history gathering and clinical assessments to differentially diagnose true TIA conditions from mimic presentations. Unfortunately, the subjective nature of TIA diagnosis has created a surprisingly high amount of variability between diagnoses made by physicians and specialist neurologists. Use of biomarker tests could offer an objective quantitative measuring tool that reduces inter-observer variation through the establishment of standardised quantitative measures and improved reproducibility. When used in combination with comprehensive clinical assessments and neurological imaging, biomarkers may offer a useful adjunct to assist a treating clinician to accurately and reliably interpret the clinical finding and confidently diagnose and treat a TIA or mimic condition. This thesis proposes a framework for undertaking an exploration of the human plasma proteome, and performs the very first proteomic pilot study that identifies candidate plasma protein biomarkers associated with TIA, which could also be used to distinguish from mimic presentations.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017.
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49

Chiu, Hui-Wen, and 邱惠雯. "1. The Antioxidant Effect of Caffeic Acid Phenethyl Ester on Human Neutrophils 2.The Proteomics Research on Neutrophil, Platelet and Vascular Smooth Muscle Cell." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/94276412368949891215.

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碩士
長庚大學
天然藥物研究所
92
1.The neutrophil is body’s first line of defense against microorganisms and a critical effector cell in both innate and humoral immunity. However, the capacity for bacterial killing carriers with it an implicity for host tissue destruction, as observed in inflammatory and autoimmune disease. Neutrophils produce potentially dangerous metabolites from superoxide in the defense against microorganisms. Alternatively, caffeic acid phenethyl ester (CAPE) was isolated from propolis that has many biological and pharmacological activities including anti-oxidation and anti-inflammatory. Although CAPE has antioxidant effects, its mechanism on the human neutrophils after fMLP treatment has not been investigated to date. Here we show that CAPE can inhibited fMLP and PMA stimulated human neutrophils superoxide production (IC50 : 7.40 ± 4.10M and 6.55 ± 1.60M). In xanthine/xanthine oxidase system, CAPE possessed superoxide scanverger effect (IC50 : 3.99 ± 1.32M) and inhibited xanthine oxidase activity (IC50 : 45.79 ± 11.79M). To further determine the mechanism of CAPE-induced inhibitory effect on the activation of fMLP-treated neutrophils. We measured the intracellular calcium level and NAD(P)H oxidase activity. Here we show that CAPE can inhibit fMLP and PMA from stimulating NAD(P)H oxidase activity, but not to inhibit fMLP from increasing intracellular calcium. However CAPE can inhibit fMLP which courses PKC translocation. Taken together, these data suggest that CAPE inhibits the fMLP-induced neutrophil activation maybe through the inhibition of cPKC and NAD(P)H oxidase activity leading to the reduction of ROS. 2.In post genomic era, simultaneous with genomics, proteomics and bio-informatics to analyze an organisms that gene and protein expression, to investigate systematic biology. However, considering splice variants and post-translational modification of protein, the number of all structurally and functionally will probable exceed 30,000 ~ 40,000 by at least 10 ~ 200 fold. Proteomics has the power to provide comprehensive structural and quantitative information on all these proteins. Proteomics uses a combination of sophisticated techniques including two-dimensional gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to characterize proteins. In this article, the development of a two-dimensional map of human neutrophil, human platelet, rabbit platelet, spontaneously hypertension (SHR) rat vascular smooth muscle cell and sprague-dawley (SD) rat vascular smooth muscle cell. In neutrophil section, a large number of cytoskeleton proteins were detected, which related to neutrophil function such as adhesion. In platelet section, a large number of blood-coagulant and cytoskeleton proteins were detected. In vascular smooth muscle cell section, a large number of muscle concentrate and growth proteins were detected. The aim of this project is to build up a complete database of such proteins, to be utilized for future studies where is used as a disease or drug treatment marker.
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50

Wei, Wei. "Microfluidics-Based Single-Cell Functional Proteomics Microchip for Portraying Protein Signal Transduction Networks within the Framework of Physicochemical Principles, with Applications in Fundamental and Translational Cancer Research." Thesis, 2014. https://thesis.library.caltech.edu/8112/1/Wei_Wei_2014_thesis.pdf.

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Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research.

The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow.

The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM).

The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model.

The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out.

In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.

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