Rozprawy doktorskie: „Serum Proteomics”

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Kabir, M. "Ovarian cancer serum biomarker discovery using proteomics". Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444306/.

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Ovarian cancer is a lethal gynaecological malignancy which is known as the silent killer. It has a poor prognosis due to the lack of major symptoms in early stage disease and hence its late detection. Cancer antigen-125, the most widely used biomarker for ovarian cancer detection, lacks appropriate sensitivity and specificity. Thus, early biomarkers of the disease are urgently required. Proteomic analysis of human serum promises to be a valuable approach for the discovery of putative biomarkers for human malignancies like ovarian cancer, which could be developed into non-invasive blood tests. In this study, serum samples from a pilot study for ovarian cancer screening which were collected prior to diagnosis were processed at Memorial Sloan Kettering Cancer Research Centre, in collaboration with Prof. Tempst's group, who had developed a novel mass spectrometry (MS)-based technology platform for the high-throughput extraction and measurement of serum peptides. Several marker peaks were identified, which when used in combination with the ovarian cancer biomarker CA-125, assisted in the discrimination of case versus healthy samples at an earlier point prior to diagnosis. Work then involved the establishment and optimisation of a similar serum profiling platform at UCL. This involved the optimisation of a liquid-handling robot to provide semi-automated high-throughput sample purification and spotting, and optimisation of spectral acquisition and processing. The reproducibility of the platform was tested and the effects of different sample handling conditions on peptide profiles examined. The method was then used to search for putative markers of ovarian cancer, using identically processed samples from women diagnosed with malignant or benign ovarian cancer and healthy controls. Finally, as a complementary approach to discover protein biomarkers, the same samples were profiled using 2D Difference Gel Electrophoresis, employing different fractionation strategies to overcome the very large dynamic range of protein expression in serum. Mass spectrometry was used to identify several previously reported and some novel putative biomarkers of ovarian cancer, which warrant further validation.

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Brandtzaeg, Ole Kristian, Elin Johnsen, Hanne Roberg-Larsen, Knut Fredrik Seip, Evan L. MacLean, Laurence R. Gesquiere, Siri Leknes, Elsa Lundanes i Steven Ray Wilson. "Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum". NATURE PUBLISHING GROUP, 2016. http://hdl.handle.net/10150/622705.

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The neuropeptide oxytocin (OT) is associated with a plethora of social behaviors, and is a key topic at the intersection of psychology and biology. However, tools for measuring OT are still not fully developed. We describe a robust nano liquid chromatography-mass spectrometry (nanoLC-MS) platform for measuring the total amount of OT in human plasma/serum. OT binds strongly to plasma proteins, but a reduction/alkylation (R/A) procedure breaks this bond, enabling ample detection of total OT. The method (R/A + robust nanoLC-MS) was used to determine total OT plasma/serum levels to startlingly high concentrations (high pg/mL-ng/mL). Similar results were obtained when combining R/A and ELISA. Compared to measuring free OT, measuring total OT can have advantages in e.g. biomarker studies.

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Grassl, Julia. "Breast cancer serum proteomics : sample processing and protein profiling by mass spectrometry". Thesis, Swansea University, 2007. https://cronfa.swan.ac.uk/Record/cronfa42525.

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The aim of this project was to develop a method for discovery of biomarkers or a protein pattern, as a signature of breast cancer. Early detection of breast cancer is crucial to increase the survival rates of patients. Little was published about biomarker discovery from serum using mass spectrometry, so over the course of the project each factor of the methodology was analysed and optimized. It was shown that standardisation of sample preparation and handling is critical for any quantitative study. The presence of albumin and other highly abundant proteins in serum interferes with proteomic analysis and so depletion techniques were investigated. Centrifugal ultrafiltration was optimised and an extensive study showed it to be a robust and efficient method to enrich the LMW proteome for subsequent biomarker discovery in serum. SELDI-ToF and MALDI-ToF MS were compared for intact protein profiling for breast cancer. In contrast to SELDI-ToF, MALDI-ToF MS had been little tested for this purpose and therefore new software was developed for peak alignment enabling comparison of multiple spectra. LMW serum samples from 8 breast cancer and 8 control individuals were analysed in each experiment. Here we detected seven potential markers in total and gained initial peptide identifications for three markers. This study also tested the use of label-free quantitation using LC-MS on serum samples from breast cancer patients; one differentially-expressed peptide was discovered. The lack of a software tool for comparison of the resulting spectra limited the detection of further markers. The profiling results showed that the use of replicates all the way from starting with the initial serum sample through to data retrieval is crucial due to variation between the biological replicates, and also to reduce any variation occurring from sample preparation.

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Koutroukides, Theodoros Alexis. "Serum proteome profiling using amine-reactive isobaric tagging mass spectrometry in schizophrenia". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607699.

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Ward, Nicholas. "Serum protein profiling using a proteomics approach in the diagnosis of colorectal cancer". Thesis, University of Essex, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510507.

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Sabounchi, Schütt Fariba. "Bronchoalveolar lavage and serum protein patterns in healthy individuals and sarcoidosis patients : a proteomics approach /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-790-8/.

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Cai, Guimei. "Evaluating the effective peak capacity of a saw-tooth gradient for reverse-phase high performance liquid chromatography separation of proteins and peptides". Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5326.

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Thesis (M.S.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains viii, 59 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 54-56).

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Mlynarek, Marcin Aleksander. "Proteomics and the identification of serum biomarkers in a mouse model of oral squamous cell carcinoma". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101731.

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Objective. To establish a clinically relevant model for the identification of protein serum biomarkers for oral squamous cell carcinoma, and to identify specific candidate proteins.
Methods. Samples of oral cancer and adjacent normal tissue were obtained and were transplanted orthotopically into tongues of immunocompromised mice. When the mice lost 20% of their weight, they were sacrificed by exsanguinations. The serum was analyzed by two separate protocols: DIGE/MALDI and MudPIT/LC/ESI. Preliminary validation was conducted on an established cancer marker.
Results. We identified over one hundred proteins as being differentially expressed between control and cancer-bearing mice (p<0.05); including EGFR, cytokeratin 10, gelsolin, titin, vitronectin, retinoblastoma protein family, bullous pemphigoid antigen, and clusterin.
Conclusion. We report a proteomic approach for the identification of serum biomarkers of oral cancer using an orthotopic mouse model. We identified several proteins that can be exploited as potential markers for diagnosis of oral squamous cell carcinoma.

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Zaman, Khadiza. "Revisiting the Neuroprotective Role of 17 Beta Estradiol: A Multi-Omics Based Analysis of the Rat Brain and Serum". Thesis, University of North Texas, 2008. https://digital.library.unt.edu/ark:/67531/metadc1248456/.

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The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of the perturbed networks were involved in cell metabolism, neural development, protein synthesis, cellular trafficking and degradation, and several stress response signaling pathways. We assessed the neuroenergetic status of the brain based on E2's response to various energy generating pathways, including glycolysis, TCA cycle, and oxidative phosphorylation, and several signaling pathways. All energetics pathways were shown to be downregulated in E2 treatment, which suggests that E2 exerts its neuroprotective role by restoring energy homeostasis in OVX rat model by regulating complex signaling and metabolic networks. Our second focus was to determine the metabolite response (amino acids and lipids) after E2 treatment in the brain and serum by employing targeted metabolomics study. We have found that in rat brain cortex there was significant upregulation of a wide number of amino acids suggesting alternate route of metabolism. Another alternate explanation is that E2 replacement replenished the amino acid pool in the tissue. Pathway enrichment analysis revealed upregulation of several pathways, including amino sugar metabolism, purine metabolism, and glutathione metabolism. By combining proteomics and metabolomics in two different biological matrices we were able to gather a vast array of information on how E2 replacement after E2 deprivation can confer neuroprotection. Our findings will help to create a foundation of basic science to be used for developing potentially effective hormone therapies.

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Zaman, Khadiza. "Revisiting the Neuroprotective Role of 17B-Estradiol (E2): A Multi-Omics Based Analysis of the Rat Brain and Serum". Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1248456/.

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The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of the perturbed networks were involved in cell metabolism, neural development, protein synthesis, cellular trafficking and degradation, and several stress response signaling pathways. We assessed the neuroenergetic status of the brain based on E2's response to various energy generating pathways, including glycolysis, TCA cycle, and oxidative phosphorylation, and several signaling pathways. All energetics pathways were shown to be downregulated in E2 treatment, which suggests that E2 exerts its neuroprotective role by restoring energy homeostasis in OVX rat model by regulating complex signaling and metabolic networks. Our second focus was to determine the metabolite response (amino acids and lipids) after E2 treatment in the brain and serum by employing targeted metabolomics study. We have found that in rat brain cortex there was significant upregulation of a wide number of amino acids suggesting alternate route of metabolism. Another alternate explanation is that E2 replacement replenished the amino acid pool in the tissue. Pathway enrichment analysis revealed upregulation of several pathways, including amino sugar metabolism, purine metabolism, and glutathione metabolism. By combining proteomics and metabolomics in two different biological matrices we were able to gather a vast array of information on how E2 replacement after E2 deprivation can confer neuroprotection. Our findings will help to create a foundation of basic science to be used for developing potentially effective hormone therapies.

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11

Zhu, Rui. "ProteinChip SELDI-TOF MS technology to identify serum biomarkers for neuroblastoma and hepatitis B virus-induced hepatocellular carcinoma". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36392431.

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Anand, Swati. "Discovery of Low-Molecular Weight Novel Serum Biomarkers for Diagnosing Preeclampsia and Alzheimer's Disease". BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6200.

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Preeclampsia (PE), a life threatening pregnancy-related disorder, is characterized mainly by new onset of hypertension and proteinuria after 20 weeks of gestation. Currently, PE cannot be predicted prior to onset of symptoms and there is no cure for the disease. There is a clear value in having biomarkers able, early in a pregnancy, to identify women at risk for PE so that proper treatment therapies could be developed. Although a number of serum candidate markers have been proposed to be altered in PE patients, their use is limited due to poor sensitivity and specificity. Therefore, there is ongoing need for better set of novel biomarkers predicting PE. Consequently, for my first project, we used a serum proteomic approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (cLC-ESI-QTOF). Our approach focuses on the less abundant (nM or lower), lower molecular weight peptides and lipids predicting PE. We got previously collected sera from pregnant women at 12–14 weeks gestation. There were 24 controls, having term uncomplicated pregnancies and 24 cases, which developed PE later in the same pregnancy. Many statistically significant serum PE biomarker candidates were found comparing cases and controls. In addition, multimarker combinations having high detection sensitivity and specificity (AUC >0.9) were developed using logistic regression analysis. For my second project, serum lipidomic analysis of sera from pregnant women was undertaken to determine if useful PE lipid biomarkers exist. A discovery study involving a shotgun lipidomic approach was performed using sera collected at 12-14 weeks of pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed PE. Lipids were extracted using organic solvent and analyzed by direct infusion into a time-of-flight mass spectrometer. Statistically significant lipid markers were found and reevaluated in a second confirmatory study having 43 controls and 37 PE cases. The initial study detected 45 potential PE markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Several multi-marker panels with AUC >0.85 and high predictive values were developed from these markers. My third project also involved the above mentioned approach for detection of novel lipid biomarkers for Alzheimer's disease. Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of age-related dementia. Currently, there are no methods to detect Alzheimer's at an early stage when treatment therapies could be applied. Therefore, there is need for detection of panel of biomarkers for detecting patients at risk to AD at an early stage. In the initial discovery set, sera from 29 different stage AD cases and 32 controls were analyzed using direct infusion mass spectrometry (ESI-TOF). This study yielded 89 potential lipid biomarkers which were evaluated in another confirmation study. Of these, 35 markers continued to be statistically significant in the second confirmatory set. Using the confirmed markers, several multi-marker panels with AUC > 0.87 were developed for any stage AD cases vs controls. Multi-marker panels with AUCs > 0.90 were developed for each specific CDR vs controls, including the earliest stage of AD. These lipidomic biomarkers are likely to distinguish AD cases regardless of the stage from controls. In conclusion, we successfully detected, validated and identified low molecular weight novel biomarkers for PE and lipid biomarkers for AD.

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Sun, Jinchun. "A MASS SPECTROMETRY-BASED STUDY OF SERUM BUTYRYLCHOLINESTERASE INHIBITION FROM PESTICIDE EXPOSURE AND ORGANOPHOSPHATE PESTICIDE-INDUCED PROTEOME ALTERATION". UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/290.

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Pesticides including organophosphates (OPs) and carbamates (CBs) are widelyused to control undesirable pests. These compounds are neurotoxic and inhibithydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase. Public healthconcerns have increased with the escalating usage of pesticides. Reliable monitoringprograms are required to detect and quantify pesticide exposure, as well as to promotean understanding of their neurotoxic properties. In this dissertation, both theanticholinergic (Part I) toxicity and neurotoxicity in neuroblastoma cells (Part II) ofpesticides were explored using mass spectrometry (MS). The high sensitivity andhigh-throughput of this technique renders it well-suited for proteomics analysis.Part I describes the study of butyrylcholinesterase (BChE) inhibition resultingfrom OP and CB exposure. The main hypothesis of Part I is that the specialmodification of BChE can provide the origin and extent of pesticide exposure. A novelmethod for detection and quantification of pesticide exposure was designed using aproteomics approach and equine BChE (eBChE) as a model system. The methodologyfeatured detection and analysis of phosphorylated or carbamylated peptides at theactive site serine residue. The developed technique was successfully applied towardsthe study of human BChE (hBChE) inhibition in vitro and in serum samples. Aspecially designed affinity column enabled an isolation of BChE from serum. EnrichedBChE was subjected to enzymatic digestion by a novel on-bead double digestionprotocol. LC/MS/MS was employed to produce a calibration system for the analysis ofhBChE inhibition, which was then applied towards quantification of the enzyme.Part II describes a proteomic study of the neurotoxicity in neuroblastoma cellscaused from chlorpyrifos (CPF), an organophosphate pesticide. The concerns of CPFexposure to pregnant women, infants and children are increasing due todevelopmentally neurotoxic effects of this chemical. The main hypothesis of Part II isthat CPF can cause protein alterations and these altered proteins can be detected usingproteomics. Systematic studies at subcellular levels evaluated proteome changes inSH-SY5Y cells exposed to CPF. Two-dimensional gel electrophoresis (2DE) wasapplied with MALDI-TOF-MS to analyze differential protein expression. Thirty sevencommon unique altered proteins were identified, which play important roles inmetabolic pathway.

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Merrell, Karen. "A Top-Down Proteomic Approach for the Discovery of Novel Serum Biomarkers of Pregnancy-Related Disease". Diss., CLICK HERE for online access, 2009. http://contentdm.lib.byu.edu/ETD/image/etd3117.pdf.

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Zhu, Rui, i 朱睿. "ProteinChip SELDI-TOF MS technology to identify serum biomarkers for neuroblastoma and hepatitis B virus-induced hepatocellular carcinoma". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B36392431.

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Drobin, Kimi. "Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum". Licentiate thesis, KTH, Proteinvetenskap, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-225980.

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Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.

QC 20180412

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17

Ritorto, Maria Stella [Verfasser]. "Cancer proteomics of mouse serum and liver tissue samples to discover candidate biomarkers for hepatocellular carcinoma (HCC) in c-Myc transgenic mice / Maria Stella Ritorto". Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2011. http://d-nb.info/101545996X/34.

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Neiman, Maja. "Bead based protein profiling in blood". Doctoral thesis, KTH, Proteomik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-117960.

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This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles. A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays.

QC 20130208

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19

Byström, Sanna. "Affinity assays for profiling disease-associated proteins in human plasma". Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-202616.

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Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer. The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided. Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD. In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies.

QC 20170302

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Stern, Ana Carolina Bassi. "Análise da influência da restrição nutricional na modulação proteica de células de leucemia mielóide crônica (K562)". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5167/tde-06022017-114158/.

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A formação de uma célula cancerígena é um processo constituído por múltiplas etapas no qual ocorrem diversas alterações genéticas e epigenéticas. O stress ambiental induzido pela restrição nutricional ao tumor causa a desregulação do metabolismo celular, além de aumentar a liberação de citosinas, quimiocinas e fatores de crescimento. Existem diversos estudos que descrevem os impactos do estresse ambiental na progressão tumoral e aquisição de resistência, contudo a maioria destes dá enfoque ao efeito da hipóxia e hipoglicemia. Apesar da restrição lipídica e sérica também serem fontes de estresse microambiental, pouco se sabe sobre os efeitos destas restrições na célula cancerígena. No presente estudo, foi avaliada a influência da restrição sérica e lipídica in vitro nas células de leucemia mielóide crônica K562 e verificadas possíveis alterações na expressão proteica das células que se encontram em restrição nutricional. Foi observado que a restrição lipídica, em todos os testes realizados, não induziu alterações significativas em relação ao controle. Na restrição plasmática, por sua vez, houve diminuição da viabilidade celular, aumento da apoptose, aumento da quantidade de células na fase G2 do ciclo celular e desenvolvimento de uma resistência adquirida a fatores de stress ambiental como pH e presença de espécies oxido redutivas e ao quimioterápico vincristina. Com a análise proteômica baseada em espectrometria de massas, para identificação e quantificação de proteínas, foi possível identificar diferenças no padrão de expressão de proteínas relacionadas as alterações supracitadas como SD1, MGST2, MGST1, GSTT1 e MGT3
The formation of a cancer cell is a multistep process in which there are several genetic and epigenetic changes. The environmental stress induced by tumor nutritional deficiency causes disruption of cell metabolism, and increases the release of cytokines, chemokines and growth factors. There are many studies describing the effects of environmental stress on tumor progression and acquisition of resistance; however, most of these focus on the effect of hypoxia and hypoglycemia. Although the lipid and serum restriction can also be sources of microenvironmental stress, little is known about the effects of these restrictions on cancer cells. In the present study, we evaluated the influence of serum lipid and restriction in chronic myelogenous leukemia cells K562 in vitro. Lipid restriction didn\'t show significant changes when compared controls. Plasmatic restriction reduced cell ciability, increased cell death and the amont of cells in G2 phase of cell cycle. Also increased cells with acquired resistance too environmental stress factors such as pH or the presence of oxide species reductive or chemotherapeutic agent vincristine. With mass spectrometrybased proteomics, it was possible to identify the change in expression of proteins related to the aforementioned effects such as SD1, MGST2, MGST1, GSTT1 e MGT3

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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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Kassa, Fikregabrail. "Proteomic analysis of serum proteins from malaria patients and identification of hemozoin-binding proteins". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66961.

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Malaria is one of the most prevalent infectious diseases worldwide and remains to be a major public health problem in most parts of the world with more than 250 million cases and over one million deaths each year. The malaria parasite, Plasmodium, infects red blood cells (RBCs) and catabolizes hemoglobin which results in the formation of hemozoin. Using proteomic approaches, we compared the serum protein profiles of malaria patients and healthy individuals in order to identify malaria biomarkers. Furthermore, we used the malarial hemozoin to identify hemozoin-binding serum proteins, which can also serve as biomarkers in the malaria context. The comparison of serum protein profiles was achieved using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Using this approach, we have identified 173 significant peaks (potential biomarkers) from two fractions (fraction 1 and 6) of CM10 and IMAC30 ProteinChips. Additionally, we used the malarial hemozoin to trap serum proteins and analyzed their potential as specific biomarkers. By coating hemozoin with serum proteins from malaria patients and healthy individuals in vitro, followed by a liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, we have identified 41 hemozoin-binding proteins. Our quantitative and functional analysis on the identified hemozoin-binding proteins using Scaffold and Blast2GO proteomic platforms revealed several malaria biomarkers such as gelsolin, apolipoprotein E, and serum amyloid A. The major function of the identified proteins was binding, which includes protein and lipid binding/transporter activities. The detection of biomarkers and the identification of hemozoin-binding proteins aid in the early detection of malaria and as diagnostic and therapeutic targets.
La malaria est l'une des maladies infectieuses les plus fréquentes dans le monde et demeure un problème de santé publique majeur dans plusieurs régions avec plus de 250 millions de cas et plus de 1 million de décès chaque année. Le parasite de la malaria, le Plasmodium, infecte les globules rouges et catabolyse l'hémoglobine ce qui résulte en la formation d'un pigment appelé hémozoïne. Utilisant une approche protéomique, nous avons comparé le profil protéique de sérums de patients atteints de la malaria avec ceux provenant d'individus sains afin d'identifier des biomarqueurs associés à la malaria. De plus, nous avons utilisé l'hémozoïne de la malaria afin d'identifier des protéines de sérums s'y liant, ces dernières pouvant également servir de biomarqueurs dans un contexte de malaria. La comparaison entre les profils protéiques de sérum a été effectuée par l'utilisation du SELDI-TOF MS (Surface enhanced laser desorption/ionization time-of-flight mass spectrometry). Au moyen de cette technique, nous avons identifié 173 pics significatifs et potentiels biomarqueurs en utilisant des surface chromatographiques (proteinChips) possédant différentes affinités (CM10 étant échangeuse de cations et IMAC fixant des protéines via un métal) présents dans deux différentes fractions (fraction 1 et 6). Également, nous avons utilisé l'hémozoïne de la malaria afin de capturer des protéines sériques et nous avons étudié leur potentielle utilisation comme biomarqueurs spécifiques. Les protéines sériques provenant de patients atteints de la malaria ou d'individus sains liant des cristaux d'hémozoïne in vitro ont été analysées par chromatographie liquide couplée à la spectrométrie de masse (LC-MS/MS) menant à l'identification de 41 protéines liant l'hémozoïne. Notre étude quantitative et fonctionnelle utilisant les programmes Scaffold et Blast2GO sur les protéin

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Lazarim, Fernanda Lorenzi 1981. "Analise proteomica de soro de ratos em diferentes situações de exercicio e uma experiencia de pesquisa em ensino". [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314076.

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Orientador: Denise Vaz de Macedo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A resposta adaptativa decorrente de um programa de treinamento está relacionada a um intenso processo de síntese protéica, cujo efeito cumulativo de várias sessões de exercício leva a alterações fenotípicas do músculo e aumento de rendimento em capacidades biomotoras diversas. Para isso é necessário um tempo adequado de recuperação entre os estímulos. Um processo contínuo de treinamento intensificado sem o tempo de recuperação adequado é denominado overtraining. Este pode culminar em basicamente dois estados diferenciados em relação ao desempenho: overreaching funcional (FOR), com manutenção ou mesmo melhora de desempenho após o descanso, e overreaching não funcional (NFOR), caracterizado pela queda no desempenho por tempo prolongado. A visualização das alterações agudas e crônicas do perfil protéico tanto de células como de fluidos pode auxiliar na compreensão dos mecanismos envolvidos nos estados FOR e NFOR, e possibilitar a identificação de marcadores que auxiliem na detecção desses estados. Nesse contexto a análise proteômica pode ser uma ferramenta bastante útil, pois permite separar, quantificar e identificar o perfil protéico de tecidos e fluidos biológicos. A presente tese está dividida em duas partes: pesquisa (Parte I) e ensino (Parte II), que refletem as experiências vividas desde a iniciação científica, sendo igualmente relevantes para minha formação acadêmica. A Parte I é constituída por três capítulos cujo objetivo principal foi investigar as alterações agudas e crônicas decorrentes do exercício físico no perfil protéico do soro de ratos através da análise proteômica. O capítulo 1 apresenta uma revisão sobre os mecanismos moleculares envolvidos na resposta adaptativa ao treinamento, as proteínas do soro, as técnicas utilizadas na análise proteômica e sua aplicabilidade nas pesquisas com exercício. O capitulo 2 apresenta as alterações agudas no perfil proteico do soro de ratos submetidos a um exercício exaustivo de média duração em esteira, 3 e 24 horas após o estímulo. As proteínas diferencialmente expressas 24 horas após o exercício corresponderam a proteínas de fase aguda sintetizadas em resposta à instalação de um processo inflamatório, indicando que a geração de microtraumas e a inflamação são partes integrantes da resposta aguda ao exercício. O capítulo 3 apresenta as alterações no perfil proteico do soro de ratos submetidos a um protocolo de indução ao continuum treinamento-overtraining, desenvolvido recentemente no nosso laboratório, e que produz animais nos estados FOR e NFOR. As proteínas diferencialmente expressas indicam um quadro antiinflamatório nos animais do grupo FOR e alterações protéicas que favoreceram os processos adaptativos envolvidos na biogênese mitocondrial e regeneração do tecido danificado. Também apresentaram melhora no perfil lipídico. O grupo NFOR apresentou alterações de proteínas de fase aguda indicando um processo inflamatório instalado e alterações de algumas proteínas que podem ter prejudicado o desencadeamento da resposta adaptativa, resultando na queda da performance. A Parte II da tese apresenta uma proposta de atividade prática, aplicada num curso de especialização com enfoque em bioquímica para alunos de Educação Física e Nutrição. Essa atividade consiste na discussão dos conceitos de Índice Glicêmico e Carga Glicêmica a partir de dados obtidos pelos próprios alunos. Utilizamos essa aula para a introdução ao estudo das vias de síntese e integração metabólica no estado alimentado
Abstract: The adaptive response to a training program is related to an intensive process of protein synthesis, which cumulative effect of multiple sessions of exercise leads to muscle phenotypic alterations and increases different physical capacities. For such adaptation an appropriate time for recovery between stimuli is required. A continuous process of intensified training without adequate recovery time is called overtraining. It can result in basically two different states concerning performance: functional overreaching (FOR), with maintenance or even improvement of performance after the recovery period, and non-functional overreaching (NFOR), characterized by performance decrement for a prolonged period. The visualization of acute and chronic changes on the protein profile of both cells and fluids may help one to understand the mechanisms involved on FOR and NFOR states, and it can enable the identification of biomarkers helping to detect these states. Within this context the proteomic analysis can be an interesting tool as it enables to separate, identify and quantify the protein profile of tissues and biological fluids. This work is divided in two parts: research (Part I), and education (Part II), which represent the experiences that I have been living since my scientific initiation and therefore, both are relevant for my education. Part I consists of three chapters in which the main goal is to investigate the acute and chronic changes in response to exercise in serum proteins profile of rats by proteomic analysis. Chapter 1 presents a review of the molecular mechanisms involved in the adaptive response to training, serum proteins, the techniques used in proteomics analysis and its applicability on exercise research. Chapter 2 presents the acute changes in serum protein profile of rats submitted to an exhaustive exercise of average duration on a treadmill, 3 and 24 hours after the stimulus. The proteins differently expressed 24 hours after the exercise were the acute-phase protein synthesized in response to installation of the inflammatory process, indicating that the generation of micro trauma and inflammation are parts of the acute response to the exercise. Chapter 3 reveals the changes in the serum protein profile of rats, submitted to an exercise protocol developed recently in our laboratory, to induce the animals through the continuum training-overtraining, leading the animals to the FOR and NFOR states. The differently expressed proteins indicate an anti-inflammatory process in the animals that were in the FOR group and protein changes which favored the adaptive processes involved in mitochondrial biogenesis and the complete recovery of tissue damage, as well as the improvement on the lipid profile. The NFOR group presented changes of acute phase proteins indicating the instalation of an inflamatory process and alterations in some proteins that may have impaired the development of the adaptive response, which results in performance decrement. Part II of this work shows a proposal for a practical activity implemented in a specialization course with focus on Biochemistry for Physical Education and Nutrition students. This activity consists in the discussion of the Glycemic Index and Glycemic Load concepts through data obtained by the students. This class is used to introduce the study of synthesis pathways and metabolic integration in the fed state
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

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Pontes, Letícia Gomes de. "Aspectos cruciais sobre doenças em búfalos sorodoadores do manejo dos animais à identificação de marcadores moleculares de sanidade /". Botucatu, 2018. http://hdl.handle.net/11449/180526.

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Orientador: Lucilene Delazari dos Santos
Resumo: O uso de animais de grande porte como sorodoadores tem se mostrado de grande importância para a produção do selante de fibrina do Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP). O objetivo deste trabalho foi certificar um plantel de bubalinos, realizar uma investigação sobre os potenciais candidatos à biomarcadores de brucelose em soro de búfalos e evidenciar o isolamento e a quantificação pioneira de exossomos (EVs) do soro de bubalinos com theileriose e babesiose. Realizaram-se os seguintes testes sorológicos: brucelose (BRU), leptospirose (LEP), febre aftosa (FMD), rinotraqueíte infecciosa bovina (IBR), diarréia viral bovina (BVD), varíola bovina (Pox) e tuberculose, língua azul (BTV), estomatite vesicular (EV) - sorotipos cocal (COCV) e alagoano (VSAV), leucose bovina (BLV), neospora (NC), toxoplasmose (TX) e testes bioquímicos laboratoriais. Todas as amostras de soro foram submetidas ao diagnóstico sorológico para detecção de brucelose, diagnóstico de reação em cadeia da polimerase para detecção de theileriose e babesiose, cromatografia líquida de afinidade, isolamento de exossomos, digestão de proteínas em solução, análise de espectrometria de massa e ferramentas de bioinformátca. Observamos um enriquecimento de 90,5% de proteínas nos soros de búfalos após este protocolo de depleção. A análise MS/MS evidenciou que as principais diferenças no proteoma dos animais com brucelose. No que se refere ao exossosmos isolados e quantififcados neste trabalho, nossa met... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The use of large animals as sorbozers has been shown to be of great importance for the production of fibrin sealant from the Center for the Study of Venoms and Poisonous Animals (CEVAP). The objective of this work was to certify a flock of buffaloes, to conduct an investigation of the potential candidates for brucellosis biomarkers in buffalo serum and to evidence the isolation and the pioneer quantification of buffalo serum exosomes (EVs) with theileriose and babesiosis. The following serological tests were carried out: brucellosis (BRU), leptospirosis (LEP), foot-and-mouth disease (FMD), infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), bovine pox (Pox) and tuberculosis, blue tongue (BTV), vesicular stomatitis (EV) - cocal (COCV) and alagoan (VSAV) serotypes, bovine leukosis (BLV), neospora (NC), toxoplasmosis (TX) and laboratory biochemical tests. All serum samples were submitted: 1) serological test; 2) Polymerase chain reaction; 3) Depletion; 4) Isolation of exosomes; 5) Mass spectrometry and 6) Analysis and interpretation of the data. The MS / MS analysis showed that the major differences in the proteome of animals with brucellosis. Regarding the isolated and quantified exosmos in this work, our methodology is strongly encouraged for the isolation and characterization of EVs in Apicomplexa infections in farm animals. We conclude that this study on different fronts such as management, infectious diseases and parasitic diseases can be used in small pro... (Complete abstract click electronic access below)
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Vidotto, Alessandra. "Marcadores protéicos do carcinoma epidermóide de cabeça e pescoço com fenótipo invasivo". Faculdade de Medicina de São José do Rio Preto, 2009. http://bdtd.famerp.br/handle/tede/125.

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The regional lymph nodes play a pivotal role in diagnosis, staging and management of head and neck squamous cell carcinomas (HNSCC). Despite their importance, detailed understanding of the probable mechanisms of lymphatic metastases has not been completely achieved. Subjects and Methods: We analyzed metastatic and normal lymph node tissues, as well as saliva and serum from sixth-two patients with HNSCC, and twenty-nine controls using two-dimensional electrophoresis, MALDI-Q-TOF and western blot. Results: Several proteins were found to be significantly increased in metastatic nodes, such as stratifin, glutathione S-transferase pi, apoliproteín A-I, alpha-1-microglobulin, disulfide isomerase, galectin, citokeratins, immunoglobulins, transtirretin, calciun-binding protein (família S100) and fat-binding protein (FABP). Among the down-regulated proteins in metastatic lymph nodes are calreticulin, tropomiosin 3, triosephosphate isomerase, piruvate quinase, anidrase carbonic, gamma actin, peroxiredoxin 2, profilin 1, gliceraldeyde 3- fosfato desidrogenase and heat shock proteins. These proteins are involved in epidermis development, cell proliferation, migration and adhesion, apoptosis, defense and inflammatory response and xenobiotic metabolism. Our data on the expression of heat shock proteins and enzymes of the glycolytic pathway suggest an effect of the lymph node environment in controlling tumor progression or in metabolic reprogramming of the metastatic cell. In saliva, 13 proteins showed an altered pattern of expression in samples patient, including over-expression of keratins, immunoglobulins, alphaamylase, PLUNC and zinc-alpha-2-glycoprotein and down-regulation of myosin. In serum samples, six proteins were over-expressed (serum albumin, alpha-1- microglobulin/bikunin precursor, apolipoprotein A-I, haptoglobin, serotransferrin, transthyretin) and two were under-expressed (hemoglobin subunit alpha, hemoglobin subunit beta) compared to the control group. Conclusion: New potential markers, such as profilin-1 and E-FABP, were identified and may be proved useful for defining the invasive phenotype of head and neck carcinomas.
O comprometimento de linfonodos regionais por células neoplásicas é atualmente o indicador mais utilizado para prognóstico em pacientes com carcinoma epidermóide de cabeça e pescoço (CECP). Apesar disso, a compreensão detalhada dos mecanismos envolvidos na formação de metástases linfáticas ainda não foi completamente atingida. Casuística e Método: Foi avaliado o perfil protéico de linfonodos metastáticos e não metastáticos, bem como de amostras de saliva e soro de 62 pacientes em diferentes estágios da doença e de 29 controles, utilizando eletroforese bidimensional, espectrometria de massas por MALDI-Q-TOF e experimentos de validação por Western blot. Resultados: Os resultados mostraram várias proteínas com expressão elevada em linfonodos metastáticos em relação aos não metastáticos, como stratifina, glutathiona S-transferase pi, apoliproteína A-I, alfa-1-microglobulina, dissulfeto isomerase, galectinas, citoqueratinas, imunoglobulinas, transtirretina e proteínas de ligação ao cálcio (família S100) e a ácidos graxos (FABP). De forma inversa, as proteínas calrreticulina, tropomiosina 3, triofosfato isomerase, piruvato quinase, anidrase carbônica, gama actina, peroxirredoxina 2, profilina 1, gliceraldeído 3-fosfato desidrogenase e proteínas de choque térmico mostraram níveis reduzidos em linfonodos metastáticos. Essas proteínas estão envolvidas em processos de desenvolvimento epidérmico, proliferação, migração e adesão celular, apoptose, resposta inflamatória e metabolismo de xenobióticos. Os dados relacionados à expressão de proteínas de choque térmico e enzimas da via glicolítica sugerem um efeito do ambiente dos linfonodos e no controle da progressão do tumor ou na reprogramação das células metastáticas. Em saliva, 13 proteínas exibiram um padrão alterado nas amostras de pacientes com câncer, incluindo expressão elevada de queratinas, imunoglobulinas, alfa-amilase, PLUNC e zinc-alfa-2-glicoproteína e expressão reduzida de miosina. Em amostras de soro, seis proteínas apresentaram expressão aumentada (albumina, alfa-1-microglobulina/bikunina precursor, apolipoproteína A-I, haptoglobina, serotransferrina e transtirretina) e duas estavam com expressão diminuída (hemoglobina alfa e hemoglobina beta), quando comparadas com o grupo controle. Conclusão: Os resultados obtidos revelaram novos marcadores potenciais, como profilina 1 e E-FABP, PLUNC e transtirretin que podem ser úteis na definição do fenótipo invasivo e no rastreamento e diagnóstico desse grupo de neoplasias.

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Lepper, Marlen Franziska [Verfasser], Anette-Gabriele [Akademischer Betreuer] Ziegler, Fabian [Gutachter] Theis i Anette-Gabriele [Gutachter] Ziegler. "Proteomic phenotyping of patient-derived peripheral blood mononuclear cells and serum in recent-onset type 1 diabetes / Marlen Franziska Lepper ; Gutachter: Fabian Theis, Anette-Gabriele Ziegler ; Betreuer: Anette-Gabriele Ziegler". München : Universitätsbibliothek der TU München, 2018. http://d-nb.info/1175091731/34.

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Jones, Christina Michele. "Applications and challenges in mass spectrometry-based untargeted metabolomics". Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54830.

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Metabolomics is the methodical scientific study of biochemical processes associated with the metabolome—which comprises the entire collection of metabolites in any biological entity. Metabolome changes occur as a result of modifications in the genome and proteome, and are, therefore, directly related to cellular phenotype. Thus, metabolomic analysis is capable of providing a snapshot of cellular physiology. Untargeted metabolomics is an impartial, all-inclusive approach for detecting as many metabolites as possible without a priori knowledge of their identity. Hence, it is a valuable exploratory tool capable of providing extensive chemical information for discovery and hypothesis-generation regarding biochemical processes. A history of metabolomics and advances in the field corresponding to improved analytical technologies are described in Chapter 1 of this dissertation. Additionally, Chapter 1 introduces the analytical workflows involved in untargeted metabolomics research to provide a foundation for Chapters 2 – 5. Part I of this dissertation which encompasses Chapters 2 – 3 describes the utilization of mass spectrometry (MS)-based untargeted metabolomic analysis to acquire new insight into cancer detection. There is a knowledge deficit regarding the biochemical processes of the origin and proliferative molecular mechanisms of many types of cancer which has also led to a shortage of sensitive and specific biomarkers. Chapter 2 describes the development of an in vitro diagnostic multivariate index assay (IVDMIA) for prostate cancer (PCa) prediction based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS) metabolic profiling of blood serum samples from 64 PCa patients and 50 healthy individuals. A panel of 40 metabolic spectral features was found to be differential with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The performance of the IVDMIA was higher than the prevalent prostate-specific antigen blood test, thus, highlighting that a combination of multiple discriminant features yields higher predictive power for PCa detection than the univariate analysis of a single marker. Chapter 3 describes two approaches that were taken to investigate metabolic patterns for early detection of ovarian cancer (OC). First, Dicer-Pten double knockout (DKO) mice that phenocopy many of the features of metastatic high-grade serous carcinoma (HGSC) observed in women were studied. Using UPLC-MS, serum samples from 14 early-stage tumor DKO mice and 11 controls were analyzed. Iterative multivariate classification selected 18 metabolites that, when considered as a panel, yielded 100% accuracy, sensitivity, and specificity for early-stage HGSC detection. In the second approach, serum metabolic phenotypes of an early-stage OC pilot patient cohort were characterized. Serum samples were collected from 24 early-stage OC patients and 40 healthy women, and subsequently analyzed using UPLC-MS. Multivariate statistical analysis employing support vector machine learning methods and recursive feature elimination selected a panel of metabolites that differentiated between age-matched samples with 100% cross-validated accuracy, sensitivity, and specificity. This small pilot study demonstrated that metabolic phenotypes may be useful for detecting early-stage OC and, thus, supports conducting larger, more comprehensive studies. Many challenges exist in the field of untargeted metabolomics. Part II of this dissertation which encompasses Chapters 4 – 5 focuses on two specific challenges. While metabolomic data may be used to generate hypothesis concerning biological processes, determining causal relationships within metabolic networks with only metabolomic data is impractical. Proteins play major roles in these networks; therefore, pairing metabolomic information with that acquired from proteomics gives a more comprehensive snapshot of perturbations to metabolic pathways. Chapter 4 describes the integration of MS- and NMR-based metabolomics with proteomics analyses to investigate the role of chemically mediated ecological interactions between Karenia brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. This integrated systems biology approach showed that K. brevis allelopathy distinctively perturbed the metabolisms of these two competitors. A. glacialis had a more robust metabolic response to K. brevis allelopathy which may be a result of its repeated exposure to K. brevis blooms in the Gulf of Mexico. However, K. brevis allelopathy disrupted energy metabolism and obstructed cellular protection mechanisms including altering cell membrane components, inhibiting osmoregulation, and increasing oxidative stress in T. pseudonana. This work represents the first instance of metabolites and proteins measured simultaneously to understand the effects of allelopathy or in fact any form of competition. Chromatography is traditionally coupled to MS for untargeted metabolomics studies. While coupling chromatography to MS greatly enhances metabolome analysis due to the orthogonality of the techniques, the lengthy analysis times pose challenges for large metabolomics studies. Consequently, there is still a need for developing higher throughput MS approaches. A rapid metabolic fingerprinting method that utilizes a new transmission mode direct analysis in real time (TM-DART) ambient sampling technique is presented in Chapter 5. The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. In terms of reproducibility, TM-DART compared favorably with traditional probe mode DART analysis, with coefficients of variation as low as 16%. TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera and was adapted for exhaled breath condensate (EBC) analysis. To determine the feasibility of utilizing TM-DART for metabolomics investigations, TM-DART was interfaced with traveling wave ion mobility spectrometry (TWIMS) time-of-flight (TOF) MS for the analysis of EBC samples from cystic fibrosis patients and healthy controls. TM-DART-TWIMS-TOF MS was able to successfully detect cystic fibrosis in this small sample cohort, thereby, demonstrating it can be employed for probing metabolome changes. Finally, in Chapter 6, a perspective on the presented work is provided along with goals on which future studies may focus.

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Lin, Ching-Hung, i 林勁宏. "Proteomics Approach for Identifying Serum Biomarkers of Geriatric Frailty". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/96641243591367657717.

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博士
國立體育大學
體育研究所
101
Elderly frailty may result in disability or increase in the risk of degenerative diseases. The frailty was previously diagnosed by body movement or questionnaire, whereas internal physiological changes can not be determined by using these assessments. If internal physiologic changes can be used to define frailty, more precise status of frailty would be accurately diagnosed. Sarcopenia, which is an important phenomenon of frailty, can be induced by inflammation, insulin resistance, and decrease in anabolic hormones. The purpose of this study was to compare differences in distinct levels of frailty by analyzing sarcopenia, physical activity level, and whole body muscle mass. Proteomics, a new analysis techniques, was also used to investigate various serum proteins in different degrees of frailty. The elderly subjects (n=18; age: yrs; height: cm; weight: kg) were divided to no frailty (NF; n=6; age: 77.50.99 yrs; height: 160.53.13 cm; weight: 62.53.01 kg), prefrailty (PF; n=6; age: 79.01.00 yrs; height: 153.34.19 cm; weight: 65.33.95 kg) and frailty (F; n=6; age: 77.73.83 yrs; height: 156.67.20 cm; weight: 62.87.14 kg) in accordance with the Chinese-Canadian Study of Health and Aging Clinical Frailty Scale. We also recruited a group of young subjects (n=6; age: 27.21.14 yrs; height: 169.21.92 cm; weight: 60.23.87 kg) to serve as the young control group. After an 8 hours fasting, fasting venous blood samples were collected, whole body muscle mass and physical activity ability was measured. The blood analyses included convention biochemical markers, inflammatory markers, insulin sensitivity, anabolic hormones and proteomics. The difference of various biomarkers of each groups were determined by using one-way ANOVA, and the correlations were examined by Pearson product moment correlation. We observed that F group had significantly lower physical activity ability among Y, NF and PF (grip power: 13.81+/-1.07 vs 36.72+/-3.53, 26.1+/-3.02, &; 21.64+/-3.78 kg; 6 min walk test: 215.20+/-42.21 vs 617.33+/-15.50, 438.33+/-42.02, &; 364.33+/-29.56 m). There was a strong positive correlation between muscle strength and muscle mass in NF and PF groups. There was no significant difference in muscle mass loss among experimental groups, but there was significant difference in strength decrease in F group. Although the older subjects without frailty showed decline in the serum level of anabolic hormones, thereby indirectly impairing their physical activity ability. By using proteomics techniques, we observed that the angiotensinogen and kininogen-1 significantly increased during normal process of frailty development (9.85+/-2.04 vs 5.04+/-0.85, 4.80+/-0.66 &; 5.50+/-0.92; 26.99+/-1.83 vs 15.30+/-1.51, 15.31+/-1.72 &; 17.94+/-3.14). This phenomenon might help to explain the possible mechanisms for the interactions between vascular degeneration and compensation, and the relationship between aging and strength decrease. In addition, during normal process of frailty development, antithrombin Ⅲ significantly increased (23.411.50 vs 17.951.43, 9.620.88 &;12.960.69), but muscle strength showed gradual decrease overtime. Our results therefore suggest that antithrombin Ⅲ may serve as an important biomarker during normal process of frailty development.

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Baum, Adrienne. "Proteomics-Basierte suche nach neuen prognostischen Serum-Biomarkern für schwere Entzündliche Erkrankungen /". 2008. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=017026728&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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Yao, Jheng-Liang, i 姚正良. "Proteomics Approach to Identify Serum Glycoproteins as Biomarkers in Human Lung Adenocarcinoma". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/80588434297067626149.

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碩士
臺灣大學
生化科學研究所
95
Lung cancer is the most common cancer in the world and has the highly lethal ratio from cancer. The survival rates of 5-year patient are very low at about 8-14%. The low survival rate is caused by that disease diagnosis is usually late, and prognosis is poor. To diagnose effectively in the early stage of cancer formation is important for increasing survival rate. Detection in the early stage of lung cancer is critical for successful clinical therapy. Presently, many reports discuss the glycosylation changes in several cancer cases such as liver cancer, lung cancer, ovarian cancer etc. The differences are including fucosylation, sialylation, and high branches formation. They approach the subject matter from gene level to discuss the glycosyltransferase expression, and from the glycan and protein level to discuss the glycoform difference. The aim of our study is to setup methods and attempts to identify serum biomarker of lung cancer. According to several reports, they did not discuss that the difference in glycoprotein resulted from protein level or glycan level. In the experiment, we combine the application of lectin and the method of proteomics for researching in serum biomarker of glycoprotein. Even if the difference is in protein level, it also has the potential to be a biomarker for cancer diagnosis. If the difference is in glycan level, we can discuss the different site and the effect in physiology. First, we use several kinds of letin to screen serum. There are some differences in the result of Con A lectin staining. The affinity column of Con A was used to separate the protein which can be bound by Con A. Then the bound protein was separated by 2-D electrophoresis or direct identified by mass. In the result, we find that several identified proteins were in immune response and were reported before in other cancer like liver cancer or gastric cancer. It is needed to discuss that the possibility of smoking and some factors inducing lung inflammation lead to lung cancer. We use Con A in the experiment. Because Con A can bind almost all proteins with N-glycosylation, the differences of identified proteins were in protein level. If we use other kinds of lectin, we can identify protein differences in glycan level. In the other side, we find the difference in the fragments of complement C3. It is needed to discuss what causes the result, cancer or not.

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31

"Untargeted Lcms Serum Metabolomics Of The Sierra Leonean Lassa Fever Patient And Metaanalysis Of The Virion Proteome". 2016.

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32

Yi-AnChen i 陳怡諳. "Structural Characterization of Bovine Serum Albumin Encapsulated Gold Nanoclusters using Mass Spectrometry-Based Proteomics Technique". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/weutdg.

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碩士
國立成功大學
化學系
105
Nowadays, the use of protein encapsulated gold nanoclusters is of high interest in many fields. Their particular properties such as luminescence in the visible range, nontoxicity, biocompatibility and feasible functionalization made them attractive for applications in biosensing, bioimaging and biolabelling. Although many synthetic strategies such as the use of redox reagents, pH, and temperature have been shown to improve the luminescence or the sensing sensitivity of the nanoclusters, molecular structure of protein protected gold nanoclusters remains un-explored. In this study, we applied liquid chromatography-mass spectrometry (LC-MS) based proteomics technique to study the structural changes of BSA by forming gold nanoclusters. In our previously study, we have synthesized bovine serum albumin protected gold nanoclusters (BSA-AuNCs). In order to improve their monodispersity, we tried to change our synthesis methods and purification methods. First, we used two-step method to synthesize red fluorescent gold nanoclusters (BSA-AuNCs_red). Secondly, we used ascorbic acid under slightly basic condition (pH =8) to form the blue fluorescent gold nanoclusters (BSA-AuNCs_blue).The products, BSA-AuNCs were first purified by dialysis membrane, and then by non-continuous sucrose gradient or ultracentrifugefilter and characterized by Fluorescent spectroscopy, Ultraviolet-visible spectroscopy, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), Circular Dichroism spectrum (CD spectrum) and Electron spectroscopy for chemical analysis (ESCA). The bottom-up proteomics technique was then applied to identify the changes of amino acid residues of BSA under native and denatured condition. The red nanoclusters exhibited luminescence mainly around 640 nm; the blue one mainly around 440 nm. MALDI-MS data show a broad range of mass distribution (3Da) with an average around 71 kDa for the red nanoclusters (about 25 Au atom) and 74 kDa for the blue nanoclusters (about 40 Au atom).The data derived from bottom-up proteomics indicated cysteine is the major species been oxidized to sulfur dioxide or sulfur trioxide by nanoclusters formation. The oxidized percentage was much higher for the blue than for the red one, resulting in more disulfide linkages were cleaved for the blue nanoclusters. These observations were consistent with the observations of ESCA. In addition, proteomics data revealed the cleaved disulfide linkage sites, among which Cys 125 appeared to be main target of oxidization for both the red and the blue nanocluster. Moreover, ESCA data indicated a higher percentage of Au(I)/Au(0) for the red nanoclusters than the blue nanoclusters. Taken together, our study indicated that nanoclusters formation may be driven by oxidation of site-specific cysteine residues, causing some disulfide linkages to break. The use of reducing agents such as ascorbic acid appeared to accelerate cysteine oxidation and disulfide cleavages even under a relatively mild pH (pH=8) environment. The bigger size of blue nanoclusters may be attributed to disulfide cleavages. It remains to study whether the oxidized cysteineor sulfate play a role in stabilizing nanoclusters and whether it affects the optical or chemical properties of the nanoclusters.

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33

Yu-ChenHsu i 徐御宸. "Structural Characterization of Bovine Serum Albumin-Capped Gold nanoclusters by Mass Spectrometry and Proteomics Techniques". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/am59f8.

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碩士
國立成功大學
化學系
106
In this paper, we reported new structural insights on the ﬂuorescent bovine serum albumin (BSA)−gold (Au) nanoclusters (BSA-AuNCs) synthesized by optimized method. Using MALDI-TOF, Au cluster signals (peaks with 197 Da-spacing) in the low mass region (〈5000 Da) as well as intact ion signals (M.W.~71k Da ~25 Au for the aged form and M.W.~68k Da ~8 Au for the normal form) were detected, suggesting the formation of BSA-capsulated Au nanoclusters. Fluorescence and ESCA results also provide evidence of formation. Using mass spectrometry and proteomics techniques, methionine, histidine, and cysteine were identified to be main oxidized amino acid in BSA-AuNCs, suggesting these three amino acids were primarily responsible for the reduction of Au ion by BSA. By observing the oxidation state of Cys. site, we can find some disulfide bond fragments in the process of synthesis will break. The ligand protected nanocluster cores were further isolated by enzyme digestion and 10 kDa cut-off filtering. Site-specific oxidization was considered to break the disulfide linkage well as on the free Cys58 of the red BSA-AuNCs were identified mainly from the non-core fraction. In contrast, multiple non-oxidized cysteine residues: Cys99, Cys339, Cys537 were enriched in the core fraction of BSA-AuNCs_red which fluorescence was quenched upon disulfide cleavage by TCEP, suggesting BSA-AuNCs_red were mainly protected by these disulfide linkages. The data indicated protein conformational changes accompanied with Au ion-induced cysteine oxidation and disulfide cleavages make room for Au attachment to disulfide group. Compared to BSA-AuNCs_red, BSA-AuNCs_blue have lesser degree of cysteine oxidation and the Au core was not mainly protected by cysteine residues. The blue fluorescence was not quenched by TCEP either. Our data revealed structural insights of BSA-AuNCs at the residue level which has never been gained before. Multiple disulfide linkages of BSA were confirmed to play important roles in the red BSA-AuNCs formation and stabilization.

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Chow, Brian Andrew. "Stress Physiology of Bears: Cortisol Dynamics and Identification of Novel Serum Proteins". Thesis, 2013. http://hdl.handle.net/10012/7406.

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There is a need to understand how free-ranging animals respond and adapt to stress. However, little is currently known regarding the physiologic adaptations to stress in bears, and there are few tools available to wildlife managers to assess the health and stress status of free-ranging animals, including ursids. The hypothalamus-pituitary-adrenal (HPA) axis plays major roles in the physiological adaptation to stress, leading to the increased secretion of glucocorticoids (e.g. cortisol in most mammals) that mediate adaptive changes in physiology and behaviour. The vast majority of glucocorticoids are bound to its primary carrier protein, corticosteroid-binding globulin (CBG), in most animals, and only the unbound fraction is bioavailable. Thus, CBG plays a major role in modulating glucocorticoid dynamics, and this protein must be characterized to build a more complete understanding of the adaptive role that the HPA axis plays in mitigating stress in bears. The overall objective of this thesis was to characterize the HPA axis activity and CBG levels in bears, and develop tools targeted towards the monitoring of the health and stress status of American black bear (Ursus americanus), grizzly bear (U. arctos), and polar bear (U. maritimus). The binding characteristics of cortisol to CBG in bears were studied via saturation binding experiments, and this information was used to estimate free cortisol concentrations based on CBG concentrations. To quantify CBG concentrations in bears, an enzyme-linked immunosorbent assay (ELISA) was developed. Grizzly bear CBG cDNA was cloned and sequenced, and an antibody was developed against a peptide sequence of the deduced amino acid sequence. The antibody showed good cross-reactivity against black, grizzly, and polar bear CBG, and the ELISA based on this antibody found differences in the mean CBG levels between species. Using this data, free cortisol levels were estimated, and mean levels were elevated in polar bears relative to black and grizzly bears. Having developed these tools, the roles that corticosteroid-binding globulin (CBG) and bioavailable cortisol played in the physiological adaptation to major life history traits and environmental challenges faced by ursids were investigated. Importantly, CBG was not modulated by the acute stress of capture and handling, despite the large differences in the magnitude of acute cortisol responses that are induced by these methods, suggesting that CBG levels may reflect the chronic health and stress status of bears. Altogether, there were few changes in CBG levels throughout much of the annual life cycle of bears, implying that CBG does not play a major adaptive role in the life history traits of bears and, instead, metabolic and environmental factors may be the key modulators of cortisol dynamics. However, CBG was not significantly associated with our measures of dietary patterns and nutrition, including body condition, seasonal dietary patterns, and fasting. The majority of the observed variation in the levels of this protein in bears remains unexplained. However, stress-induced free cortisol levels were negatively associated with urea to creatinine ratio (an indicator of dietary protein content and fasting status in grizzly and polar bears, respectively) and positively associated with lactation in hibernating black bears, suggesting that the variation in adrenal function may be playing an important role in the adaptation to adverse environmental conditions and/or metabolic stress in bears. In addition to serum cortisol dynamics, other proteins were also hypothesized to play adaptive roles in maintaining the hibernating phenotype in bears. Changes in the serum proteome during hibernation in black bears were assessed as a means to discover novel proteins that may be indicative of metabolic stress in bears. The serum proteomes of active and hibernating black bears were compared and analyzed for significant changes by two-dimensional electrophoresis and tandem mass spectrometry. Proteins involved with immune-related function were significantly altered during hibernation, leading to the proposal that the serum protein changes are essential for maintaining immune competence, wound healing, and bone structure. Altogether, this thesis developed a method to quantify CBG and estimated free cortisol concentrations in bears, and characterized their roles in the physiological adaptations associated with the major life history traits and environmental challenges faced by ursids. Also, novel serum proteins were identified as potential markers of immune function and health status in bears. These tools may be tremendously useful for wildlife managers and conservationists in determining how chronic stressors, including anthropogenic activities and climate change, may impact the stress and health performances of individual and populations of free-ranging bears.

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Streich, Jan-Hendrik. "Rheumatoide Arthritis: Proteomische Analyse von Serum und synovialen Fibroblasten zur Detektion von Biomarkern". Doctoral thesis, 2009. http://hdl.handle.net/11858/00-1735-0000-0006-AD60-1.

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Yang, Q., M. Zhang, Dean J. Harrington, G. W. Black i I. C. Sutcliffe. "A proteomic investigation of Streptococcus agalactiae reveals that human serum induces the C protein β antigen and arginine deiminase". 2011. http://hdl.handle.net/10454/11568.

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No
Streptococcus agalactiae is a major neonatal pathogen. Disease progression is characterised by bacterial adaptation from commensal maternal vaginal colonisation to environments associated with neonatal disease, including exposure to blood. To explore this adaptation in vitro, we have used proteomics to identify proteins differentially expressed following growth on Todd Hewitt agar in the presence or absence of 10% v/v human serum. Twelve differentially expressed proteins were identified. Notably, the C protein β antigen and arginine deiminase proteins were upregulated following growth in the presence of human serum, consistent with previous studies implicating these two proteins in the pathogenesis of S. agalactiae disease.

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37

Gkanatsiou, Eleni. "Development of an assay to monitor the role of Serum Amyloid P-component in Alzheimer's Disease". Thesis, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-297654.

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Alzheimer’s Disease is the most common form of dementia, affecting 48 million people worldwide. Despite this fact, only 45% of the patients have received the diagnose. The reason behind this is the fact that the cause of the disease is still unclear. Several hypotheses have been suggested, with main focus in the imbalance between the production and the clearance of Αβ in the brain (formation of plaques) or hyperphosphorylation of the tau protein (formation of tangles). In order to have a better understanding of what is actually happening in the brain, more biomarkers need to be developed. Keeping this in mind, we tried to develop a method to monitor the protein levels of SAP in the brain. SAP is a glycoprotein, normally produced by the liver in acute phase immune responses. SAP has been correlated with AD in the 1980s and quite recently it has been shown that SAP is elevated in AD patients, but not in individuals with plaques and no dementia. For this reason, we developed a mass spectrometry based targeted quantification method for monitoring SAP in the brain, as well as C9, a blood contamination reference protein. Our method is robust enough to be further used in large studies, in order to investigate the role of SAP in AD.

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38

Gabuza, Kwazikwakhe. "Identification of differentially expressed proteins in obese rats fed different high fat diets using proteomics and bioinformatics approaches". 2013. http://hdl.handle.net/11394/3954.

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Philosophiae Doctor - PhD
Obesity is a medical condition in which an energy imbalance leads to excessive accumulation of body fat. Obesity leads to a reduction in life expectancy through its association with chronic diseases of lifestyle. The prevalence of obesity is rapidly increasing throughout the world. It is now accepted that most cases of obesity result from an interaction between genetic and environmental factors. This rapid increase in obesity generally leads to an increase in morbidity and mortality from chronic diseases such as cardiovascular disease, type 2 diabetes, osteoarthritis and cancer of which obesity is a risk factor. There is a lack of information in molecular research to explain how obesity predisposes individuals to these diseases. Proteomics is a molecular tool and a set of techniques used to identify changes at protein level from a diseased state. This study aims to identify differentially expressed proteins in serum of obese rats fed different isocaloric diets using proteomics.

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39

Makawita, Shalini. "Integrative Preoteomic Analysis of Cell Line Conditioned Media and Pancreatic Juice for the Identification of Candidate Pancreatic Cancer Biomarkers". Thesis, 2011. http://hdl.handle.net/1807/32919.

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Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

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40

Prates, João Alexandre Martins Forreta. "Pinpointing new protein and phosphoprotein biomarkers in rheumatoid arthritis by high-resolution label-free mass spectrometry analysis of liquid biopsies". Master's thesis, 2019. http://hdl.handle.net/10362/90818.

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Rheumatoid arthritis is an autoimmune inflammatory disease that attacks the joints, leading to joint destruction, if left untreated. The disease affects 0.5 to 1 % of the population in developed countries and causes great impairment to those affected and may even lead to early mortality, since the disease is usually correlated to cardiovascular events. Premature diagnosis and treatment are of the utmost importance, since it has been proved that people who began treatment within 3 months of disease onset had better outcomes, usually being able to avoid cartilage destruction in the joints. This work has the primary goal of identifying potential biomarkers in serum samples of patients with rheumatoid arthritis. Another objective was to check the phosphoproteome for differently phosphorylated peptides. In order to achieve these goals, we employed liquid chromatography-mass spectrometry to perform Label-Free Quantification of the non-phosphorylated fraction of the proteome, as well as for identifying the peptide sequences and phosphorylation present on the phosphoproteome of the different conditions. We compared serum samples from rheumatoid arthritis to healthy donors and to patients with systemic lupus erythematosus, another autoimmune disease characterised by chronic inflammation. We were able to identify 43 proteins, that were differently expressed between rheumatoid arthritis and healthy subjects. 12 of these were specific to rheumatoid arthritis. We were also able to identify 41 peptides that possessed different phosphorylation patterns between rheumatoid arthritis and healthy subjects. It was also possible to identify a kinase that appears to be active in rheumatoid arthritis, but not in healthy subjects.

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41

Hou, Hsiang-Lin, i 侯湘伶. "Proteomic Analysis of Aging-Related Biomarkers in Mouse Serum". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/90634557435628275145.

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碩士
實踐大學
食品營養與保健生技學系碩士班
101
Due to the growing populations of aging, development of biological markers for prediction of aging-related chronic diseases and prognosis tracking becomes an important issue for current biomedical research. The normal aging process is inevitable and universal. It is characterized by a general decline in physiological functions with accumulation of multiple deleterious effects. Based on the free radical theories related to the aging processes, the major source of the oxidative lesions comes from generation and accumulation of oxidants in the mitochondria within cells, becoming one of the factors leading to aging and death. In this study, we applied proteomic strategies to detect and compare serum proteins between 7 months (middle-aged) and 24-month-old (aged) mice in order to explore and isolate biomarkers of normal aging processes. The results showed that thirteen proteins were detected at different levels in expression significantly. Among these proteins, five are related to acute reactions and three are involved in the immune responses. Overall, the inflammation responses appeared to go up during aging. Further post-translational modification analysis on serum albumin in mice indicated there are four different kinds of modification related to oxidative stress including Nitration, Carbonylation, Homocysteine and 4-Hydroxy-2-nonenal; the modification ratio was significantly increased in the aged mice. Totally, all the results of this study will generate specific serum aging-related biomarkers to survey aging-related chronic diseases and prognosis tracking following treatment.

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42

Tien, Chao-Chen, i 田兆真. "Study on the proteomic analysis of rabbit serum with hepatic failure". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/05146776390923024410.

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碩士
國立中正大學
化學工程研究所
90
Abstract After the human genome has been completely sequenced, the related research of proteomics becomes more important. In this study, the technology of proteomics was used to analyze rabbit serum proteins with hepatic failure and serum proteins from healthy rabbit. The results can be useful to design a bioartificial liver and to help medical doctors for diagnosing the hepatic failure. In this study, copolymer particles of glycidyl methacrylate (GMA) and styrene were prepared by the method of dispersion copolymerization. The resultant non-porous copolymerized beads were chemically modified to introduce primary amino group, which were ready for the attachment of dye (Cibacron Blue F3GA). The prepared dye-immobilized beads were used to remove albumin from the rabbit serum. After the affinity depletion, we can figure out whether there are other proteins shielded by albumin or not. In the analysis of two-dimensional gel electrophoresis, we use 17 cm immobilized pH gradient gel (IPG) strips to absorb all proteins in rabbit serum sample. After the electrophoresis procedures of isoelectric focusing and SDS-PAGE, rabbit serum proteins could be well separated according to their pI points and molecular weights. After the silver stain, the two-dimensional gel was scanned by using an image scanner and protein spots were analyzed by the PDquest software. Comparing serum proteins from diseased rabbit with those from healthy one, some marker proteins that changed with hepatic failure were asserted. The marker spots in the two-dimensional get were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Matching their peptide mass fingerprints with database identified four marker proteins. They are Serum amyloid A-3 protein precursor (SAA3), Serum amyloid A-2 protein precursor (SAA2), Hypothetical protein and Putative serum amyloid A protein. SAA3 and SAA2 are members of the SAA family of acute-phase or inflammatory response proteins. Results obtained in this work show that the expression level of these proteins decreased, or even disappeared, during the process of hepatic failure.

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43

Chen, Ta-Jen, i 陳達人. "Utilizing artificial neural networks in serum proteomic analysis for prostate cancer detection". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/25176474922681169976.

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44

Wang, Kuo-Hui, i 王國慧. "Comparative Study of Serum Proteomic in Elite Sprinters and Long Distance Runners". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/93890241046958835939.

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博士
中國文化大學
體育學系運動教練碩博士班
101
Proteomics is the performance of genomic complement proteins, and proteomics is the protein level on functional genomics. In this study, proteomic strategies to detect and relatively elite sprinter (sprint runner group, SR), long distance runners (long-distance runner group, LDR) and theuntrainedcontrol group (control group, CON) between serum proteins. Purposes: To identify the sprint and long distance runners marathon specific serum protein and comparison of two different types of track athletes difference between the serum proteome. Methods: Serum protein fractionations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then analyzed by a quantitative nano-LC-MS/MS-based proteomic profiling. The one-way analysis of variance (ANOVA) followed by Scheffe post hoc comparison was used to determine whether there is any significant difference of each protein level between three groups, the significance level (alpha value) was set at .05. Results: (1) Of the 307 identified proteins, there were 26 unique proteins in the SR group, and 18 unique proteins in the LDR group. (2) The expression levels of 7 coagulation function-associated proteins (vitronectin, serum paraoxonase/arylesterase 1, fibulin-1, complement C3, vitamin K-dependent protein, inter-alpha-trypsin inhibitor heavy chain H3 and von Willebrand factor) were significantly decreased in the group LDR compared to group SR. (3) The expression levels of 2antioxidantproteins,afamin and glutathione peroxidase 3, were also significantly decreased in the group LDR compared to group SR. (4) The expression levels of 7 immune function-related proteins (Ig gamma-3 chain C region, Ig lambda-like polypeptide 5, clusterin, complement C1s subcomponent, complement factor B, complement C4-A, complement C1q subcomponent subunit A) were significantly decreased in the group LDR compared to group SR. Conclusion: This study results clearly identified the potential serum protein markers for elite sprinters and long distance runners. The changes in regulation of coagulation, antioxidant or immune function-specific proteins may also provide further clinical applications for these two different track athletes.

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45

Chen, Li-chiu, i 陳麗秋. "Identification of serum tumor markers for malignant disease using high-throughput proteomic technologies". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/73930771765341250337.

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碩士
長庚大學
醫學生物技術研究所
92
Background: Malignant tumors are at the top leading cause of death in Taiwan in the past decades. Currently, there is no serum marker for detecting oral or nasopharyngeal cancers (ORC or NPC), neither available serum marker with high sensitivity and specificity for breast, lung, and prostate cancers (BC, LC, and PC). Methods: Affinity purification with magnet beads and MALDI-TOF mass spectrometry analysis were used to screen and identify potential serum tumor markers for BC, LC, PC, ORC, and NPC. Compiled protein profile of each cancer group was compared to normal samples, and the differential spectra were statistically analyzed using bioinformatic softwares. Results: Since Cu bead can discriminate most differential spectra between normal and tumor group, it was used for protein profiling. Among the five tested malignant diseases except breast cancer, several markers with high sensitivity (>80%) and specificity (>90%) were found. They are 4 LC-specific markers with molecular weight of 2878±5, 3809±5, 4975±5, and 8610±5; 2 PC-specific markers with molecular weight of 1892±5 and 2022±5; 1 ORC-specific marker with molecular weight of 2664±5; and 1 NPC-specific marker with molecular weight of 2020±5. After combination of two NPC-specific markers, the detecting sensitivity was greatly increased (94%0. Finally, two most sensitive and specific markers were identified with a PC and NPC marker (2020 Da) and an ORC marker (2658 Da). They are complement C3 precursor and alpha fibrinogen, respectively. Conclusion: High-throughput proteomic approach could greatly facilitate the discovery of novel and better serum markers. 4 LC-specific markers, 2 PC-specific markers, 1 ORC-specific marker and 1 NPC-specific marker were found. The high specificity and sensitivity achieved by the use of these tumor-specific markers show great potential for the detection of the malignant diseases.

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46

Mohan, Kumar D. "Proteomic Approaches to Study Glioma Development, Progression and Therapy". Thesis, 2013. http://etd.iisc.ernet.in/2005/3306.

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Astrocytoma, the tumor of astrocytic origin, accounts for about 60 % of the primary brain tumors. As per World Health Organization grading system, astrocytoma is classified as circumscribed astrocytoma (Grade I; pilocytic astrocytoma) and diffusely infiltrating astrocytoma. Grade I tumor is biologically benign and can be cured by surgical resection of the tumor. The diffusely infiltrating astrocytoma is further subclassified into grade II/diffuse astrocytoma (DA), grade III/anaplastic astrocytoma (AA) and grade IV/glioblastoma (GBM). Aggressiveness of the disease increases as the tumor progresses from lower grade to higher grade. In particular, GBMs are the most malignant and aggressive human cancers. For a newly diagnosed GBM patient, the current treatment option is surgical resection of the tumor followed by radiation and temozolomide therapy. Despite the treatment is multimodal (surgery+radiation+temozolomide) the median survival of GBM patients remain very low at 14.6 months. Although numerous markers with potential utility in prognosis and treatment of GBMs have been reported, they are yet to be translated into clinical utility. Our knowledge of understanding the complete biology of GBMs needs further comprehensive studies towards the identification of markers with potential utility to prognose/treat the GBM patients efficiently. Therefore, with an immense need to develop new biomarkers/therapeutic strategies in order to improve the diagnosis, prognosis and existing treatment of the GBM, the current work is designed to study the following aspects on glioma

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Huang, Yun-Ju, i 黃韻如. "Studies of Differential Expressions of Serum Proteins in Patients with Endometriosis Using Proteomic Approaches". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/19696854415331456215.

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碩士
臺北醫學大學
生物醫學材料研究所
93
Endometriosis is a disease characterized by the presence of endometrial tissue of ectopic sites. The etiology of endometriosis is believed to be the dissemination of endometrial cells in the peritoneal cavity through retrograde menstruation. Endometriosis is the main cause of infertility in women at reproductive age. However, there is no efficient and low invasive procedure for diagnosis, unless laparoscopy or laparotomy, the invasive operation is applied to confirm the occurrence of endometriosis. This study aims to identify the potential serum markers for endometriosis diagnosis using proteomic approaches. By monitoring the aberrant protein expression in endometriosis compared to the controls, several different protein expressions have been identified including α1-antitrypsin, serotransferrin, haptoglobin-1. We then use the serum samples collected from 237 subjects grouped as: (1) control (n=24), (2) pregnant (n=31), (3) adenomyosis (n=10), (4) ovarian and pelvic endometriosis (n=43) and (5)endometriosis following GnRH analog treatment (n=90) by immuno-dot blot and western blot for analysis to confirm the specificity and sensitivity of the candidate protein with endomtriosis. Our data showed that α1-antitrypsin was significantly expressed in sera of women with endometriosis. Further statistics analysis indicated that α1-antitrypsin could be a potential marker for diagnosis of endometriosis. Identification of serum marker is substantial for disease diagnosis and the follow-up treatment. This proteomic approach will progress for the development of diagnosis tool and therapeutic strategies for endometriosis.

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Chang, Wan-Ching, i 張捥晴. "Serum proteomic profiles between diabetic patients and healthy adults with Tai-Chi exercise by Nano LC-ESI technology". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/47870735118954428231.

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碩士
國立中山大學
生物科學系研究所
100
We have previously used a two-dimensional fluorescence difference gel electrophoresis protein expression with matrix-assisted laser desorption ionization of mass spectrometry to identify the serum proteomic profiles before and after the Tai Chi exercise in normal adults (Yang & Chang, et al. Clin Chem 56:127, 2010). However, the high abundant serum proteins in seyal samples might interfere the discovery of low abundance proteins in two-dimensional electrophoresis, but these low abundant proteins may play an important role on human physiology. Therefore, we looked for another way to resolve this complex issue. After multiple attempts, we chose a commercial affinity column to exclude 14 kinds of high-abundance proteins before analyses of serum proteomic displays. This column could be fit into a fast liquid chromatography separation of purified proteins and eluted for low abundant proteins. The low abundant proteins were first expressed by one-dimensional gel electrophoresis of proteins followed by a series of gel cut down for in-gel digestion by trypsin and subject to nano-liquid chromatography with electrospray ionization tandem mass spectrometry analysis (nano-LC ESI MS/MS ). The results obtained by software analysis were subject to its functional pathways analysis. We analysed 3 comparisons of the protein displays including differences between normal adults before and after exercise, differences of normal adults and diabetic patients before and after exercise. Experiments were next performed to validate the most significant difference of proteins between each category by enzyme-linked immunoassay. Results showed that dipeptidyl peptidase 4 (DPP4) and neutrophil gelatinase-associated lipocalin (NGAL) proteins were significantly higher in diabetic patients than in normal adults ( P values were 0.011 and less than 0.001), while the prolactin-inducible protein (PIP) was higher in normal adults than in diabetic patients after exercise (P value of 0.042). To our knowledge, decrease of DPP4 in type 2 diabetes has been shown to reduce blood sugar and improve the immunity; and NGAL has been confirmed to be an indicator for early diagnosis of acute kidney injury. Therefore, we have identified certain functional proteomic markers in normal and diabetic patients after Tai Chi exercise. This study model with exclusion of high-abundance serum proteins is a useful mode for identifying immune and metabolic marker with and without.

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Lai, Li-Jung, i 賴麗絨. "Proteomic analysis of bovine serum albumin subjected to glycation and anti-glycative activity enhancement of miso fermented with resveratrol supplementation". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/4e9dfk.

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博士
國立嘉義大學
食品科學系研究所
106
Advanced glycation end products (AGEs) produced and accumulated in the human body leading to subsequent abnormal physiological functions is of concern for the public. In the recent years, most in vitro determinations of glycation was conducted at 37oC of body temperature. However, it takes quite long period for each determination. In this study, bovine serum albumin-fructose (BSA-FRU) was used as the reaction system reacted at an elevated temperature with an attempt to develop a time-saving and effective procedure for glycation assessment. Measurement of the produced fluorescent AGEs revealed that the reaction at 50oC for 12 h was equivalent to that produced by reaction at 37oC for 4 days. After reaction by BSA with fructose at 50°C for 24 h for glycation and subjected to SDS-PAGE analysis, the protein patterns showed that 66 KDa-band tended to aggregate to form the 97 KDa-band and cross-linked proteins due to glycation. Additionally, the 50 KDa-band decreased along with appearance of the detected 97 KDa-band of glycated protein. When bands of 50 KDa and 97 KDa were cut and subjected to proteomic analysis and MASCOT NCBInr database search, both sequences were mostly matched with that of gi | 1351907. Both are belonging to BSA is apparent. As estimated, AGEs formation at 50oC for 24 h is approximately equivalent to that formed at 37oC for 5 weeks. Further comparison of the detected peptide sequences with those reported in the literature addressed on formation and disappearance of AGEs-precursor and AGEs during glycation, the peptide segments of KPLLEK (aa 304-309) and QIKKQTALVEL (aa 545-555 containing K548 ) susceptible to be initiated for modification and subsequent glycation to form cross-linking and glycated peptide fragments. Thus, it is of merit that BSA-FRU reaction at 50oC is time effective to determine fluorescent intensity along with SDS-PAGE to check disappearance of the 50 KDa-band and formation of the 97 KDa-band. In further comparison with the sequences of human serum albumin, common presence of these two glycation-sensitive peptide fragments might be of potency to be used in development of rapid approaches in monitoring status of human blood glycation status. Multi-functional and biological activities of resveratrol have attracted extensive attention. In this study, miso was supplemented with resveratrol with doses of 0, 500, 1000 and 2500 ppm for fermentation. During fermentation, microbial enumeration and chemical analysis revealed that resveratrol did not affect the fermentation of miso. It is noticed that antioxidant and anti-glycative activities increased significantly with an increase of time of fermentation. Additional dose dependent antioxidant and anti-glycative activities were detected in the resveratrol-supplemented miso products. After subjection of the 8-week-fermented products to sensory evaluation, all products supplemented with resveratrol up to 1000 ppm were accepted. In particular, the products supplemented with 1000 ppm after cook were organoleptically accepted and bearing potential antioxidant and anti-glycative activities could be regarded as a functional and of merit traditional food.

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Schwedler, Sina [Verfasser]. "Voruntersuchungen zur mehrdimensionalen Proteomanalyse mittels nativer chromatographischer Trennungen anhand des Modell-Proteoms "humanes Serum" / von Sina Schwedler". 2007. http://d-nb.info/986918024/34.

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