Relevant bibliographies by topics / Protein microarray

Academic literature on the topic 'Protein microarray'

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Published: 4 June 2021
Last updated: 14 March 2023

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Journal articles on the topic "Protein microarray"

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Shao, Weiping, Zhimin Zhou, Isabelle Laroche, Hong Lu, Qiuling Zong, Dhavalkumar D. Patel, Stephen Kingsmore, and Steven P. Piccoli. "Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling." Journal of Biomedicine and Biotechnology 2003, no. 5 (2003): 299–307. http://dx.doi.org/10.1155/s1110724303209268.

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Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/ml range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.
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ZHANG, YONG. "INTEGRATION OF NANOPARTICLES WITH PROTEIN MICROARRAYS." International Journal of Nanoscience 05, no. 02n03 (April 2006): 189–94. http://dx.doi.org/10.1142/s0219581x0600422x.

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A variety of DNA, protein or cell microarray devices and systems have been developed and commercialized. In addition to the biomolecule related analysis, they are also being used for pharmacogenomic research, infectious and genetic disease and cancer diagnostics, and proteomic and cellular analysis.1 Currently, microarray is fabricated on a planar surface; this limits the amount of biomolecules that can be bounded on the surface. In this work, a planar protein microarray chip with nonplanar spot surface was fabricated to enhance the chip performance. A nonplanar spot surface was created by first coating the silica nanoparticles with albumin and depositing them into the patterned microwells. The curve surfaces of the nanoparticles increase the surface area for immobilization of proteins, which helps to enhance the detection sensitivity of the chip. Using this technique, proteins are immobilized onto the nanoparticles before they are deposited onto the chip, and therefore the method of protein immobilization can be customized at each spot. Furthermore, a nonplanar surface promotes the retention of native protein structure better than planar surface.2 The technique developed can be used to produce different types of microarrays, such as DNA, protein and antibody microarrays.
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Chatterjee, Deb K., Kalavathy Sitaraman, Cassio Baptista, James Hartley, Thomas M. Hill, and David J. Munroe. "Protein Microarray On-Demand: A Novel Protein Microarray System." PLoS ONE 3, no. 9 (September 24, 2008): e3265. http://dx.doi.org/10.1371/journal.pone.0003265.

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Joos, Thomas. "Protein microarray technology." Expert Review of Proteomics 1, no. 1 (June 2004): 1–3. http://dx.doi.org/10.1586/14789450.1.1.1.

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Stoll, Dieter. "Protein microarray technology." Frontiers in Bioscience 7, no. 1-3 (2002): c13. http://dx.doi.org/10.2741/stoll.

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Joos, T. O. "PROTEIN MICROARRAY TECHNOLOGY." Shock 21, Supplement (March 2004): 1. http://dx.doi.org/10.1097/00024382-200403001-00002.

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Doerr, Allison. "Protein microarray velcro." Nature Methods 2, no. 9 (September 2005): 642. http://dx.doi.org/10.1038/nmeth0905-642a.

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Hall, David A., Jason Ptacek, and Michael Snyder. "Protein microarray technology." Mechanisms of Ageing and Development 128, no. 1 (January 2007): 161–67. http://dx.doi.org/10.1016/j.mad.2006.11.021.

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Templin, Markus F., Dieter Stoll, Monika Schrenk, Petra C. Traub, Christian F. Vöhringer, and Thomas O. Joos. "Protein microarray technology." Trends in Biotechnology 20, no. 4 (April 2002): 160–66. http://dx.doi.org/10.1016/s0167-7799(01)01910-2.

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Templin, Markus F., Dieter Stoll, Monika Schrenk, Petra C. Traub, Christian F. Vöhringer, and Thomas O. Joos. "Protein microarray technology." Drug Discovery Today 7, no. 15 (August 2002): 815–22. http://dx.doi.org/10.1016/s1359-6446(00)01910-2.

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Dissertations / Theses on the topic "Protein microarray"

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Klenkar, Goran. "Protein Microarray Chips." Doctoral thesis, Linköping : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8904.

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Zhou, Ye. "Microcontact printing for protein microarray applications /." Linköping : Univ, 2004. http://www.bibl.liu.se/liupubl/disp/disp2004/tek886s.pdf.

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Voelker, Alden Earl. "Selective Fusion-Tag-Catalyzed Protein Immobilizations for Microarray and Biosensor Applications." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1370448393.

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Bove, Elia <1978&gt. "Identification of surface protein complexes of Streptococcus pyogenes through protein microarray technology." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1686/2/BoveElia_Tesi.pdf.

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A systematic characterization of the composition and structure of the bacterial cell-surface proteome and its complexes can provide an invaluable tool for its comprehensive understanding. The knowledge of protein complexes composition and structure could offer new, more effective targets for a more specific and consequently effective immune response against a complex instead of a single protein. Large-scale protein-protein interaction screens are the first step towards the identification of complexes and their attribution to specific pathways. Currently, several methods exist for identifying protein interactions and protein microarrays provide the most appealing alternative to existing techniques for a high throughput screening of protein-protein interactions in vitro under reasonably straightforward conditions. In this study approximately 100 proteins of Group A Streptococcus (GAS) predicted to be secreted or surface exposed by genomic and proteomic approaches were purified in a His-tagged form and used to generate protein microarrays on nitrocellulose-coated slides. To identify protein-protein interactions each purified protein was then labeled with biotin, hybridized to the microarray and interactions were detected with Cy3-labelled streptavidin. Only reciprocal interactions, i. e. binding of the same two interactors irrespective of which of the two partners is in solid-phase or in solution, were taken as bona fide protein-protein interactions. Using this approach, we have identified 20 interactors of one of the potent toxins secreted by GAS and known as superantigens. Several of these interactors belong to the molecular chaperone or protein folding catalyst families and presumably are involved in the secretion and folding of the superantigen. In addition, a very interesting interaction was found between the superantigen and the substrate binding subunit of a well characterized ABC transporter. This finding opens a new perspective on the current understanding of how superantigens are modified by the bacterial cell in order to become major players in causing disease.
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Bove, Elia <1978&gt. "Identification of surface protein complexes of Streptococcus pyogenes through protein microarray technology." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1686/.

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A systematic characterization of the composition and structure of the bacterial cell-surface proteome and its complexes can provide an invaluable tool for its comprehensive understanding. The knowledge of protein complexes composition and structure could offer new, more effective targets for a more specific and consequently effective immune response against a complex instead of a single protein. Large-scale protein-protein interaction screens are the first step towards the identification of complexes and their attribution to specific pathways. Currently, several methods exist for identifying protein interactions and protein microarrays provide the most appealing alternative to existing techniques for a high throughput screening of protein-protein interactions in vitro under reasonably straightforward conditions. In this study approximately 100 proteins of Group A Streptococcus (GAS) predicted to be secreted or surface exposed by genomic and proteomic approaches were purified in a His-tagged form and used to generate protein microarrays on nitrocellulose-coated slides. To identify protein-protein interactions each purified protein was then labeled with biotin, hybridized to the microarray and interactions were detected with Cy3-labelled streptavidin. Only reciprocal interactions, i. e. binding of the same two interactors irrespective of which of the two partners is in solid-phase or in solution, were taken as bona fide protein-protein interactions. Using this approach, we have identified 20 interactors of one of the potent toxins secreted by GAS and known as superantigens. Several of these interactors belong to the molecular chaperone or protein folding catalyst families and presumably are involved in the secretion and folding of the superantigen. In addition, a very interesting interaction was found between the superantigen and the substrate binding subunit of a well characterized ABC transporter. This finding opens a new perspective on the current understanding of how superantigens are modified by the bacterial cell in order to become major players in causing disease.
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Qureshi, Aaron M. "A combinatorial design of a protein-binding DNA microarray." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/2081.

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Thesis (M.S.) -- University of Maryland, College Park, 2004.
Thesis research directed by: Dept. of Mathematics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Sundberg, Mårten. "Protein microarrays for validation of affinity binders." Licentiate thesis, KTH, Proteomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-48256.

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Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.
QC 20111117
Development and applications of protein microarrays
The Swedish Human Proteome Resource (HPR) program
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Scietti, Luigi Angelo Domenico <1986&gt. "Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6994/1/Luigi_Scietti_PhD_thesis_final.pdf.

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Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.
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Scietti, Luigi Angelo Domenico <1986&gt. "Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6994/.

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Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.
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Riba, Michela. "Mitochip, microarray of human mitochondrial protein genes, development and applications." Thesis, Open University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599927.

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Books on the topic "Protein microarray"

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Dev, Kambhampati, ed. Protein microarray technology. Weinheim: Wiley-VCH, 2004.

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Wu, Catherine J., ed. Protein Microarray for Disease Analysis. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0.

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Protein microarray for disease analysis: Methods and protocols. New York, NY: Humana Press, 2011.

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Wang, Lin. New Antibody Microarray Tube for Cellular Localization and Signaling Pathways. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009.

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Microarray analysis. Hoboken, NJ: Wiley-Liss, 2003.

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Amaratunga, Dhammika, and Javier Cabrera, eds. Exploration and Analysis of DNA Microarray and Protein Array Data. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2003. http://dx.doi.org/10.1002/9780470317129.

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Javier, Cabrera, ed. Exploration and analysis of DNA microarray and protein array data. Hoboken, NJ: John Wiley, 2004.

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Amaratunga, Dhammika. Exploration and analysis of DNA microarray and protein array data. Hoboken, NJ: John Wiley, 2003.

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R, Müller U., and Nicolau Dan V, eds. Microarray technology and its applications. Berlin: Springer, 2005.

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Small molecule microarrays: Methods and protocols. New York, NY: Humana Press, 2010.

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Book chapters on the topic "Protein microarray"

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McAllister-Williams, R. Hamish, Daniel Bertrand, Hans Rollema, Raymond S. Hurst, Linda P. Spear, Tim C. Kirkham, Thomas Steckler, et al. "Protein Microarray." In Encyclopedia of Psychopharmacology, 1075. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-540-68706-1_1553.

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Clarke, Charlotte H., and Eric T. Fung. "Protein Microarray Technology." In Springer Protocols Handbooks, 451–61. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-60327-375-6_29.

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McAllister-Williams, R. Hamish, Daniel Bertrand, Hans Rollema, Raymond S. Hurst, Linda P. Spear, Tim C. Kirkham, Thomas Steckler, et al. "Protein-Binding Microarray." In Encyclopedia of Psychopharmacology, 1075. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-540-68706-1_4483.

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Sitaraman, Kalavathy, and Deb K. Chatterjee. "Protein–Protein Interactions: An Application of Tus-Ter Mediated Protein Microarray System." In Protein Microarray for Disease Analysis, 185–200. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0_12.

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Barderas, Rodrigo, Sanjeeva Srivastava, and Joshua LaBaer. "Protein Microarray-Based for." In Methods in Molecular Biology, 3–6. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1562-1_1.

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Zong, Yaping, Shanshan Zhang, Huang-Tsu Chen, Yunfei Zong, and Yaxian Shi. "Forward-Phase and Reverse-Phase Protein Microarray." In Microarrays, 363–73. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1007/978-1-59745-303-5_18.

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Winkler, Dirk F. H., Heiko Andresen, and Kai Hilpert. "SPOT Synthesis as a Tool to Study Protein–Protein Interactions." In Protein Microarray for Disease Analysis, 105–27. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0_8.

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Sibani, Sahar, and Joshua LaBaer. "Immunoprofiling Using NAPPA Protein Microarrays." In Protein Microarray for Disease Analysis, 149–61. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0_10.

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Schäferling, Michael, and Stefan Nagl. "Förster Resonance Energy Transfer Methods for Quantification of Protein–Protein Interactions on Microarrays." In Protein Microarray for Disease Analysis, 303–20. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0_19.

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Knickerbocker, Tanya, and Gavin MacBeath. "Detecting and Quantifying Multiple Proteins in Clinical Samples in High-Throughput Using Antibody Microarrays." In Protein Microarray for Disease Analysis, 3–13. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-043-0_1.

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Conference papers on the topic "Protein microarray"

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Simion, Monica, Lavinia Ruta, Irina Kleps, Carmen Mihailescu, Teodora Ignat, Dana Stan, Florin Craciunoiu, Mihaela Miu, and Adina Bragaru. "Surface Functionalization for Protein Microarray." In 2007 International Semiconductor Conference (CAS 2007). IEEE, 2007. http://dx.doi.org/10.1109/smicnd.2007.4519665.

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Wang, Xiao, Jinghua Gu, Jianhua Xuan, Ayesha N. Shajahan, Robert Clarke, and Li Chen. "Sampling-Based Subnetwork Identification from Microarray Data and Protein-Protein Interaction Network." In 2012 Eleventh International Conference on Machine Learning and Applications (ICMLA). IEEE, 2012. http://dx.doi.org/10.1109/icmla.2012.221.

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Montagu, Jean, Herman DeWeerd, Nathan Tyburczy, Natalia Rodionova, and Peter Maimonis. "Obstacles to the production of protein microarray cassettes." In SPIE MOEMS-MEMS: Micro- and Nanofabrication, edited by Wanjun Wang. SPIE, 2009. http://dx.doi.org/10.1117/12.806639.

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Yang, Rusong, Lian Wei, Ying Feng, Xiujian Li, and Quan Zhou. "Recent progress in making protein microarray through BioLP." In Fourth International Conference on Optical and Photonics Engineering, edited by Anand K. Asundi, Xiyan Huang, and Yi Xie. SPIE, 2017. http://dx.doi.org/10.1117/12.2266876.

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Simion, Monica, Lavinia Ruta, Irina Kleps, Carmen Mihailescu, Adina Bragaru, Mihaela Miu, and Teodora Ignat. "Biohybrid surface preparation for protein/DNA microarray applications." In 2008 International Semiconductor Conference. IEEE, 2008. http://dx.doi.org/10.1109/smicnd.2008.4703368.

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Young Bun Kim, Jean Gao, Johanne V. Pastor, and Kevin Rosenblatt. "Signaling biomarker pattern discovery using reverse phase protein microarray." In 2008 8th IEEE International Conference on Bioinformatics and BioEngineering (BIBE). IEEE, 2008. http://dx.doi.org/10.1109/bibe.2008.4696738.

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Wang, Xuefeng, Ming Zhao, and David D. Nolte. "A novel concept for protein microarray: land-contrast BioCD." In SPIE BiOS: Biomedical Optics, edited by Ramesh Raghavachari and Rongguang Liang. SPIE, 2009. http://dx.doi.org/10.1117/12.809358.

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Bhatti, Sabah A., Ryoji Hirota, and Narufumi Suganuma. "Validation Of Protein Microarray For Diagnosis Of Human Mesothelin." In American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a4808.

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Chen, Yu-Feng, Fan-Gang Tseng, and Ching-Chang Chieng. "Crosstalk of Parallel Components for Microfluidic Systems." In ASME 2004 2nd International Conference on Microchannels and Minichannels. ASMEDC, 2004. http://dx.doi.org/10.1115/icmm2004-2408.

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Numerical simulations based on first principle equations is used to study the crosstalk phenomena and elimination mechanism for the parallel microchannel systems as microarray. The cases of this study focus on the surface tension driven flow include inkjet printing system, stamping by stamper protein microarray with embedded microchannels and the filling of H-shaped parallel microchannels. The parallel arrangements of microfluid components in terms of configuration as well as the parallel operation mode are the key issues to ensure the least crosstalk.
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Wang, Tao, Rui Zhang, Ke-li Xing, Zi-yu Guo, Li-li Nie, and Yun-fang Jia. "Effect of Surface Modification for Protein Microarray on Immobilizing Probe." In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5515667.

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Reports on the topic "Protein microarray"

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Zangar, Richard, and Susan M. Varnum. Protein Microarray Technology for the Noninvasive Diagnosis and Prognosis of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, July 2001. http://dx.doi.org/10.21236/ada396500.

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Zangar, Richard C., and Susan M. Varnum. Protein Microarray Technology for the Noninvasive Diagnosis and Prognosis of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, July 2002. http://dx.doi.org/10.21236/ada409625.

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Zangar, Richard C. Protein Microarray Technology for the Noninvasive Diagnosis and Prognosis of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, July 2003. http://dx.doi.org/10.21236/ada425180.

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Splitter, Gary, and Menachem Banai. Microarray Analysis of Brucella melitensis Pathogenesis. United States Department of Agriculture, 2006. http://dx.doi.org/10.32747/2006.7709884.bard.

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Original Objectives 1. To determine the Brucella genes that lead to chronic macrophage infection. 2. To identify Brucella genes that contribute to infection. 3. To confirm the importance of Brucella genes in macrophages and placental cells by mutational analysis. Background Brucella spp. is a Gram-negative facultative intracellular bacterium that infects ruminants causing abortion or birth of severely debilitated animals. Brucellosis continues in Israel, caused by B. melitensis despite an intensive eradication campaign. Problems with the Rev1 vaccine emphasize the need for a greater understanding of Brucella pathogenesis that could improve vaccine designs. Virulent Brucella has developed a successful strategy for survival in its host and transmission to other hosts. To invade the host, virulent Brucella establishes an intracellular niche within macrophages avoiding macrophage killing, ensuring its long-term survival. Then, to exit the host, Brucella uses placenta where it replicates to high numbers resulting in abortion. Also, Brucella traffics to the mammary gland where it is secreted in milk. Missing from our understanding of brucellosis is the surprisingly lillie basic information detailing the mechanisms that permit bacterial persistence in infected macrophages (chronic infection) and dissemination to other animals from infected placental cells and milk (acute infection). Microarray analysis is a powerful approach to determine global gene expression in bacteria. The close genomic similarities of Brucella species and our recent comparative genomic studies of Brucella species using our B. melitensis microarray, suqqests that the data obtained from studying B. melitensis 16M would enable understanding the pathogenicity of other Brucella organisms, particularly the diverse B. melitensis variants that confound Brucella eradication in Israel. Conclusions Results from our BARD studies have identified previously unknown mechanisms of Brucella melitensis pathogenesis- i.e., response to blue light, quorum sensing, second messenger signaling by cyclic di-GMP, the importance of genomic island 2 for lipopolysaccharide in the outer bacterial membrane, and the role of a TIR domain containing protein that mimics a host intracellular signaling molecule. Each one of these pathogenic mechanisms offers major steps in our understanding of Brucella pathogenesis. Strikingly, our molecular results have correlated well to the pathognomonic profile of the disease. We have shown that infected cattle do not elicit antibodies to the organisms at the onset of infection, in correlation to the stealth pathogenesis shown by a molecular approach. Moreover, our field studies have shown that Brucella exploit this time frame to transmit in nature by synchronizing their life cycle to the gestation cycle of their host succumbing to abortion in the last trimester of pregnancy that spreads massive numbers of organisms in the environment. Knowing the bacterial mechanisms that contribute to the virulence of Brucella in its host has initiated the agricultural opportunities for developing new vaccines and diagnostic assays as well as improving control and eradication campaigns based on herd management and linking diagnosis to the pregnancy status of the animals. Scientific and Agricultural Implications Our BARD funded studies have revealed important Brucella virulence mechanisms of pathogenesis. Our publication in Science has identified a highly novel concept where Brucella utilizes blue light to increase its virulence similar to some plant bacterial pathogens. Further, our studies have revealed bacterial second messengers that regulate virulence, quorum sensing mechanisms permitting bacteria to evaluate their environment, and a genomic island that controls synthesis of its lipopolysaccharide surface. Discussions are ongoing with a vaccine company for application of this genomic island knowledge in a Brucella vaccine by the U.S. lab. Also, our new technology of bioengineering bioluminescent Brucella has resulted in a spin-off application for diagnosis of Brucella infected animals by the Israeli lab by prioritizing bacterial diagnosis over serological diagnosis.
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5

Litovchick, Larisa. Functional Characterization of the Protein Product of the Prostate Carcinoma Gene Fusion TMPRSS2:ERG Using the Proteomic and Microarray Analyses. Fort Belvoir, VA: Defense Technical Information Center, July 2009. http://dx.doi.org/10.21236/ada509751.

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6

Ghanim, Murad, Joe Cicero, Judith K. Brown, and Henryk Czosnek. Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels. United States Department of Agriculture, February 2010. http://dx.doi.org/10.32747/2010.7592654.bard.

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Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.
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Meir, Shimon, Michael S. Reid, Cai-Zhong Jiang, Amnon Lers, and Sonia Philosoph-Hadas. Molecular Studies of Postharvest Leaf and Flower Senescence. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7592657.bard.

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Original objectives: To understand the regulation of abscission by exploring the nature of changes of auxin-related gene expression in tomato (Lycopersicon esculatumMill) abscission zones (AZs) following organ removal, and by analyzing the function of these genes. Our specific goals were: 1) To complete the microarray analyses in tomato flower and leaf AZs, for identifying genes whose expression changes early in response to auxin depletion; 2) To examine, using virus-induced gene silencing (VIGS), the effect of silencing target genes on ethylene sensitivity and abscission competence of the leaf and flower AZs; 3) To isolate and characterize promoters from AZ-specific genes to be used in functional analysis; 4) To generate stable transgenic tomato plants with selected genes silenced with RNAi, under the control of an AZ-specific promoter, for further characterization of their abscission phenotypes. Background: Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many ornamentals and other fresh produce. The process is initiated by changes in the auxin gradient across the AZ, and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the initiation of the abscission process, as the AZ becomes sensitized to ethylene. The present project was focused on elucidating these early molecular regulatory events, in order to gain a better control of the abscission process for agricultural manipulations. Major conclusions, solutions, achievements: Microarray analyses, using the Affymetrix Tomato GeneChip®, revealed changes in expression, occurring early in abscission, of many genes with possible regulatory functions. These included a range of auxin- and ethylene-related transcription factors (TFs), other TFs that are transiently induced just after flower removal, and a set of novel AZ-specific genes. We also identified four different defense-related genes, including: Cysteine-type endopeptidase, α- DOX1, WIN2, and SDF2, that are newly-associated with the late stage of the abscission process. This supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses. To facilitate functional studies we implemented an efficient VIGS system in tomato, and isolated two abscission-specific promoters (pTAPG1 and pTAPG4) for gene silencing in stable transformation. Using the VIGS system we could demonstrate the importance of TAPGs in abscission of tomato leaf petioles, and evaluated the importance of more than 45 genes in abscission. Among them we identified few critical genes involved in leaf and flower abscission. These included: PTRP-F1, PRP, TKN4, KNOTTED-like homeobox TF, KD1, and KNOX-like homeodomain protein genes, the silencing of which caused a striking retardation of pedicel abscission, and ERF1, ERF4, Clavata-like3 protein, Sucrose transporter protein, and IAA10 genes, the silencing of which delayed petiole abscission. The importance of PRPand KD1 genes in abscission was confirmed also by antisense–silencing using pTAPG4. Experiments testing the effects of RNAi silencing of few other genes are still in progress, The analysis of the microarray results of flower and leaf AZs allowed us to establish a clear sequence of events occurring during acquisition of tissue sensitivity to ethylene, and to confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes in both AZs. Implication, both scientific and agricultural: Our studies had provided new insights into the regulation of the abscission process, and shaded light on the molecular mechanisms that drive the acquisition of abscission competence in the AZ. We pointed out some critical genes involved in regulation of abscission, and further expanded our knowledge of auxin-ethylene cross talk during the abscission process. This permits the development of novel techniques for manipulating abscission, and thereby improving the postharvest performance of ornamentals and other crops.
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Lichter, Amnon, Gopi K. Podila, and Maria R. Davis. Identification of Genetic Determinants that Facilitate Development of B. cinerea at Low Temperature and its Postharvest Pathogenicity. United States Department of Agriculture, March 2011. http://dx.doi.org/10.32747/2011.7592641.bard.

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Botrytis cinerea is the postharvest pathogen of many agricultural produce with table grapes, strawberries and tomatoes as major targets. The high efficiency with which B. cinerea causes disease on these produce during storage is attributed in part due to its exceptional ability to develop at very low temperature. Our major goal was to understand the genetic determinants which enable it to develop at low temperature. The specific research objectives were: 1. Identify expression pattern of genes in a coldenriched cDNA library. 2. Identify B. cinerea orthologs of cold-induced genes 3. Profile protein expression and secretion at low temperature on strawberry and grape supplemented media. 4. Test novel methods for the functional analysis of coldresponsive genes. Objective 1 was modified during the research because a microarray platform became available and it allowed us to probe the whole set of candidate genes according to the sequence of 2 strains of the fungus, BO5.10 and T4. The results of this experiment allowed us to validate some of our earlier observations which referred to genes which were the product of a SSH suppression-subtraction library. Before the microarray became available during 2008 we also analyzed the expression of 15 orthologs of cold-induced genes and some of these results were also validated by the microarray experiment. One of our goals was also to perform functional analysis of cold-induced genes. This goal was hampered for 3 years because current methodology for transformation with ‘protoplasts’ failed to deliver knockouts of bacteriordopsin-like (bR) gene which was our primary target for functional analysis. Consequently, we developed 2 alternative transformation platforms, one which involves an air-gun based technique and another which involves DNA injection into sclerotia. Both techniques show great promise and have been validated using different constructs. This contribution is likely to serve the scientific community in the near future. Using these technologies we generated gene knockout constructs of 2 genes and have tested there effect on survival of the fungus at low temperature. With reference to the bR genes our results show that it has a significant effect on mycelial growth of the B. cinerea and the mutants have retarded development at extreme conditions of ionic stress, osmotic stress and low temperature. Another gene of unknown function, HP1 is still under analysis. An ortholog of the yeast cold-induced gene, CCH1 which encodes a calcium tunnel and was shown to be cold-induced in B. cinerea was recently cloned and used to complement yeast mutants and rescue them from cold-sensitivity. One of the significant findings of the microarray study involves a T2 ribonuclease which was validated to be cold-induced by qPCR analysis. This and other genes will serve for future studies. In the frame of the study we also screened a population of 631 natural B. cinerea isolates for development at low temperature and have identified several strains with much higher and lower capacity to develop at low temperature. These strains are likely to be used in the future as candidates for further functional analysis. The major conclusions from the above research point to specific targets of cold-induced genes which are likely to play a role in cold tolerance. One of the most significant observations from the microarray study is that low temperature does not induce ‘general stress response in B. cinerea, which is in agreement to its exceptional capacity to develop at low temperature. Due to the tragic murder of the Co-PI Maria R. Davis and GopiPodila on Feb. 2010 it is impossible to deliver their contribution to the research. The information of the PI is that they failed to deliver objective 4 and none of the information which relates to objective 3 has been delivered to the PI before the murder or in a visit to U. Alabama during June, 2010. Therefore, this report is based solely on the IS data.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.

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The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Sessa, Guido, and Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695876.bard.

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The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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