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Protein Microarray: "Theory" to "Real Practice"Ng, Jin Kiat, Ajikumar, Parayil Kumaran, Lee, Jim Yang, Stephanopoulos, Gregory, Too, Heng-Phon 01 1900 (has links)
Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems. / Singapore-MIT Alliance (SMA)
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Advances in protein microarray technology for glycomic analysisPropheter, Daniel Champlin 13 October 2011 (has links)
The cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins.
This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins.
Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale. / text
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Stationary and temporal structure of antibody titer distributions to human influenza A virus in southern VietnamNhat, Nguyen Thi Duy January 2017 (has links)
Seroepidemiology aims to understand population-level exposure and immunity to infectious disease. Serological results are normally presented as binary outcomes describing the presence or absence of antibody, despite the fact that many assays measure continuous quantities. A population's antibody titers may include information on multiple serological states - naiveté, recent infection, non-recent infection, childhood infection - not just seropositivity or seronegativity. In the first part of this thesis, I investigate 20,152 general-population serum samples from southern Vietnam collected between 2009 and 2013 from which I report antibody titers to the influenza virus HA1 protein using a continuous titer measurement from a protein microarray assay. I describe titer distributions to subtypes 2009 H1N1 and H3N2, and using a model selection approach for mixture distributions, I determine that 2009 H1N1 is best described by four titer subgroups while H3N2 is best described by three titer subgroups. For H1N1, my interpretation is that the two highest-titer subgroups correspond to recent and historical infection, which is consistent with pandemic attack rates. For H3N2 however, right-censoring of titers makes interpretations difficult to validate. To move beyond this stationary interpretation of titers, I developed two methods for analyzing this serum collection as a time series. First, I attempted to analyze mixture categories in individual time windows. This approach did not lead to a consistent temporal picture of titer change; results differed by site and were sensitive to assumptions in the mixture fitting. Second, I attempted to fit a hybrid-dynamical model with free incidence parameters. This inference was robust to parameter assumptions, consistent across sites, and in agreement with the incidence reported in Vietnam's influenza surveillance network. In addition, my new approach showed evidence that there was a second silent wave of the 2009 influenza pandemic that was not recorded in national surveillance, which is this thesis' main novel result.
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The Structure and Stability of Alpha-Helical, Orthogonal-Bundle Proteins on SurfacesWei, Shuai 29 June 2010 (has links) (PDF)
The interaction of proteins with surfaces is a major problem involved in protein microarrays. Understanding protein/surface interactions is key to improving the performance of protein microarrays, but current understanding of the behavior of proteins on surfaces is lacking. Prevailing theories on the subject, which suggest that proteins should be stabilized when tethered to surfaces, do not explain the experimentally observed fact that proteins are often denatured on surfaces. In an attempt to develop some predictive capabilities with respect to protein/surface interactions, it was asked in previous works if the stabilization/destabilization of proteins on surfaces could be correlated to secondary structure and found that no link existed. However, further investigation has revealed that proteins with similar tertiary structure show predictable stabilization patterns. In this research, it is reported how five, alpha-helical, orthogonal-bundle proteins behave on the surface compared to the bulk. By measuring stabilization using melting temperatures and the Gibbs energies of folding, it is shown that the stability of proteins tethered to surfaces can be correlated to the shape of the loop region where the tether is placed and the free rotation ability of the part of proteins near surfaces. It is also shown that any destabilization that occurs because of the surface is an enthalpic effect and that surfaces always stabilize proteins entropically. Furthermore, the entropical stabilization effect comes from unfolded states of the tethered protein, while the enthalpical destabilization effect is from the folded states of protein. A further analysis of surface induced change of folding mechanism is also studied with a multi-state protein 7LZM in this research. The result showed that by tethering a protein on a surface, the melting temperature of part of the protein changed, which leads to a miss of state.
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Protein-Surface Interactions with Coarse-Grain Simulation MethodsWei, Shuai 19 March 2013 (has links) (PDF)
The interaction of proteins with surfaces is a major process involved in protein microarrays. Understanding protein-surface interactions is key to improving the performance of protein microarrays, but current understanding of the behavior of proteins on surfaces is lacking. Prevailing theories on the subject, which suggest that proteins should be stabilized when tethered to surfaces, do not explain the experimentally observed fact that proteins are often denatured on surfaces. This document outlines several studies done to develop a model which is capable of predicting the stabilization and destabilization of proteins tethered to surfaces. As the start point of the research, part of this research showed that the stability of five mainly-alpha, orthogonal-bundle proteins tethered to surfaces can be correlated to the shape of the loop region where the tether is placed and the free rotation ability of the part of proteins near surfaces. To test the expandability of the protein stability prediction pattern derived for mainly-alpha, orthogonal-bundle proteins, same analysis is performed for proteins from other structure motifs. Besides the study in these small two-state proteins, a further analysis of surface-induced change of folding mechanism is also studied with a multi-state lysozyme protein 7LZM. The result showed that by tethering a protein on a surface, the melting temperature of a part of the protein changed, which leads to an avoidance of the meta-stable state. Besides the change of folding mechanism, by tethering the lysozyme protein to a certain site, the protein could both keep a stable structure and a good orientation, allowing active sites to be available to other proteins in bulk solution. All the work described above are done with a purely repulsive surface model which was widely used to roughly simulate solid surfaces in protein microarrays. For a next-level understanding of protein-surface interactions, a novel coarse-grain surface model was developed, parameterized, and validated according to experimental results from different groups. A case study of interaction between lysozyme protein 7LZM and three types of surfaces with the novel model has been performed. The results showed that protein stabilities and structures are dependent on the types of surfaces and their different hydrophobicities. This result is consistent with previously published experimental work.
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New micropatterning techniques for the spatial addressable immobilization of proteinsFilipponi, Luisa, n/a January 2006 (has links)
Bio-microdevices are miniaturised devices based on biologically derived components
(e.g., DNA, proteins, and cells) combined or integrated with microfabricated substrates.
These devices are of interest for numerous applications, ranging from drug discovery, to
environmental monitoring, to tissue engineering. Before a bio-microdevice can be fully
developed, specific fabrication issues need to be addressed. One of the most important
is the spatial immobilization of selected biomolecules in specific micro-areas of the
device. Among the biomolecules of interest, the controlled immobilization of proteins to
surfaces is particularly challenging due to the complexity of these macromolecules and
their tendency to lose bioactivity during the immobilization step. The present Thesis
reports on three novel micropatterning techniques for the spatial immobilization of
proteins with bioactivity retention and improved read-out of the resulting micropatterns.
The technologies developed are based on three different micropatterning approaches,
namely 1) direct-writing UV laser microablation (proLAB), 2) a novel microcontact
printing method (�CPTA) and 3) a replica molding method combined with bead selfassembly
(BeadMicroArray). The first two technologies, proLAB and �CPTA, are an
implementation of existing techniques (laser ablation and �CP, respectively), whereas
the third, i.e., the BeadMicroArray, is a totally new technique and type of patterning
platform.
'ProLAB' is a technology that uses a micro-dissection tool equipped with a UV laser
(the LaserScissors�) for ablating a substrate made of a layer of ablatable material, gold,
deposited over a thin polymer layer. The latter layer is transparent to the laser but
favours protein adsorption. In the present work microchannels were chosen as the
structure of interest with the aim of arranging them in 'bar-codes', so to create an
'information-addressable' microarray. This platform was fabricated and its application
to specific antigen binding demonstrated.
The second technique that was developed is a microstamping method which exploits the
instability of a high-aspect ratio rubber stamp fabricated via soft-lithography. The
technique is denominated microcontact printing trapping air (�CPTA) since the collapsing of a rubber stamp made of an array of micro-pillars over a plane glass surface
resulted in the formation of a large air gap around the entire array. The method can be
successfully employed for printing micro-arrays of proteins, maintaining biological
activity. The technique was compared with robotic spotting and found that microarrays
obtained with the �CPTA method were more homogeneous and had a higher signal-tonoise
ratio.
The third technique developed, the BeadMicroArray, introduces a totally new platform
for the spatial addressable immobilization of proteins. It combines replica molding with
microbead self-assembling, resulting in a platform where diagnostic beads are entrapped
at the tip of micropillars arranged in a microarray format. The fabrication of the
BeadMicroArray involves depositing functional microbeads in an array of V-shaped
wells using spin coating. The deposition is totally random, and conditions were
optimised to fill about half the array during spin coating. After replica molding, the
resulting polymer mold contains pyramid-shaped posts with beads entrapped at the very
tip of the post. Thanks to the fabrication mode involved, every BeadMicroArray
fabricated contains a unique geometric code, therefore assigning a specific code to each
microarray. In the present work it was demonstrated that the functionality of the beads
after replica molding remains intact, and that proteins can be selectively immobilized on
the beads, for instance via biorecognition. The platform showed a remarkable level of
selectively which, together with an efficient blocking towards protein non-specific
adsorption, lead to a read-out characterized by a very good signal-to-noise. Also, after
recognition, a code was clearly visible, therefore showing the encoding capacity of this
unique microarray.
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Development and Application of Lysate Microarray Technology for Quantitative Analysis of Human DiseaseYe, Albert Shanbuo 28 August 2013 (has links)
Reductionist biology has yielded tremendous insight into the basis of biochemistry and genetic disease. However, the remarkable failure of reductionist biology to explain complex problems, especially cancer, has led to the development of systems biology. The vast complexity of biological systems remains the most difficult problem in biology today. In order to understand this complexity, we need tools to massively multiplex measurements of a signaling network. Therefore, we developed lysate microarray technology to fill this need. In this work, we discuss three ways in which lysate microarrays were applied to human disease. In the first work, we discuss a key stage in malaria development. The liver-stage malaria parasite represents a promising target for intervention, and we present the first use of lysate microarray technology as a screening tool for host-parasite interactions in an infectious disease. We identified three cancer-related pathways that are modified in malaria infection, and studied the p53 pathway in depth. Our finding that the parasite downregulates p53 and that treatment with Nutlin-3 strongly decreases parasite load may lead to the development of a prophylactic malaria vaccine. In the second work, we began by screening drug combinations and varying dosing schedule in triple-negative breast cancers (TNBCs). We systematically explored stimulation space and collected a large lysate microarray dataset, which was used for statistical analysis. We identified a sensitization effect when a growth factor signaling inhibitor was presented before a genotoxic agent. This sensitization was generalizable among a subset of TNBCs and may generally be important for cancers driven by growth factor signaling, as we found the effect extends to nonTNBC cancers. We hope this data will be useful in guiding cancer treatment strategies in patients. In the third work, we study the changing role of the DNA Damage Response (DDR) as a cell line evolves towards cancer. We used the MCF10A progression series and studied how these cell lines respond to genotoxic agents. We identified differences in cell fates after treatment, and collected a large lysate microarray dataset for statistical analysis. Early analysis of the data indicates gross rewiring within the DDR between the MCF10A cell lines.
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Regulation of FGF Receptor 1 by Nedd4-1Persaud, Avinash 19 June 2014 (has links)
The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1.
To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation.
Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography.
Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.
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Regulation of FGF Receptor 1 by Nedd4-1Persaud, Avinash 19 June 2014 (has links)
The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1.
To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation.
Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography.
Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.
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3D-Microstructured Protein Chip for Cancer Diagnosis / Diagnostic du cancer par puces à protéines 3DYang, Zhugen 20 July 2012 (has links)
Un système est dit robuste s'il est possible de garantir son bon comportement Le cancer est en passe de devenir la première cause de décès dans le monde avec un nombre de cas de cancer qui a pratiquement doublé sur les trente dernières années. Le diagnostic du cancer est d’autant plus important qu’il est maintenant reconnu que, plus la prise en charge du patient est rapide, plus les traitements thérapeutiques sont efficaces. Ce diagnostic doit être précis, fiable, et établi dans les premiers stades de la maladie afin d’augmenter significativement les chances de succès du/des traitements. Les techniques conventionnelles pour le diagnostic du cancer sont essentiellement basées sur des techniques d’imagerie (radiographies, IRM…) associés à des tests cytologiques et biochimiques. Avec le développement récent des technologies de biologie moléculaire (et notamment en protéomique), de nombreux marqueurs tumoraux ont été identifiés et sont utilisés dans des tests d’immunoassay pour le diagnostic voire pronostic du cancer en oncologie clinique. Cependant, le faible taux de marqueurs tumoraux dans le sérum de patient, ainsi que leur grande diversité, sont un challenge important pour l’établissement d’un diagnostic d’autant plus que les techniques de détection souffrent souvent d’un manque de sensibilité et de sélectivité. De plus, du fait de la diversité et de la variabilité des cancers, aucun marqueur tumoral n’est suffisamment spécifique pour permettre un diagnostic précis. Aussi, afin d’augmenter la fiabilité et la précision du diagnostic, il est nécessaire d’utiliser plusieurs marqueurs tumoraux. Dans ce contexte, grâce à leur capacité d’analyse haut débit en parallèle et le faible volume d’échantillon nécessaire, les technologies de puces à protéines (protein microarray)présentent de nombreux avantages pour l’identification de marqueurs tumoraux associés à la réponse humorale. Comme les marqueurs tumoraux sont souvent présents dans les échantillons en très faible quantité (à l’échelle sub micro-molaire), il y a un besoin urgent de développer des puces à protéines avec une détection ultrasensible de marqueurs tumoraux. La spécificité du diagnostic sera fortement liée au choix des protéines que l’on veut détecter(notées protéines cibles) et par conséquent au choix des protéines sondes que l’on va immobiliser sur le support. Un des paramètres critiques dans le développement de puces à protéines sensibles est la chimie de surface qui détermine le mode d’immobilisation de la protéine sonde sur le support et influence son activité biologique et donc sa capacité à reconnaitre et interagir avec la protéine cible que l’on cherche à détecter. Comme de nombreuses études suggèrent qu’un seul biomarqueur n’est pas suffisamment spécifique et sensible, la recherche d’une combinaison pertinente de biomarqueurs est un axe important pour l’amélioration d’un tel diagnostic. L’objectif de ce travail de thèse est donc le développement d’un outil original basé sur la technologie de puces à protéines fonctionnalisées avec différentes chimies de surface pour la détection sensible et spécifique de biomarqueurs tumoraux afin d’améliorer le diagnostic du cancer. Deux types de puces à protéines seront développés pour des applications différentes. Une première puce, avec comme protéines sondes des anticorps, sera développée pour la détection de biomarqueurs tumoraux impliqués dans le cancer colorectal. Une deuxième puce, où les protéines sondes seront des antigènes, sera étudiée en vue de l’identification de réponses autoimmunes de patientes atteintes d’un cancer du sein. [...] / Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients’ and 28 healthy donors’ sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.
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