Dissertations / Theses on the topic 'Protein patterning'
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1
Deb, Joyita. "Protein-hormone interactions patterning the gynoecium." Thesis, University of East Anglia, 2015. https://ueaeprints.uea.ac.uk/54301/.
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The gynoecium is among the most intricately patterned organs of the plant, comprisingdifferent tissue sub-structures, all with the purpose of facilitating propagation to the next generation. It is therefore representative of the complexity involved in the initiation and establishment of organ patterning and presents a unique model to study the mechanisms coordinating development. As with all other organs, the interplay between genetic and hormonal factors specifies carpel development. However, although much is known about the genetic components involved in carpel development, our understanding of hormonal regulation and the cross-talk between these two pathways is limited. Thus, the aim of this thesis has been to address this issue by obtaining an integrated view of the genetic and hormonal regulatory mechanisms which act to coordinate gynoecium development. It has done so using broadly two approaches: first, by characterising the transcription factor interactions which pattern the carpel, and second, by elucidating the cross-talk between these interactions and the plant hormone auxin. Further, it has also studied the role of auxin in carpel morphogenesis. Observations from this research have uncovered a novel auxin co-receptor complex formed by the transcription factors IND and ETT. The co-receptor binds the IAA molecule directly and exhibits specificity for IAA over the synthetic analogues NAA and 2,4-D. This coreceptor functions to coordinate the development of the style and stigmatic tissues of the carpel, possibly via the regulation of PID kinase. Further, this work has also identified novel roles in protein-protein dimerisation for the domains involved in this interaction. Analyses also indicate that this novel auxin signalling pathway may also be conserved in the Brassicaceae through the ETT orthologues in this family. Finally, this project has analysed how ETT’s role as an auxin receptor could be translated into precise spatiotemporal regulation of its target genes to specify the boundaries necessary for gynoecium patterning. Together, the results from this work have posed new questions as to the signalling mechanisms through which auxin coordinates its varied and numerous functions in plants.
2
Patel, Nikin. "The immobilization and micro-patterning of protein." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262955.
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Cai, Yangjun. "Simple Alternative Patterning Techniques for Selective Protein Adsorption." University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1257386752.
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Sharma, Rajan. "Protein-mediated patterning of DNA scaffolds for nanoscale electronics." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521527.
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Veiseh, Mandana. "Protein and cell patterning for cell-based biosensor applications /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/10563.
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Williams, Sophie Elizabeth. "Nanoscale surface patterning as a means of controlling protein immobilisation." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55766/.
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It would be desirable to synthesise a molecularly imprinted polymer with specific high-affinity protein recognition sites as a durable, cost-effective replacement for antibodies in biotechnology. A novel protein imprinting approach was proposed as an outline for these investigations. The focus of this project was consideration of fundamental aspects of surface nanometer-scale patterning and protein-surface interactions with the aim of preparing an ordered array of surface protein. This was in part achieved during the course of the work. An equilibrium dialysis method was validated for the measurement of ligand-protein binding parameters. Human serum albumin (HSA) and ethacrynic acid (ETH) were chosen as the ligand-protein pair to be surface-immobilised. Molecular modelling suggested a good fit for ethacrynic acid in the covalent HSA binding 'cleft', however, the covalent HSA-ETH complex was not successfully isolated. A derivative of the ligand, ETH-glycine, was synthesised to a very high purity but a low yield. A gas-phase silanisation method was developed to deposit functional aminopropyletriethoxysilane (APS) groups onto silicon wafer surfaces. The dispersion of APS could not be sufficiently controlled, by changing the evaporative distance or the APS evaporation concentration, and hence it was not possible to bring about gold nanoparticle (AuNP) patterning at the nanometer scale using this approach. However AuNP patterning could be achieved by incubating APS monolayer surfaces with different dilutions of a commercially available AuNP solution. Subsequent development of a protein imprinting strategy would require that non-specifically adsorbed HSA can be removed from PNA silicon surfaces. This was found to be difficult to achieve using mild conditions. Controlled gas-phase deposition of APS could not be used to directly facilitate dispersed ligand attachment. AuNP patterning can potentially be used as an indirect method for controlling surface dispersion of immobilised ligand. Controlled surface orientation and patterning of HSA, using the specific interaction with ETH, remains a significant challenge.
7
Ericsson, Emma. "Biosensor surface chemistry for oriented protein immobilization and biochip patterning." Licentiate thesis, Linköpings universitet, Sensorvetenskap och Molekylfysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-88102.
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This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization. Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance. Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.
8
Zamparini, Andrea. "The homeodomain protein Hex and the regulation of early embryonic patterning." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413801.
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Zhang, Feng. "Chemical Vapor Deposition of Silanes and Patterning on Silicon." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2902.
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Self assembled monolayers (SAMs) are widely used for surface modification. Alkylsilane monolayers are one of the most widely deposited and studied SAMs. My work focuses on the preparation, patterning, and application of alkysilane monolayers. 3-aminopropyltriethoxysilane (APTES) is one of the most popular silanes used to make active surfaces for surface modification. To possibly improve the surface physical properties and increase options for processing this material, I prepared and studied a series of amino silane surfaces on silicon/silicon dioxide from APTES and two other related silanes by chemical vapor deposition (CVD). I also explored CVD of 3-mercaptopropyltrimethoxysilane on silicon and quartz. Several deposition conditions were investigated. Results show that properties of silane monolayers are quite consistent under different conditions. For monolayer patterning, I developed a new and extremely rapid technique, which we termed laser activation modification of semiconductor surfaces or LAMSS. This method consists of wetting a semiconductor surface with a reactive compound and then firing a highly focused nanosecond pulse of laser light through the transparent liquid onto the surface. The high peak power of the pulse at the surface activates the surface so that it reacts with the liquid with which it is in contact. I also developed a new application for monolayer patterning. I built a technologically viable platform for producing protein arrays on silicon that appears to meet all requirements for industrial application including automation, low cost, and high throughput. This method used microlens array (MA) patterning with a laser to pattern the surface, which was followed by protein deposition. Stencil lithography is a good patterning technique compatible with monolayer modification. Here, I added a new patterning method and accordingly present a simple, straightforward procedure for patterning silicon based on plasma oxidation through a stencil mask. We termed this method subsurface oxidation for micropatterning silicon (SOMS).
10
Corbett, Sybilla Louise. "Nanoscale patterning of complex DNA structures with the bacterial protein Recombinase A." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15373/.
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The use of DNA as a structural material has been intensively developed since its inception in the early 1980s. The potential of DNA structures in the field of materials science is hampered by current approaches to augmentation. It is not currently possible to alter the targeting of heterogenous additional elements to structures once they have been made. The post hoc patterning of DNA architectures is therefore of great importance. The bacterial protein Recombinase A (RecA) may be able to provide this function. This thesis will discuss the patterning of DNA structures with RecA. RecA has been shown to pattern linear dsDNA strands with high levels of efficiency. To test the potential of RecA to pattern more complex DNA, novel strategies for creating DNA topologies have been explored. This work has produced DNA strands containing regions of base pair mismatching and with terminal three-way junctions. A method has also been developed for the creation of a 200 base product with unpaired branched junctions, using four synthetic oligomers in a scaffolded cycling ligation reaction with a heat stable ligase. A method to create longer DNA strands with three-way junctions at the termini has also been developed. RecA patterning of a structure with internal mismatches was carried out. Mismatches proximal to the patterning area led to an increase in patterning efficiency with an increase in mismatch length. When the mismatch was separated from the patterning region a more complex relationship was observed, with intermediate-length mismatches resulting in a decrease in pattering efficiency. The introduction of a nick in the phosphate backbone proximal to the patterning region also increased patterning efficiency. Two further DNA structures were produced on which patterning did not prove possible. The ligase chain reaction was shown to produce DNA strands that could be incorporated into a structure with central base pairing and terminal single stranded DNA regions. Attempts to create three-way junctions from these structures were not successful. A second structure was created through treatment of double stranded DNA from the polymerase chain reaction. Single strands of DNA were produced that could be annealed to produce terminal three-way junctions. Atomic force microscopy demonstrated the correct annealing of this structure. However, it did not prove possible to pattern these structures with RecA. Recombinant RecA production through bacterial induction produced soluble protein at a high yield. There was some evidence of DNA contamination and the purified protein showed low activity.
11
Freisinger, Christina M. "Regulator of G protein signaling 3 modulates Wnt5b calcium dynamics and somite patterning." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/667.
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The process of vertebrate development requires communication among many cells of the embryo in order to define the body axis (front/back, head/tail or left/right). The Wnt signaling network plays a key role in a vast array of cellular processes including body axis patterning and cell polarity. One arm of the Wnt signaling network, the non-canonical Wnt pathway, mediates intracellular Ca2+ release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GAPs and are uniquely situated to control the amplitude of a Wnt signal. I hypothesize that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this modulation is essential for developmental patterning events. To this end, this thesis is focused on the effects G protein regulation has on intracellular Ca2+ release dynamics and developmental patterning events.
I combine cellular, molecular and genetic analyses with high resolution Ca2+ imaging to provide new understanding of the role of RGS proteins on Wnt mediated Ca2+ release dynamics and developmental patterning events. In chapter 2, I describe endogenous Ca2+ dynamics from the very first cell divisions through early somitogenesis in zebrafish embryos. I find that each phase of zebrafish development has a distinct pattern of Ca2+ release, highlighting the complexity of Ca2+ ion and cellular physiology.
In Chapter 3, I identify rgs3 as potential modulator of Ca2+ dynamics and Chapter 4 expands upon these observations by providing data supporting that Rgs3 function is necessary for appropriate frequency and amplitude of Ca2+ release during somitogenesis and that Rgs3 functions downstream of Wnt5 activity. My results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development.
In Chapter 5, I report the identification and characterization of Rgs3b, a paralogue to Rgs3, in zebrafish. I describe results indicating that Rgs3b is poised to interact with Wnt11 indicating that individual RGS genes may have unique roles in modulating Wnt/Ca2+ signaling in different tissues or different stages. In conclusion, this thesis provides data supporting that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this is a substantial breakthrough in understanding how RGS proteins function to fine-tune known signaling pathways
12
Mughal, Muhammad Zeeshan. "Nano-patterning of hydrogenated amorphous carbon (a-C:H) surfaces for control of protein absorption." Thesis, Ulster University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603545.
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The thesis presents an investigation of the nano-patterning of hydrogenated amorphous carbon (a-C:H) surfaces to control protein adsorption. The relevant literature is first reviewed, noting the link between protein adsorption and mis-folding and its relevance to bio-compatibility and nano-toxicity. It then identifies how nano-topography influences protein adsorption, the debates and conflicts in the literature regarding this effect and the issues associated with controlling nanotopography independently of local surface composition. The first experimental chapter deals with the preparation and analysis of a-C:H patterns made by focused ion beam (FIB) milling and atomic force microscopy (AFM) nanoindentation. These methods resulted in nano-patterns with 2 nm height amplitude and 60 nm spacing, hence of size commensurate with that of proteins. The challenges associated with the production of such patterns are discussed, particularly the analysis and simulation of the implanted gallium profile in the FIB patterns. Advanced AFM techniques were used to investigate the possible compositional nature of the patterns. The Interleave/Lift method detected an onset of long range interactions between a protein coated tip and the patterned surfaces at a 38 nm tip-surface distance for both patterning methods. A hill/valley compositional contrast was also noted, stronger in the case of the FIB pattern. Short range adhesive tip/surface forces were mapped with the digital pulse force method (DPFM). Again, this showed stronger compositional contrast for the FIB pattern. These effects were interpreted as arising from the electrostatic interactions between the negatively charged protein coated tip and the patterned surfaces. Using SRIM modelling and the measured contrast values, a negative charge per adsorbed protein of 1.3-3e was estimated. The second half of the thesis investigates protein adsorption on a-C:H for four different protein/solvent systems; bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) in de-ionized (DI) water or phosphate buffer saline (PBS) solutions. Fourier transform infrared (FTIR) analysis revealed that there is a significant change in the secondary structure of the proteins once they adsorbed onto a-C:H, corresponding to an increase of the 0 -sheet component, often associated with exposure of the buried hydrophobic groups. This is also consistent with the large surface footprint of the adsorbed proteins, measured by AFM microscopy. Adsorption on FIB-patterned surfaces reveals changed adsorption behaviours, with significant increases in adsorbed foot-prints for all systems expect for the BSA-DI system where this footprint decreases slightly. Finally, adsorption experiments were carried out on patterns made by the FIB and AFM techniques. This comparison indicates that, for the FIB pattern, BSA adsorbs preferentially in the valleys whereas, for the AFM pattern, it resides on the hills. This effect, consistent with the previous analysis, was attributed to the buried charges in the valleys of the FIB pattern. Overall, the work presented in this thesis showed that nano-patterned a-C:H model surfaces are useful to study and control protein adsorption, suggesting that, in the case studied here, nano-topography modifies qualitatively the adsorption process. In addition, the methods developed here can be extended to other patterning techniques and protein systems to study independently the influence of topography and composition on protein adsorption.
13
Filipponi, Luisa, and n/a. "New micropatterning techniques for the spatial addressable immobilization of proteins." Swinburne University of Technology, 2006. http://adt.lib.swin.edu.au./public/adt-VSWT20060905.113858.
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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.
14
Reuther, Cordula. "Patterning planar surfaces with motor proteins: Towards spatial control over motile microtubules." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-20916.
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A major challenge in nanotechnology is the spatially controlled transport of cargo on the nanometer scale. The use of a nanoscale transport system based on molecular motors and filaments of the cytoskeleton proved as a promising approach to this problem. Therefore, the objective of this work was to pattern planar surfaces with motor proteins in a way that allows controlled and guided movement of microtubule-shuttles. The first part of the work was in particular focused on generating nanometer–sized tracks of motor proteins on unstructured surfaces. Specifically, microtubules themselves were used as biological templates for the stamping and alignment of motor proteins. Compared to other soft lithography techniques like microcontact printing this approach circumvented protein denaturation due to drying and conformational changes caused by mechanical stress. Given the large persistence length of microtubules their encounters with the boundaries of the nanotracks are limited to shallow approach angles. This way, the generated structures proved very efficient for the guiding of microtubules without topographical barriers. Topography-free guiding, as demonstrated in this work, is expected to significantly ease the design and fabrication of microtubule-transport systems and opens up the possibility to transport cargo of unlimited size, i.e. without any constraints by the dimensions of topographic guiding channels. Moreover, the biotemplated patterning is a promising tool for in vitro studies on the individual and cooperative action of motor proteins. In particular it might be helpful for the reconstitution of complex subcellular machineries in synthetic environments. As an example, microtubule-microtubule sliding by the biomolecular motor ncd has been shown to induce directional sliding between antiparallel microtubules and static cross-linking between parallel ones. The second part of the work explored an in-situ patterning technique for motor proteins to enable user-defined pattern designs, and investigated the achievable resolution. Photothermal patterning, based on localized light-to-heat conversion combined with a thermoresponsive polymer layer, was presented as a novel method. Specifically, the conformation of poly(N-isopropylacrylamide) (PNIPAM) molecules in aqueous solution was switched between the swollen state at T < 30°C (protein-repelling conformation) to the collapsed state at T > 33°C (protein-binding conformation) by optical signals of visible light to generate heat in a highly-localized manner. Upon heating of a light-absorbing layer on the substrate, the surface-grafted PNIPAM molecules collapsed locally and allowed motor proteins in solution to bind in the illuminated areas. To confirm the successful patterning of kinesin-1 molecules and their functionality microtubule-based gliding motility assays were performed. It was shown that the microtubules bind to the patterned kinesin-1 molecules and are transported exclusively in the patterned areas. While the achieved pattern sizes were currently in the range of ten micrometers, finite element modeling (implemented in COMSOL) showed that increased optical intensities possibly combined with cooling of the sample allow to significantly scale down the pattern dimensions. The produced patterns can be reversibly activated and deactivated at high and low temperature, respectively. Moreover, sequential patterning of multiple kinds of proteins on the same surface will be possible in a similar way without the need for specific linker molecules or elaborate surface preparation. Another advantage of the method is the use of visible light, which is versatile as any wavelength can be applied. In addition visible light is in comparison to other UV-based photopatterning techniques non-damaging to proteinsDer räumlich kontrollierte Transport von nanoskaligen Objekten ist eine große Herausforderung auf dem Gebiet der Nanotechnologie. Ein auf molekularen Motoren und Filamenten des Zellskeletts basierendes Nanotransportsystem hat sich dabei als ein viel versprechender Ansatz erwiesen. Das Ziel der vorgelegten Arbeit war es daher, ebene Oberflächen so mit Motorproteinen zu strukturieren, dass eine kontrollierte und geführte Bewegung von Mikrotubuli-Transportern ermöglicht wird. Der erste Teil der Arbeit war insbesondere darauf fokussiert, Motorprotein-Spuren im Nanometerbereich zu erzeugen. Im zweiten Teil der Arbeit wurde eine Strukturierungsmethode zur Realisierung von benutzerdefinierten Musterdesigns untersucht und die erreichbare Auflösung bestimmt. Für die Nanometerstrukturierung von Oberflächen mit funktionalen Motorproteinen wurde ein neuer Ansatz demonstriert. Mit der Anwendung von Biotemplaten, wie hier der Mikrotubuli, konnte ein hoch-lokalisiertes und orientiertes Anbinden von Proteinen an Oberflächen sowie gleichzeitig geringer Proteindenaturierung erreicht werden. Durch spezifisches Stempeln beziehungsweise Binden von Motoren wurden Muster aus funktionellen Proteinen mit hoher Oberflächendichte hergestellt. Die erzeugten Motor-Spuren haben gezeigt, dass Nanometerstrukturierungen möglich sind und ohne topographische Barrieren zu zuverlässiger Führung von Mikrotubuli führen können. Bisher konnte die nicht-topographische Strukturierung von Oberflächen mit Kinesin-1-Motoren nur im Mikrometerbereich demonstriert werden. Wegen der hohen Steifigkeit der Mikrotubuli war die thermische Energie des Systems in diesen Fällen nicht ausreichend, um die führende Spitze der Mikrotubuli zurück auf das Gebiet mit den strukturierten Motoren zu biegen. Dieses Problem wird durch die kleine Breite der hier demonstrierten Motor-Nanospuren verhindert, da das Auftreffen der Mikrotubuli mit den Grenzlinien auf extrem flache Winkel begrenzt ist. Interessanterweise haben sich Spuren des nicht-prozessiven Motors Kinesin-14 für das Führen und den Transport im Nanometerbereich als noch zuverlässiger herausgestellt als Kinesin-1-Spuren. Es ist zu erwarten, dass nicht-topographisches Führen, wie es in dieser Arbeit gezeigt wurde, das Design und die Herstellung von Mikrotubuli-Transportsystemen deutlich vereinfacht und die Möglichkeit eröffnet, Cargo mit unlimitierter Größe, d.h. ohne Einschränkungen durch die Abmessungen der topographischen Führungskanäle, zu transportieren. Zusätzlich ist die biotemplierte Strukturierung ein viel versprechendes Werkzeug um das individuelle und das kooperative Arbeiten von Motorproteinen in vitro untersuchen und komplexe subzelluläre Maschinerien in synthetischer Umgebung rekonstituieren zu können. Dies wurde am Beispiel des gerichteten Gleitens des biomolekularen Motors Kinesin-14 gezeigt, der ein gerichtetes Gleiten zwischen antiparallelen Mikrotubuli und statisches Vernetzen zwischen parallelen Mikrotubuli hervorruft. Mit dem Ansatz des biotemplierten Strukturierens ist es jedoch nicht einfach möglich, benutzerdefinierte Spuren zu erzeugen. Daher wurde die photothermische Proteinstrukturierung als eine neue Methode für die frei programmierbare, hochauflösende und schnelle Herstellung von strukturierten Proteinoberflächen eingeführt. Auf diese Weise wurden Kinesin-1-Muster durch licht-induziertes Heizen einer licht-absorbierenden Substratschicht erzeugt. Die thermisch schaltbaren poly(N-isopropylacrylamid) (PNIPAM) Moleküle auf der Oberfläche kollabierten lokal und erlaubten es den Motorproteinen, in den beleuchteten Gebieten aus der Lösung an die Oberfläche zu binden. Die Bewegung gleitender Mikrotubuli bestätigte anschließend die erfolgreiche Strukturierung der Kinesin-1-Motoren und deren Funktionalität, da die Mikrotubuli an die strukturierten Motoren banden und ausschließlich in den strukturierten Gebieten transportiert wurden. Neben der Proteinstrukturierung wurde die lokalisierte Licht-zu-Wärme-Umwandlung kombiniert mit einer thermisch schaltbaren Polymerschicht auch für die lokale Aktivierung von Kinesin-1-Motoren auf der Oberfläche genutzt. Ein Vorteil der photothermischen Proteinstrukturierung ist die Möglichkeit, sichtbares Licht zu verwenden, da jede beliebige Wellenlänge angewendet werden kann und sichtbares Licht, im Vergleich zu anderen UV-basierten Photostrukturierungsmethoden, Proteine nicht schädigt. Modellierungen mit Hilfe der Finite-Elemente-Methode (implementiert in der Software COMSOL) haben gezeigt, dass die Lichtintensität und die Oberflächentemperatur speziell eingestellt werden müssen, um definierte Strukturgrößen zu erzielen. Während die derzeitig erzeugten Muster Größen im Bereich von zehn Mikrometern hatten, könnten durch höhere optische Intensitäten kombiniert mit Kühlung der Probe die Größenordnungen signifikant reduziert werden. Die reale experimentelle Auflösung wird jedoch auch von der Schaltcharakteristik des Polymers und der Proteinbindungsdynamik abhängen. Die hergestellten Muster können reversibel bei hohen beziehungsweise niedrigen Temperaturen aktiviert und deaktiviert werden. Zusätzlich können auf die gleiche Weise verschiedene Proteinsorten sequentiell auf einer Oberfläche strukturiert werden, ohne dass spezifische Bindungsmoleküle oder aufwändige Oberflächenpräparationen notwendig wären. Die Möglichkeit, Proteine reversibel an die Oberfläche zu binden, um geschriebene Muster wieder löschen zu können, wäre eine Weiterentwicklung und würde die Anwendungsmöglichkeiten der photothermischen Strukturierungsmethode erweitern. Außerdem würden optisch schaltbare Polymere das direkte Strukturieren von Motoren mit Licht ermöglichen und daher die Methode vereinfachen
15
Gharbiah, Maey Monir. "Patterning the Mud Snail Ilyanassa obsoleta: The Role of Cell Signaling and Asymmetric Protein Localization." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195869.
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The polar lobe of Ilyanassa is asymmetrically partitioned into the D lineage of cells. Two of these cells, 3D and 4d, induce proper axial cell fate patterning in the embryo. Based on known embryological data in Ilyanassa, I hypothesized that Notch signaling would be required for this induction. I found that Notch signaling is required for cell fates induced by 4d and is temporally required well after 4d induction. Based on these results, I hypothesize that Notch signaling is involved in a reciprocal induction between the micromeres and the macromeres (endoderm) resulting in the maintenance of micromere fate induction and endoderm specification.Loss of the polar lobe results in the loss of cell fate induction by 3D/4d. Therefore, I hypothesized that proteins are asymmetrically bequeathed to the inducing D lineage cells by the polar lobe. To test this hypothesis, I compared global protein differences between two cell stage intact embryos, lobeless embryos, and isolated polar lobes by 2-Dimensional Electrophoresis analysis. I found several (12) quantitative differences between these samples including four spots enriched in the polar lobe isolates. I identified voltage-dependent anion-selective channel (VDAC) as one of the candidate proteins enriched in polar lobe isolates. I propose that VDAC is asymmetrically distributed by the polar lobe to the D cell and that it may function in D cell induction and mesendoderm fate specification.Lastly, I identify an acetylated tubulin antigen as a marker for cilia. I describe the pattern of cilia differentiation in the developing larvae that results in the formation of two ciliary bands, the prototroch and the metatroch, required for locomotion and feeding. These ciliary bands are conserved among annelid and mollusc larvae. Interestingly, the metatroch is derived from third quartet derivatives in the annelid Polygordius and from second quartet derivatives in the mollusc Crepidula. I provide evidence that the metatroch is derived from the first quartet derivatives in the mollusc Ilyanassa. Thus while the larval metatroch is conserved, its clonal origin is not. Based on these results, I provide support for the hypothesis that the metatroch is not homologous between annelids and molluscs or even among molluscs.
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Thuault, SeÌbastien. "Role of class III G-protein-coupled receptors in patterning epileptiform activity in the hippocampus." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400396.
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Yost, Cynthia Haycox. "Regulation of the dorsal-ventral axis in Xenopus embryos by intracellular components of the Wnt pathway /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/9224.
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Borukhovich, Ian. "Intrinsic Local Balancing of Hydrophobic and Hydrophilic Residues in Folded Protein Sequences." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/596407.
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Protein sequences may evolve to avoid highly hydrophobic local regions of sequence, in part because such sequences promote nonnative aggregation. Hydrophobic local sequences are avoided in proteins even in buried regions, where native structure requirements tend to favor them. In this dissertation, I describe three explorations of this hydrophobic suppression. In Chapter 2, I examine the occurrence of hydrophobic and polar residues in completely buried β-strand elements, and find evidence for hydrophobic suppression that decreases as a β-strand becomes more exposed. In Chapter 3, I present a generalized study of the tendency of local sequences to deviate from the hydropathy (hydrophobicity) expected based on their solvent exposure. First, I examined the hydropathy of local and nonlocal sequence groups over a large range of solvent exposures, within folded protein domains in the ASTRAL Compendium database; second, I calculated the tendency of residues within 10 positions of a nonpolar or polar reference residue to deviate from the hydropathy expected based on their structural environment. Both analyses suggested that protein sequences exhibit 'local hydropathic balance' across a range of 6-7 residues, meaning that polar and nonpolar residues are more dispersed in the sequence than expected based on solvent exposure patterns. This balance occurs in all major fold classes, domain sizes and protein functions. An unexpected finding was that it partly arises from a tendency of buried or exposed residues to be flanked by polar or nonpolar residues, respectively. This relationship may result from evolutionary selection for folding efficiency, which might be enhanced by reduced local competition for buried or exposed sites during folding. Finally, in Chapter 4, I present several exploratory analyses, including a decision-tree approach, to visualize the influence of a large number of sequence-structure properties on residue hydrophobicity. Overall, the work in this dissertation confirms that hydrophobic suppression and local hydropathic balance in general are intrinsic properties of folded proteins. I speculate that local hydropathic balance results from selection for reduced aggregation propensity, increased folding efficiency and increased native state specificity. The concept of local hydropathic balance might be used to improve the properties of designed and engineered proteins.
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Dhir, Vipra. "APPLICATION OF POLYELECTROLYTE MULTILAYERS FOR PHOTOLITHOGRAPHIC PATTERNING OF DIVERSE MAMMALIAN CELL TYPES IN SERUM FREE MEDIUM." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2601.
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Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offers a simple, versatile and silicon compatible approach that overcomes chemical surface patterning limitations, such as short-term stability and low protein adsorption resistance. In this study, direct photolithographic patterning of PAA/PAAm and PAA/PAH polyelectrolyte multilayers was developed to pattern mammalian neuronal, skeletal and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a negative surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell-selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent, while providing a little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods.M.S.
Department of Mechanical, Materials and Aerospace Engineering
Engineering and Computer Science
Materials Science & Engr MSMSE
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Johnson, Chrisopher W. A. "Design and development of a site specific protein patterning technique for use in a microfluidic antibody separation device." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/157341/.
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The rapid quantification of the concentration of different immunoglobulins classes from patient serum is required to diagnose patients in the early stages of sepsis. Microfluidic point of care technology can improve diagnostics by decreasing the analysis time, and integrating parallel analysis in a single portable device. The design of a novel method to fabricate surfaces presenting multiple micron scale protein motifs, for integration within a microfluidic channel device, is described in this thesis. Initial research focussed on conjugating protein motifs on silicon <100> substrates in micron and submicron scale patterns. A method involving the UV-initiated conjugation of a heterobifunctional linker, undecylenic acid N-Hydroxysuccinimde ester (UANHS), to a hydrogen terminated silicon surface was investigated. A photolithographic mask and phase mask were used to form micron and submicron UANHS motifs respectively, on silicon. The conjugation of protein with UANHS motifs was investigated to determine how reproducible the patterns were. The conjugation of streptavidin, streptavidin-FITC, NeutrAvidin, single domain protein L and multidomain protein L to silicon surfaces, upon reaction with UANHS, were investigated. Fluorescently labelled probes that associated with the protein motifs were used to confirm successful conjugation of protein to the silicon. Micron scale motifs of streptavidin, streptavidin-FITC, NeutrAvidin and single domain protein L could be formed reproducibly on silicon. Using a phase mask 140 nm motifs of streptavidin-FITC, conjugated to silicon, were achieved. Also an alternative method to pattern multiple proteins onto glass surfaces was investigated. A 500,000 MW dextran was modified to incorporate an aryl azide moiety, which was subsequently immobilised on glass surfaces. A method to synthesise and characterise the aryl azide conjugated dextran was investigated, as well as methods to characterise and improve the reproducibility of the aryl azide conjugated dextran layer immobilised on the glass surface. Two photolithographic masks and glass surfaces with alignment marks were fabricated. The masks were used to form micron scale protein motifs, via a photoinitiated conjugation reaction, on the aryl azide conjugated dextran surface. An in-house alignment system was built and a method to produce adjacent protein motifs was investigated. Two adjacent micron scale patterns of multidomain protein L and protein A were achieved. The surface density of conjugated protein L was investigated and a density of ~1.16x1011 molecules/cm2 was confirmed. This approach offers a method to attach high density micron scale protein motifs, aligned with micron scale resolution, which is vital to the realisation of a microfluidic point of care device.
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Wissner-Gross, Zachary Daniel. "Symmetry Breaking in Neuronal Development." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10639.
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Many physical systems break symmetry in their evolution. Biophysical systems, such as cells, developing organisms, and even entire populations, are no exception. Developing neurons represent a striking example of a biophysical system that breaks symmetry: neurons cultured in vitro begin as cell bodies with several tendrils (“neurites”) growing outward. A few days later, these same neurons invariably have the same new morphology: exactly one of the neurites (the “axon”) has grown hundreds of microns in length, while the others (the “dendrites”) are much shorter and are more branched. Previous work has shown that any of the neurites can become the axon, and so neurons must break symmetry during their development. The mechanisms underlying neuronal symmetry breaking and axon specification have recently attracted attention, with multiple groups proposing biophysical models to explain the phenomena. In this thesis, we perform the first analytical comparisons of these models by conducting multiple phenotypic and morphological studies of neurite growth in developing neurons. Studying neurite dynamics is technically challenging because neurites have unpredictable morphologies. In Chapter 4, we study neurite competition and neuronal symmetry breaking in hundreds of neurons by optically patterning micron-wide stripes to which the neurons adhere, and on which they grow exactly two neurites. We then use our measurements to test the accuracy of the models in the simple case when a neuron has exactly two neurites. In Chapter 5, we no longer constrain neuronal morphology. One characteristic of symmetry breaking systems is how the system’s complexity affects the symmetry breaking. We find that a majority of the models predict that neurons with more neurites break symmetry much slower than neurons with fewer neurites. Experimentally, we find that neurons with different neurite counts break symmetry at the same rate, consistent with previous observations. We then determine why the models disagree in their predictions, and rectify the models using our own experimental data. In particular, we find that neurons with higher neurite counts have higher concentrations of key proteins involved in symmetry breaking, so that neurons, regardless of neurite count, can break symmetry at the same rate.Physics
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Braun, Michelle M. "Anteroposterior patterning of the vertebrate forebrain : a role for Wnt signaling /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10666.
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Alexa, Kristen M. "Endoderm Patterning in Zebrafish: Pancreas Development: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/450.
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The pancreas is located below the liver and adjacent to the small intestine where it connects to the duodenum. It consists of exocrine and endocrine components. The exocrine portion makes enzymes which are deposited in the duodenum to digest fats, proteins, and carbohydrates. Exocrine tissue also makes bicarbonates that neutralize stomach acids. The endocrine portion produces hormones such as insulin and glucagon which are released into the blood stream. These hormones regulate glucose transport into the body's cells and are crucial for energy production. The pancreas is associated with diseases such as cancer, diabetes, Annular pancreas and Nesidioblastosis. Annular pancreas and Nesidioblastosis are congenital malformations associated with excess endocrine tissue of the pancreas and its structures. Understanding the development of the pancreas might lead to insight of these diseases.
The pancreas arises from the endoderm. In zebrafish, Nodal signaling activates mix-type and gata genes that then function together to regulate sox32 expression which is necessary and sufficient to induce endoderm formation. Interestingly, sox32 is exclusive to zebrafish and works synergistically with pou5f1 to regulate its own expression and turn on sox17 expression. sox17is evolutionarily conserved from zebrafish to mouse and is necessary for endoderm formation.
Signals from within the endoderm and the surrounding mesoderm specify regions in the endoderm to develop into the pancreas and other endodermal organs. Sonic hedgehog (shh) expression in the foregut establishes the anterior boundary of the pancreas primordium while cdx4 expression establishes the posterior boundary, but what regulates these factors is unclear. We determined that two Three Amino Acid Loop Extension (TALE) homeodomain transcription cofactors, Meis3 and Pbx4, regulate shh expression in the anterior endoderm. Disrupting either meis3 or pbx4 reduces shh expression in the anterior endoderm. As a result, anterior ectopic insulin expression occurs outside the normal pancreatic domain. Therefore, we discovered upstream regulatory factors of shhexpression in the anterior endoderm, which is necessary for patterning the endoderm and pancreas primordium.
We performed an ENU (N-ethyl-N-nitrosurea) haploid screen to look for endocrine pancreas mutants and to find other factors involved in pancreas development and patterning. From the screen, we characterized two mutants. We identified an aldh1a2 mutant, aldh1a2um22, which blocks the production of Retinoic Acid (RA) from vitamin A. While RA is known to be necessary for differentiation of the pancreas and liver, we also found it to be necessary for intestine differentiation. Two other aldh family genes exist in the zebrafish genome, but our data suggests that aldh1a2is the only Aldh that functions in endoderm differentiation and it is maternally deposited.
From the screen, we discovered a second mutant, 835.4, that spontaneously arose within the background. pou5f1 expression is normal in mutant embryos, but sox32 expression is reduced and sox17 expression is lost. Downstream endoderm genes of sox17 are also lost and as a result no endodermal organs develop. Rescue experiments indicate that the mutation is located between sox32 and sox17 in the endoderm pathway. We currently have not been successful at mapping this mutation and therefore are unable to rule out the possibility that it lies in the sox17 gene. However, our data suggest that the mutation occurs in a new gene that is necessary for sox17 expression, potentially working with sox32 and/or pou5f1.
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Reynolds, Nicholas. "Nanometre Scale Patterning and Site Specific Immobilisation of Photosynthetic Membrane Proteins." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512018.
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Liu, Man-Chi S. M. Massachusetts Institute of Technology. "Rapid 3-D laser microprinting of bioscaffolds and patterning of proteins." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/92217.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 68-72).
Tissue engineers have been developing biological substitutes to regenerate or replace damaged tissue. Tissues contain both exquisite microarchitectures and chemical cues to support cell migration, proliferation and differentiation. The majority of tissue engineering strategies use porous scaffolds containing chemical cues for culturing cells. However, these methods are unable to truly recapitulate the complexity of the in-vivo environment, limiting the effective regeneration. Several techniques have been developed to create three-dimensional patterns of proteins and 3-D print the architectures of bio-scaffolds for studying and directing cell development. Scott has developed a rapid 3-D laser microprinting system', which is able to simultaneously print the defined architecture of scaffolds and internal patterns of proteins inside scaffolds with high-speed and high-resolution. The object of this thesis is to further develop the technique of rapid 3-D laser microprinting by researching on the biological activity and functions of printed scaffolds and printed proteins. First, we constructed branched collagen microchannels containing microprinted patterns of P-selectin, a protein involved in leukocyte recruitment from blood vessels. We showed that leukocyte rolling occurred on P-selectin patterned collagen channels. Second, we presented a 3-D printed microvasculature by seeding endothelial cells into a printed collagen scaffold with capillary-like microarchitecture. Next, we performed leukocyte rolling assay within the printed microvasculature by printing the patterns of protein cues to activate the endothelium. Last, we created a 3-D microprinted collagen scaffolds for guiding and homing of cells. Cells were guided by printed P-selectin patterns and trapped in specific locations inside collagen scaffolds. All the work demonstrated that printed protein cues retain their biological activity, and the combination of printed scaffolds and patterned protein cues provides potential application for drug screening assays in biomimetic environments and cell delivery for regenerative medicine. We believe that this rapid printing technology will enable highly engineered therapeutic scaffolds for regenerative medicine applications.
by Man-Chi Liu.
S.M.
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Sjödal, My. "Specification of the lens and olfactory placodes and dorsoventral patterning of the telencephalon /." Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1347.
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Shah, Mirat. "pH-sensitive resist materials for combined photolithographic patterning of proteins and fluid lipid bilayers." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/43208.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2008.Includes bibliographical references (p. 33-34).
Photolithography of a pH-sensitive photoresist polymer was performed to pattern both lipid bilayers and proteins onto the same surface. The motivation behind this was to create a substrate mimicking an array of antigen- presenting cells. The substrate would consist of signaling ligand, biotin anti- CD3, bound to a lipid bilayer in a regular array of patches. The fluidity of the lipid bilayer would impart mobility to the signaling ligand. It was found that under appropriate substrate fabrication conditions, lipid bilayers and their associated ligand do segregate to the desired signaling patches. Additionally, the bilayer in these regions is fluid, and is potentially bioactive. This bodes well for our system as a future platform to study the actions of the helper T cell and antigen- presenting cell at the immunological synapse.
by Mirat Shah.
S.B.
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Jones, Robert Andrew. "Thermal Deposition and Electron Beam Patterning Techniques for Biopolymer Thin Films: DNA Complex and Proteins." Cincinnati, Ohio : University of Cincinnati, 2007. http://rave.ohiolink.edu/etdc//view?acc_num=ucin1196103818.
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Thesis (Ph.D.)--University of Cincinnati, 2007.Advisor: Dr. Andrew J. Steckl. Title from electronic thesis title page (viewed Jan. 26, 2010). Keywords: e-beam lithography; biopolymer; evaporation; DNA; Proteins. Includes abstract. Includes bibliographical references.
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Brannon, Mark K. "Wnt pathway-mediated transcriptional regulation of the Xenopus dorsoanterior organizing gene siamois /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/9256.
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Meyer, Néva P. "The repressor form of Gli3 plays a critical role in dorsoventral fate specification in the developing spinal cord /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/5055.
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Jordan, Katherine C. "Patterning the Drosophila eggshell and embryo through the interaction of the epidermal growth factor receptor and notch pathways /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5036.
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Persson, Madelen. "The role of transcriptional repression in Shh signalling and patterning of the ventral neural tube /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-7349-834-3/.
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Villefranc, Jacques A. "Two Distinct Modes of Signaling by Vascular Endothelial Growth Factor C Guide Blood and Lymphatic Vessel Patterning in Zebrafish: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/557.
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Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish.
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Kim, Soyoung. "A Study of Cell Polarity and Fate Specification in Early C. Elegans Embryos: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/385.
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Asymmetric cell divisions constitute a basic foundation of animal development, providing a mechanism for placing specific cell types at defined positions in a developing organism. In a 4-cell stage embryo in Caenorhabditis elegansthe EMS cell divides asymmetrically to specify intestinal cells, which requires a polarizing signal from the neighboring P2 cell. Here we describe how the extracellular signal from P2 is transmitted from the membrane to the nucleus during asymmetric EMS cell division, and present the identification of additional components in the pathways that accomplish this signaling.
P2/EMS signaling involves multiple inputs, which impinge on the Wnt, MAPK-like, and Src pathways. Transcriptional outputs downstream of these pathways depend on a homolog of β-catenin, WRM-1. Here we analyze the regulation of WRM-1, and show that the MAPK-like pathway maintains WRM-1 at the membrane, while its release and nuclear translocation depend on Wnt/Src signaling and sequential phosphorylation events by the major cell-cycle regulator CDK-1 and by the membrane-bound GSK-3 during EMS cell division. Our results provide novel mechanistic insights into how the signaling events at the cortex are coupled to the asymmetric EMS cell division through WRM-1.
To identify additional regulators in the pathways governing gut specification, we performed suppressor genetic screens using temperature-sensitive alleles of the gutless mutant mom-2/Wnt, and extra-gut mutant cks-1. Five intragenic suppressors and three semi-dominant suppressors were isolated in mom-2 suppressor screens. One extragenic suppressor was mapped to the locus ifg-1, eukaryotic translation initiation factor eIF4G. From the suppressor screen using cks-1(ne549), an allele of the self-cleaving nucleopore protein npp-10 was identified as a suppressor of cks-1(ne549)and other extra-gut mutants.
Taken together, these results help us better understand how the fate of intestinal cells are specified and regulated in early C. elegans embryos and broaden our knowledge of cell polarity and fate specification.
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Su, Vivian F. "Quantitative Analysis of Hedgehog Gradient Formation Using an Inducible Expression System: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/304.
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The Hedgehog (Hh) family of proteins are secreted growth factors that play an essential role in the embryonic development of all organisms and the main components in the pathway are conserved from insects to humans. These proteins affect patterning and morphogenesis of multiple tissues. Therefore, mutations in the Hh pathway can result in a wide range of developmental defects and oncogenic diseases. Because the main components in the pathway are conserved from insects to humans, Drosophilahas been shown to provide a genetically tractable system to gain insight into the processes that Hh is involved in.
In this study, the roles of Hh cholesterol modification and endocytosis during gradient fonnation are explored in the Drosophila larval wing imaginal disc. To exclude the possibility of looking at a redistribution of preexisting Hh instead of Hh movement, a spatially and temporally regulated system has been developed to induce Hh expression. Functional Hh-GFP with and without the cholesterol-modification was expressed in a wild-type or shi-tslendocytosis mutant background. The Gal80 system was used to temporally express (pulse) the Hh-GFP transgenes to look at the rate of Hh gradient formation over time and determine whether this process was affected by cholesterol modification and/or endocytosis.
Hh with and without cholesterol were both largely detected in punctate structures and the spreading of the different forms of Hh was quantified by measuring distances of these particles from the expressing cells. Hh without cholesterol showed a greater range of distribution, but a lower percentage of particles near the source. Loss of endocytosis blocked formation of intracellular Hh particles, but did not dramatically alter its movement to target cells. Staining for Hh, its receptor Ptc and cortical actin revealed that these punctate structures could be classified into four types of Hh containing particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cholesterol is specifically required for the formation of cytoplasmic particles lacking Ptc. While previous studies have shown discrepancies in the localization of Hh following a block in endocytosis, Hh with and without cholesterol is detected at both apical and basolateral surfaces, but not at basal surfaces. In the absence of cholesterol and endocytosis, Hh particles can be observed in the extracellular space.
Through three-dimensional reconstruction and quantitative analysis, this study concludes that the cholesterol modification is required to restrict Hh movement. In addition, the cholesterol modification promotes Ptc-independent internalization. This study also observes that Dynamin-dependent endocytosis is necessary for internalization but does not play an essential role in Hh distribution. The data in this thesis supports the model in which Hh movement occurs via planar diffusion.
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Klattenhoff, Carla Andrea. "piRNA Function and Biogenesis in the Drosophila Female Germline: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/395.
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The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline.
We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. We conclude that piRNA based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline.
We have also identified a new member of the piRNA pathway. We show that mutations in rhino, which encodes a rapidly evolving Heterochromatin Protein 1 (HP1) chromo box protein, lead to germline specific DNA break accumulation, trigger Chk2 kinase dependent defects in axis specification, and disrupt germline localization of Piwi proteins. Mutations in rhino and the piRNA pathway gene armitage disrupt silencing of all major transposon families, but do not alter expression of euchromatic or heterochromatic protein coding genes. Deep sequencing studies show that rhino mutations significantly reduce or eliminate anti-sense piRNAs derived from the majority of transposable elements in the Drosophila genome, and lead to a dramatic reduction in piRNAs derived from major piRNA production clusters on chromosomes 2R and 4. Rhino protein localizes to distinct nuclear foci, and associates with the chromosome 2R and 4 clusters by chromatin immunoprecipitation. The Rhino HP1 homologue is therefore required for piRNA biogenesis, transposon silencing, and maintenance of germline genome integrity.
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Romero, Catalina. "Spatio-temporal control of the cytosolic redox environment in C. elegans." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11122.
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Compartmentalization of redox reactions is essential to all life forms. Protein activity can respond to changes in the local redox environment through the reversible oxidation of cysteine thiols. For the majority of cysteines in the proteome, this interaction takes place through equilibration with the glutathione pool; this raises the question whether this redox pool acts as a buffer, or instead as a sensitive media, transducing information from a local physiological state into protein function.
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Durst, Steffen [Verfasser], and P. [Akademischer Betreuer] Nick. "Actin, Auxin, and Plant Patterning : the role of actin-binding proteins and super-resolution microscopy in tobacco cells (Nicotiana tabacum L. cv. Bright Yellow 2) / Steffen Durst. Betreuer: P. Nick." Karlsruhe : KIT-Bibliothek, 2012. http://d-nb.info/1022123920/34.
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Gezelius, Henrik. "Studies of Spinal Motor Control Networks in Genetically Modified Mouse Models." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-109889.
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Du, Tingting. "Dissecting Small RNA Loading Pathway in Drosophila melanogaster: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/356.
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In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type.
Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways.
Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.
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Portran, Didier. "Etude de mécanismes moléculaires et de lois physiques qui régissent l'auto-organisation des microtubules en réseaux ordonnés et complexes in vitro." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00848199.
Full textAbstract:
Le cytosquelette de microtubule (MT) est essentiel dans de nombreux processus cellulaire. Il est notamment impliqué dans le trafic intracellulaire, la division cellulaire, la modification et le maintien de la forme de la cellule. En fonction du type cellulaire ou de son état de différenciation, les réseaux de MTs vont adopter des architectures différentes. Ces organisations sont définies par des contraintes géométriques intracellulaires et l'activité moléculaire de nombreuses protéines associées aux MTs (MAPs). Parmi ces protéines, des membres de la famille des MAP65s ont été identifiés. In vitro, elles forment des ponts entre les MTs pour les organiser en faisceaux. Le but de mon travail de thèse a été d'étudier in vitro le rôle de MAP65s dans l'auto-organisation d'un réseau de faisceaux de MTs. Dans un premier temps, j'ai mis au point un système biomimétique utilisant la technique de " micro-patterning " qui imposent une géométrie d'assemblage pour les MTs dans des limites qui se rapprochent de celles observées dans les cellules. Cette méthode permet de contrôler précisément l'assemblage des MTs à partir de zones dont les formes, la taille et la distribution des unes par rapport aux autres sont définies. Pour valider cette technique, j'ai reconstitué des réseaux qui miment des architectures cellulaires (i.e modules du fuseau mitotique). Dans un deuxième temps j'ai étudié le rôle de MAP65s dans l'auto-organisation de réseaux de faisceaux de MTs, et plus particulièrement l'étape de co-alignement entre MTs dynamiques et dispersés. J'ai montré que MAP65-1 de plante et son orthologue chez la levure, Ase1, diminuent fortement la longueur de persistance de MTs isolés ou organisés en faisceaux. Cet assouplissement leur permet de se déformer et donc de se co-aligner pour former des faisceaux lorsqu'ils se rencontrent à des angles de rencontre élevés. L'augmentation de flexibilité est du à l'interaction du domaine de liaison de MAP65-1/Ase1 avec la lattice des MTs. Ces résultats suggèrent que la diminution de la rigidité des MTs contrôle dans les cellules l'issue des évènements des rencontres entre MTs. De façon plus générale, la modulation des propriétés mécaniques des MTs par des MAPs représente un nouveau mécanisme pour réguler la plasticité des réseaux de MTs dans les cellules eucaryotes.
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Lu, Cheng-Wei, and 呂承衛. "Sub-micron Electrically Programmable Protein Patterning Technology." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/64661910372853507808.
Full textAbstract:
碩士國立臺灣大學
電子工程學研究所
96
Bio-sensor is one of the fundamental technologies for Proteomics. With the technology of the Micro- and Nano- Electromechanical Systems, it can be easily to manufacture the miniature portable device, that include the surface plasmon resonance detector, piezoelectric quartz crystal, ion-selective field effect transistor and eletrochemical bio-sensor. Protein patterning is a method which makes those devices more selective and sensitive. Protein patterning technology is to make proteins or bio-molecules be defined on some specific areas. Using some kinds of the patterning technologies to let the bio-molecules immobilized on a substrate, and then make the assays or reagents to be interact with the patterned bio-molecules, with this method it can be fleetly and enormously analysed the interact between the unknown lysate, bio-molecules and chemicals. The patterned object could be the proteins, DANs and all kinds of bio-molecules. The application of the protein patterning can be used widely, including the genome, cancer research, drug discovery, clinical diagnostics, cell studies, tissue engineering and bio-molecule identification. Nowadays, the protein patterning technologies are hard to achieve the high resolution and programmable patterns, moreover those methods might cause the patterned proteins physical or chemical damages. So this study is using the micro- and nano-electromechanical systems to manufacture the submicron electrically programmable protein patterning device, and utilizing the fluorescence labeling process and fluorescent microscope to analyse the patterned result. This method can not only make high resolution programmable patterns effectively without denaturing those proteins, but also sustain their activities to make sure the feasible of applications.
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Lin, Chih-Hao, and 林志豪. "Statistical-Mechanics of Protein Patterning Driven by Surface Potential Modulation." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/76863109988227574304.
Full textAbstract:
碩士臺灣大學
電子工程學研究所
98
Bio-microsystem creates the opportunity of on-chip screening, biodetection, and cell monitoring for emerging bio-diagnostic applications and innovative bio-scientific discovery. One of the fundamental techniques to achieve these bio-devices and bio-microsystem is the protein patterning. Traditionally, this is achieved by microfabrication techniques such as photolithography technology, inkjet printing and micro-contact printing. However, most of the existing patterning methods require complicated processes, and hardly achieve submicron protein patterns. In this thesis, we have developed a protein patterning technique by controlling the surface electric potential. Besides, we also develop the statistical model to describe the protein patterning results. Based on these results, the insight understanding of the interaction between protein and the device surface has been established.
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Wylie, Ryan Gavin. "Three-dimensional Immobilization of Proteins within Agarose Hydrogels using Two-photon Chemistry." Thesis, 2011. http://hdl.handle.net/1807/31977.
Full textAbstract:
Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.
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Gonçalves, Carla Alexandra Carvalho. "Role of the Bone Morphogenetic Protein Pathway in Definitive Endoderm Patterning." Dissertação, 2015. https://repositorio-aberto.up.pt/handle/10216/82293.
Full text
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Gonçalves, Carla Alexandra Carvalho. "Role of the Bone Morphogenetic Protein Pathway in Definitive Endoderm Patterning." Master's thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/82293.
Full text
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Noel, John. "Microtubule Patterning and Manipulation Using Electrophoresis and Self-Assembled Monolayers." 2009. http://hdl.handle.net/1969.1/ETD-TAMU-2009-05-767.
Full textAbstract:
We developed new methods for controlling and studying microtubules (MTs) outside the
complex workings of the living cell. Several surface treatments for preventing MT
fouling on surfaces were analyzed and, for the first time, a self-assembled monolayer
(SAM) was developed which prevented MT adsorption in the absence of passivating
proteins. The morphology and thickness of the SAM was measured to determine the
mechanism of formation and origin of the MT-resistant behavior. The SAM was
integrated into electron beam lithography for patterning and manipulating MTs using
electrophoresis. Reversible MT adsorption and patterning and alignment of single MTs
were achieved. We characterized the mechanism for the MT migration under electric
field with a focus on the electrodynamics of the flow cell and the forces acting on the
MT, along with the time dependence of the process.
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Marshall, Shannon Alicia. "Stability and Conformational Specificity in Protein Design: Models for Binary Patterning and Electrostatics." Thesis, 2002. https://thesis.library.caltech.edu/6993/1/Marshall_sa_2002.pdf.
Full textAbstract:
Binary patterning (the arrangement of hydrophobic and polar amino acids) and electrostatics are important determinants of the stability and conformational specificity of designed proteins. We have developed methods to to select the optimal binary pattern and model electrostatics in protein design studies. The Genclass method of binary patterning uses a solvent accessible surface generated from backbone coordinates of the target fold and "generic" side chains, constructs whose size and shape are similar to an average amino acid. Each position is classified according to the solvent exposure of its generic side chain. The method was tested by analyzing several proteins in the Protein Data Bank and by experimentally characterizing homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, additional hydrophobic residues induce aggregation. The optimal variant was further characterized by nuclear magnetic resonance spectroscopy. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.
Electrostatic interactions are critical determinants of protein structure and function. Computational protein design algorithms typically use fast methods based on Coulomb's law to model electrostatic interactions. These methods fail to accurately account for desolvation and solvent screening, which strongly attenuate electrostatic interactions in proteins. Using the current force field, we designed a 25-fold mutant with moderate stability similar to the wild type protein. Incorporating two classes of electrostatic interactions using simple rules yielded a nine-fold mutant of the initial design that is over 3 kcal mol^(-1) more stable. The simple electrostatic model used in the ORBIT force field is unable to predict the experimentally determined stabilities of the designed variants. Finite difference Poisson-Boltzmann (FDPB) methods have substantially better predictive power, but are far too slow for problems with high combinatorial complexity. We have developed new strategies for modeling electrostatics in protein design problems that utilize one- and two-body decomposable FDPB methods. Computational results indicate that this method has the accuracy and speed required for design calculations.
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Bhagawati, Maniraj. "Photolithographic surface functionalization for spatio-temporally controlled protein immobilization." Doctoral thesis, 2012. https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2012012710014.
Full textAbstract:
Exploiting the functional diversity of proteins for fundamental research and biotechnological applications requires their functional organization into micro- and nanostructures while preserving their functional integrity to the highest possible level. My PhD research aimed to establish generic techniques based on photolithography which could be used to control the spatial as well as temporal organization of recombinantly expressed proteins on surfaces. My thesis describes in detail four strategies that I developed for achieving this goal. In the first approach a photo-induced Fenton reaction was used to selectively destroy tris(nitrilotriacetic acid) (tris-NTA) moieties on a surface. UV-irradiation through a photomask allowed localized photo-destruction and targeting of His-tagged proteins to non-irradiated regions. Photo-destruction could also be achieved by scanning selected regions with the UV laser of a confocal laser scanning microscope (CLSM) thus allowing flexible creation and modification of protein patterns. The second strategy was based on the photosensitive nitroveratryloxycarbonyl (NVOC) protection group, which was used to cage amine groups on a surface. Sequential uncaging by UV-irradiation through a photomask followed by reactions with biotin and coenzyme A was used to pattern streptavidin and ybbR-tagged proteins into microstructures. In the third approach a photo-fragmentable Histidine peptide was used to block tris-NTA surfaces against binding of His-tagged proteins. UV-irradiation through a photomask or by using a UV laser in a CLSM cleaved the peptide into short fragments which quickly dissociated from the surface due to loss in multivalency. His-tagged proteins could be efficiently targeted into irradiated regions even from a complex cell lysate. Sequential uncaging and immobilization allowed the construction of multiplexed protein patterns with a high degree of temporal control. The fourth strategy used combined peptide tags comprising of a His-tag as well as a Halo- or ybbR-tag to achieve rapid covalent immobilization of recombinant fusion proteins on surfaces functionalized with specific ligands. In combination with a photo-fragmentable histidine peptide as described above, stable spatio-temporal organization of proteins carrying these combined tags was possible. The techniques developed in this thesis enabled the photolithographical micropatterning of recombinant proteins carrying specific peptide or protein tags on surfaces in a functional manner. Owing to the generic nature of immobilization strategies, coupled with the ease of patterning, highly versatile applications of these methods both in fundamental research as well as bio-technological and analytical applications can be envisioned.
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Park, Jinseon. "Characterization Of The Local Electrical Environment In An Electrically-guided Protein Patterning System Incorporating Antifouling Self-assembled Monolayer." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-08-8520.
Full textAbstract:
In earlier research in our lab, the manipulation of microtubules on gold patterned silicon wafers was achieved by E-beam lithography, Poly (ethylene glycol) self assembled monolayers (PEG-SAMs) and electrophoresis. To develop a technique for delicate single microtubule manipulation, further studies need to be done on PEG-SAMs and electrophoresis. As a foundation of this goal, we examined the electric field in an aqueous solution between two planar electrodes and the compatibility of the antifouling property of PEG-SAMs with the electric field. For this purpose, the distribution of microbeads was analyzed using a Boltzmann distribution. The amount of adsorbed microtubules on a PEG-SAM was examined to test the compatibility of the antifouling property of a PEG-SAM with concomitant exposure to electric field. It is shown that the product of the electric field and the effective charge of the microbead does not have a linear relation with the applied electric potential but an exponentially increasing function with respect to the potential. The antifouling property of the PEG-SAM was not retained after an exposure to the electric field.