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Cloning, overexpression and biophysical characterization of grd/grl/wrt domains from<em> Caenorhabditis elegans</em> in<em> Escherichia coli</em>Lindberg, Marie January 2008 (has links)
<p>Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.</p>
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Synthesis and Physicochemical Characterization of Diamond-Like Semiconductors and Intermetallic Compounds Using High Temperature Solid-State Synthesis, Polychalcogenide Flux Synthesis and the Solid-State Microwave Synthetic MethodLekse, Jonathan 09 December 2011 (has links)
Diamond-like semiconductors are interesting materials to study due to the wide variety of technologically useful properties that these materials possess. These normal valence compounds have structures that are based on that of diamond, either the cubic or hexagonal polymorph. Though there are a finite number of possible compounds, due to isovalent and isoelectronic principles, the total number of potential compounds is quite extensive. Quaternary diamond-like semiconductors provide a unique opportunity, because much of the previous research has focused on binary and ternary systems leaving quaternary systems, relatively unexplored. Additionally, quaternary diamond-like semiconductors possess a greater degree of compositional flexibility compared to binary and ternary materials, which could result in the ability to more carefully tune desired physical properties.
<br>In order to prepare the new materials, Li2ZnGeS4, Li2ZnSnS4, Li2CdGeS4, Li2CdSnS4 and Ag2MnSnS4, several synthetic methods have been employed, including high-temperature solid-state synthesis, polychalcogenide flux synthesis and solid-state microwave synthesis. The solid-state microwave synthetic method was itself studied using a number of target systems such as the ternary diamond-like semiconductor, AgInSe2. Additionally, several intermetallic compounds, such as Ag3In, AuIn2 and Bi2Pd were prepared using this procedure. Solid-state microwave synthesis is not as well known as some of the other synthetic methods that were employed in this work possibly due to a lack of understanding of the method, training and equipment. Despite these problems, the method has the potential to save time, energy and cost due to the unique nature of microwave heating. In an attempt to gain a better understanding of this synthetic method and its capabilities, the solid-state microwave synthetic method was used to prepare diamond-like semiconductors and intermetallic compounds. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation
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Asteroseismic probing of the internal structure of main-sequence starsMiglio, Andrea 27 November 2007 (has links)
No description available.
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Les astrocytomes de bas-grade: caractérisation moléculaire et implications cliniques / Low-grade astrocytomas: molecular characterization and clinical implicationsRorive, Sandrine 20 January 2010 (has links)
La malignité des astrocytomes est établie sur base de critères morphologiques définis au sein de la classification de l’Organisation Mondiale de la Santé (OMS). Ce système de gradation, qui s’échelonne de I à IV, constitue actuellement l’outil pronostique le plus fiable. Par facilité, les cliniciens regroupent les astrocytomes de grade I (astrocytomes pilocytiques) et les astrocytomes diffus de grade II sous le terme d’« Astrocytomes de bas-grade » par opposition aux astrocytomes de haut-grade, constitués des astrocytomes anaplasiques (grade III) et des glioblastomes (GBM ; grade IV). Cette terminologie conduit à des prises en charge cliniques inadéquates car elle englobe des tumeurs très différentes en terme d’agressivité : les astrocytomes de grade I, majoritairement non infiltrants, non évolutifs et indolents et les astrocytomes diffus de grade II, toujours infiltrants et évolutifs, progressant systématiquement en astrocytomes de haut-grade et entraînant le plus souvent le décès prématuré du patient. Bien que ces tumeurs soient définies par la classification de l’OMS comme des entités clinicopathologiques distinctes, peu de données sont disponibles dans la littérature pour expliquer leurs particularités biologiques et la pratique quotidienne montre que les différencier peut être difficile.
Le but des études entreprises au cours de ce travail de thèse est d’apporter une contribution à la compréhension des mécanismes de tumorigenèse qui différencient l’astrocytome de grade I des astrocytomes diffus (grade II-IV), de manière à identifier des voies biologiques qui permettraient, au moins en partie, d’expliquer ces différences de comportement.
Au cours de la première partie de ce travail, nous avons caractérisé les profils d’expression génomique des astrocytomes de grade I et de grade II, en comparant les données d’expression de gènes (évaluées par des technologies de micropuces d’ADN) de travaux publiés entre 2000 et 2005. L’expression des gènes identifiés a été validée par des analyses de RT-PCR quantitative sur une série indépendante d’astrocytomes de grade I, II et IV. Les fonctions biologiques des protéines codées par chacun de ces gènes ont fait l’objet de recherches bibliographiques détaillées afin de proposer un modèle permettant d’approcher les différences de comportement de ces tumeurs. Cette analyse nous a permis d’identifier TIMP4 (tissue inhibitor of metalloproteinases 4) et IGFBP2 (insulin-like growth factor binding protein 2) comme gènes candidats pour améliorer la caractérisation biologique et clinique des astrocytomes de grade I par rapport aux astrocytomes diffus. TIMP4 et IGFBP2 codent respectivement pour un inhibiteur endogène des métalloprotéinases matricielles (MMPs) et une protéine de liaison capable d’inhiber l’action des « insulin-like growth factors » (IGFs, dont IGFI et IGFII), des facteurs impliqués dans la croissance et la migration des astrocytes normaux et tumoraux.
Sur base de la surexpression de TIMP4 et d’IGFBP2 dans les astrocytomes de grade I, en comparaison aux astrocytomes diffus de grade II, nous avons posé l’hypothèse suivante : « L’absence d’agressivité des astrocytomes de grade I, en comparaison aux astrocytomes diffus (grade II-IV) pourrait en partie être liée à l’inhibition par TIMP-4 de la protéolyse des complexes IGFBP2-IGFII au sein de ces tumeurs ». Cette protéolyse, qui diminue l’affinité d’IGFBP2 pour IGFII, pourrait contribuer à libérer IGFII dans la matrice extracellulaire (MEC), favoriser la liaison d’IGFII à son récepteur IGF-IR et stimuler la croissance et la migration des cellules astrocytaires tumorales. Pour tester cette hypothèse, nous avons réalisé différentes analyses biochimiques afin i) de caractériser les actions protéolytiques de MMP-2, MMP-9 et MT1-MMP sur le complexe IGFBP2-IGFII, ii) d’identifier la libération d’IGFII lors du clivage de ce complexe, et iii) d’étudier l’action inhibitrice de TIMP-4. A l’aide d’un modèle cellulaire in vitro (lignée astrocytaire tumorale LN229), nous avons ensuite observé l’influence de la protéolyse du complexe IGFBP2-IGFII sur la croissance et la motilité cellulaire. Cette étude a montré : (1) la protéolyse du complexe IGFBP2-IGFII par MMP-9, (2) l’inhibition partielle de cette protéolyse par TIMP-4, (3) la libération d’IGFII résultant de cette protéolyse et (4) les effets stimulants de la libération d’IGFII sur la croissance et la motilité des cellules LN229. Cette étude souligne le rôle important de la protéolyse des complexes IGFBP2-IGFII dans l’agressivité des astrocytomes diffus. Elle confirme les effets stimulants propres d’IGFII, d’IGFBP2 et de MMP-9 sur la motilité et/ou la croissance des cellules astrocytaires tumorales. Enfin, elle identifie un rôle inhibiteur potentiel de TIMP-4 sur la protéolyse du complexe IGFBP2-IGFII, qui pourrait contribuer à expliquer le caractère plus indolent des astrocytomes de grade I en comparaison aux astrocytomes diffus.
Au cours de la troisième partie de ce travail, nous avons caractérisé l’expression de TIMP-4 et de son récepteur potentiel, la tétraspanine CD63, sur une série de 471 gliomes, dont 354 astrocytomes de grade I à IV par la méthode d’immunohistochimie quantitative appliquée aux « tissue-microarrays ». Pour chaque patient, les variables cliniques suivantes ont été collectées : âge, localisation tumorale et multifocalité, comportement infiltrant de la tumeur, étendue de la résection chirurgicale, grade histologique, type de traitement adjuvant, et suivi, évalué en termes de récidive tumorale et de durée de survie spécifiquement liée à la tumeur. Cette troisième étude confirme la surexpression de TIMP-4 dans les astrocytomes de grade I, en comparaison aux astrocytomes diffus de grade II, et montre que CD63 suit un profil d’expression similaire. Par conséquent, nous proposons l’utilisation de la co-expression TIMP-4/CD63 comme un nouveau marqueur diagnostique de l’astrocytome pilocytique de grade I dans la prise en charge anatomo-pathologique des « Astrocytomes de bas-grade ». Cette étude souligne également l’intérêt d’utiliser TIMP-4 et CD63 pour différencier le phénotype astrocytaire du phénotype oligodendroglial des gliomes diffus. Enfin, ce travail identifie CD63 et le profil de co-expression TIMP-4/CD63 comme nouveaux marqueurs pronostiques indépendants associés à une évolution défavorable des astrocytomes diffus et des oligoastrocytomes.
Ce travail nous a donc permis, à partir de données de micropuces d’ADN, d’identifier TIMP4 et IGFBP2 comme gènes d’intérêt dans l’étude des astrocytomes. A partir de ces deux gènes, nous avons posé une hypothèse visant à expliquer le caractère non infiltrant des astrocytomes de grade I. Les tests in vitro menés dans le cadre de cette hypothèse confirment l’intérêt des protéines TIMP-4, IGFBP2 et IGFII dans la tumorigenèse des astrocytomes. Enfin, la caractérisation clinique de l’expression de TIMP-4 et de CD63, son récepteur potentiel, valide l’intérêt clinique que ces protéines représentent pour la prise en charge des patients porteurs d’un gliome. Il reste toutefois la nécessité d’approfondir nos connaissances sur les voies de signalisation utilisées par TIMP-4 et/ou CD63. Ces recherches permettraient de proposer de nouvelles stratégies thérapeutiques visant à améliorer le traitement de ces tumeurs et ainsi pallier au pronostic sombre des patients porteurs de gliomes diffus.
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The Macroinvertebrate Community in a Nature-like Fishway with Habitat Compensation PropertiesGustafsson, Stina January 2012 (has links)
Nature-like fishways are often constructed to restore connectivity in streams exploited for hydropower. They also have the potential to compensate for important habitats that have been degraded or lost. The aim of this thesis was to improve knowledge of the concept of nature-like fishway design, with special focus on their habitat compensation potential. This was done by comparing a nature-like fishway with four different habitat types, termed the biocanal, to six nearby natural reference creeks. In paper I the macroinvertebrate colonization of the biocanal was studied and the community composition was compared to that of the natural reference creeks. Furthermore, the potential for the biocanal to contain a diverse macroinvertebrate fauna was investigated. The results showed that the species composition of the benthic fauna in the biocanal was approaching that of the reference creeks and that the different habitat types in the biocanal contributed to the increased family diversity. In paper II the functional organization of the biocanal was studied using the functional feeding group approach. The results showed that after two years, the functional organization in the biocanal resembles that of the natural reference creeks.
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ECR Plasma Deposition Of Carbon - Studies On DLC Coatings And Carbon NanotubesPatra, Santanu Kumar 10 1900 (has links)
Recent developments in the field of nano-structured materials for technological as well as scientific prospective are quite interesting. In this context carbon plays a dominant role. Few examples such as carbon nanotubes (CNTs), fullerene, nanostructured diamond, as well as, amorphous carbon film, particularly, diamond-like carbon (DLC) coating are the areas of today’s research. This thesis deals with ECR plasma deposition of carbon in two different forms, i.e., Diamond-like carbon (DLC) and carbon nanotubes (CNTs) In the case of DLC coatings the chemical vapor deposition (CVD) and sputtering CVD configuration has been used. The carbon nanotubes have been grown using CVD configuration.
DLC films were deposited by ECR-rf CVD mode, as well as, ECR sputtering mode. In case of CVD films, about 0 — 100 Watts rf bias was employed in steps of 20 Watts, corresponding to effective negative self bias voltage of 15 — 440 V. CH4 and C2H2 have been used as source gas for CVD films. Microwave power was optimized at 300 Watts. In case of sputtering, a cylindrical graphite target (diameter 9 cm and length 6 cm) kept at the exit of the Ar plasma was biased with -200 V. Films were deposited on floating substrate (temperature ~100 oC). Films were deposited on Si, quartz, and steel substrates and characterized by FT-IR, Raman, UV-Visible, Photoluminescence spectroscopy (PL), spectroscopic ellipsometry. Nanoindentation was used to evaluate the film’s elastic property. Pin-on-disk measurement was used to study the tribological property of the films. Electrical properties of the films deposited on Si [p-(100), 10 Ω cm] were studied using picoammeter / source measuring instrument by two probe method.
FT-IR analysis showed sp3C-H absorption peak at 2930 cm-1 for the CVD films, while sputtered films did not show any C-H absorption. Raman spectroscopy was used to evaluate bonding aspects as well as hydrogen content of the films. Comparison of sp3C : sp2C among the films was done based on I(D) / I(G) of the Raman peaks, while hydrogen content was estimated based on background slope of the Raman spectra. It was observed that increase in rf bias induces more sp2C while hydrogen content decreases. An optimum substrate bias of 40 Watts was predicted from the Raman spectra. For sputtered films Raman spectra indicated the formation of nanocrystal diamond in a-C matrix. UV-Visible-NIR optical transmission spectroscopy was used to determine the band gap (Tauc), E0, of the films. It showed that increase in rf bias increases the absorption coefficient α. The films deposited from CH4 with a substrate bias of 0 and 20 Watts (i.e., high hydrogen content in the film) followed (hνα)1/2 = const. (hν –E0), while other films hνα = const. (hν –E0) ( h is Plank constant ν is frequency of light). E0 varied from 1.1 — 2.5 eV. It was assumed that for π--π* transition follows root relation while π--σ * transition follows linear relation. Spectroscopic ellipsometry was used to determine optical constants, film thickness, and interface thickness. Deposition rate found out to be ~100 nm / mints for C2H2, ~10 nm / mints for CH4, and ~2.5 nm /mints for sputtered films. Formation of interface layer of thickness about 5 —30 nm due to high energy ion bombardment takes place for the films deposited at 40 Watts rf bias or higher. Band gap and related phenomena was revisited from the data that was obtained from this instrument which reasonably matches with the earlier results. PL experiments were carried out at room temperature using lamp excitation source as well as laser excitation source (457.9 nm wavelength). In case of lamp excitation source any wavelength from 200 —900 nm region can be selected. PL spectra showed that there are two sources of PL signal, one from nanocrystal diamond and other from sp2C phase. To obtain PL signal from diamond UV excitation wavelength was required. This diamond phase is highly efficient emitter as compared to sp2C phase. Based on the closeness of diamond’s optical centre labeling of the peaks was done. For CVD films N3 ( 457 nm), H4 (495 nm), H3 (520 nm), [N-V]0 (~590 nm) optical centers of diamond was observed. For sputtered films [N-V]0 (2.08 eV), H3 (2.38 eV), H4 (2.50 eV), N3 (2.81eV), N3 (2.96 eV), 3.3 eV ( undocumented peak), 5RL ( 4.14 eV) optical centers of diamond as well as band-edge emission (5.01 eV ) was observed.
Nanoindentation technique was used to estimate the elastic property and related phenomena of the films. It shows that the films are having hardness of 5—17 GPa and reduced modulus of 20 —120 GPa depending on the deposition parameters. All the films show highly elastic response at lower load, i.e., at low indentation depth where elastic recovery is 85—95 %. At higher load substrate effect comes into the picture. Further morphology in and around the region was evaluated using scanning probe microscopy (SPM). It was shown that substrate effect comes into picture that is based on film’s thickness as well as its elastic property. Films were further characterized by pin-on-disk experiments. C2H2 based films were used because of high deposition rate. Since 40 Watts, 60 Watts, and 100 Watts films adhere well with steel only on these films tribological test was possible. A hardened bearing-steel was used as substrate and a 2 mm diameter cylindrical pin made of tool steel was use as pin. Studies were carried out with three different loads of 20, 40, and 60 N. Friction coefficient varied from 0.02 — 0.04 and wear rate was found to be 10-6 — 10-9 mm3 / N m. A sputtered film of 0.1 μ m on the top of the CVD film, in many respects, enhances the tribological properties. It was shown that certain amount of wear is required for low friction of DLC.
Electrical characterization of the films deposited from CH4 showed that they are highly insulating with resistivity of 1013 —1011 Ω-cm, and current conduction mechanism has been found to be predominantly space charge limited conduction (SCLC). Similar to the observations of Tauc’s relation, the film deposited with 0 and 20 Watts bias behave differently and followed the relation , where as, all other films exhibited the relation ( α, n are constants). It signifies that for 0 and 20 Watts rf biased films traps are uniformly distributed across the band gap while for others it decreases from the conduction band. For 0 and 20 Watts rf biased films no Ohmic current was observed at a detection level of 10-11 A. 40 Watts and higher rf biased films showed that three distinct regions in the I-V curves; initially Ohmic region, next to it SPLC region, and finally breakdown region. Increase in rf bias causes increase in Ohmic current. Film deposited from C2H2 showed diode-like behavior with higher conduction current limited by resistive control, and the resistivity of the films was ~ 109 — 105 Ω-cm. Difference in resistivity between the films deposited from CH4 and C2H2 was explained by considering the impurities in the source gas resulting in nitrogen doping concentration. Increase in Ohmic current for the CH4 films was explained by assuming the widening of the σ--σ * gap. Similar diode-like behavior was observed with the sputtered film.
The last part of the work deals with the growth mechanism of aligned CNTs and their field emission (FE) properties. Nanotubes were grown at 700 0C on Ni coated (thickness 40 nm, 70 nm, and 150 nm) Si substrate using a mixture of CH4 and H2 gas. Microwave power of 500 Watts was optimized for nanotube growth. Nickel nanoparticle formation mechanism from a continuous Ni film was explained by considering the stress that is generated due to the difference in thermal expansion coefficients of Si and Ni at 700 oC. Though the thicker film such as 150 nm does not form nanoparticle due to stress, hydrogen induced fragmentation of the film due the brittleness of the film even causes formation of finer nanoparticles. A substrate bias in the range 0— 250 V was used to align the nanotubes. Perfectly aligned CNTs were obtained at -250 V substrate bias. The density of the tubes varied from 108 —109 / cm2 while its length was 0.5 — 2 μ m. Due to hydrogen induced fragmentation of the films, 150 nm Ni thick film showed smallest diameter 2 — 5 nm CNTs. 40 nm films showed nanotube diameter of 10 — 30 nm and 150 — 300 nm while 70 nm showed 10 — 30 nm diameter nanotubes. Diameter of the nanotubes was estimated using transmission electron microscopy (TEM). Field emission analysis of these CNTs was done using Fowler-Nordheim (F-N) plot and the investigation revealed that the field emission properties strongly depend on density and aspect ratios. The non-linearity in the F-N plot or current saturation phenomena was explained in terms of change in work function due to heating effect during FE which was pronounced in case of longer nanotube. Suitable efficient cold-cathode emitters for a particular usage (assuming that the variables are applied field and emission current) could be designed from the obtained results. An ammonia gas sensor using thick nonaligned CNTs was realized. For this purpose a thick film of CNTs (~ 0.5 μm) was deposited. This sensor can detect 100 ppm level of ammonia. About 1.5 — 4.5 % change of resistance depending on ammonia concentration (100 —1000 ppm) was observed.
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Cloning, overexpression and biophysical characterization of grd/grl/wrt domains from Caenorhabditis elegans in Escherichia coliLindberg, Marie January 2008 (has links)
Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.
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Effects of Different Signalling Pathways on Regulation of 'GLK' GARP Transcription Factors in 'Arabidopsis thaliana'Ponomareva, Ekaterina 17 April 2012 (has links)
GLK1 and GLK2 transcription factors have been suggested to be involved in the regulation of chloroplast development, organic nitrogen signaling, disease resistance and circadian rhythmicity (Waters et al. 2009; Gutiérrez et al. 2008; Savitch et al. 2007; Sprott et al. 2010). This implies that multiple factors may play roles in regulation of GLK genes. In the present study, transcriptional regulation of GLK1 and GLK2 in Arabidopsis by various endogenous and environmental stimuli was investigated with the objective of elucidating the primary signalling pathway affecting expression of these two genes. Collectively, results of GLK1 and GLK2 expression in response to the experimental treatments of Arabidopsis point to the regulation of the two genes by changes in photosynthetic metabolism and reactive oxygen species (ROS) levels, and by organic nitrogen signalling. Changes in ROS levels and organic nitrogen signalling may also affect the two genes indirectly by interfering with or altering photosynthetic metabolism.
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Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p InteractomesWheaton, Sarah 31 May 2011 (has links)
The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.
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Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p InteractomesWheaton, Sarah 31 May 2011 (has links)
The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.
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