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Kowanetz, Katarzyna. "Adaptor Proteins in Regulation of Receptor Endocytosis." Doctoral thesis, Uppsala University, Ludwig Institute for Cancer Research, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4477.
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Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed that numerous endocytic regulatory proteins, among them ASAP1 and Dab2, interact with CIN85 via their PxxxPR motifs. The SH3 domains of CIN85 are able to cluster and exchange its effectors at subsequent stages of EGFR endocytosis, thus participating in the control of receptor internalization, recycling and degradation in the lysosome. We proposed that CIN85 functions as a scaffold molecule implicated in control of multiple steps in downregulation of RTKs.
Furthermore, we identified two novel Cbl- and ubiquitin-interacting adaptor proteins named Sts-1 and Sts-2 (Suppressors of T-cell receptor signaling). Ligand-induced and Cbl-mediated recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization and subsequent block of receptor degradation followed by prolonged mitogenic signaling pathways. Our results indicate that Sts-1 and Sts-2 represent a new class of negative regulators of Cbl functions in receptor endocytosis.
In conclusion, this thesis describes novel mechanisms by which Cbl, coupled to its effectors, orchestrates trafficking of RTKs. Detailed understanding of how these processes are controlled under physiological as well as under pathological conditions may be important for future therapeutic approaches.
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Standen, Claire. "Molecular studies of the Fe65 and X11 adaptor proteins." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401885.
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Beauparlant, Stephen Lewis. "Functional characterization of the p97 adaptor protein UBXD1." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/213118.
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Molecular Biology and GeneticsPh.D.
p97 is a member of the AAA family of proteins (ATPase Associated with various cellular Activities). It is a highly conserved and abundant protein and functions in numerous ubiquitin-mediated processes including ERAD. Endoplasmic Reticulum Associated Degradation is the process by which misfolded/ubiquitinated proteins translocate out of the ER and migrate to the proteasome for degradation. p97 maintains substrate misfolding and mediates its exit from the ER and trafficking to the 26S proteasome. It also plays important roles in protein trafficking, the cell-cycle, apoptosis and homeotypic Golgi Apparatus and Endoplasmic Reticulum membrane fusion after mitosis. In addition, p97 plays a role in the aggresome-autophagy degradation pathway, which handles the ubiquitin-mediated destruction of aggregate-prone, misfolded, cytosolic proteins. p97 mutation is the causative alteration in the disorder, IBMPFD, which is marked by defects in autophagy. This broad diversity of function is mediated through p97's interaction with a large group of adaptor proteins. Many of these adaptors harbor both p97 interaction motifs and ubiquitin association domains. However, more than half of known p97 adaptors do not. Their function is largely unknown. UBXD1 is one known adaptor for p97 that does not have a ubiquitin association domain (UBA), and has been shown to have decreased interaction with IBMPFD mutant p97R155H and p97A232E. Recently, it has been suggested to perform a role in protein trafficking, specifically in monoubiquitinated caveolin-1 internalization and trafficking to the endosome. A novel high abundance UBXD1 interacting partner has been identified via solution-based mass spectrometric analyses. ERGIC-53, the namesake of the ER-Golgi Intermediate Compartment, has been shown to be involved in bi-directional trafficking between the ER and Golgi. The association between UBXD1 and ERGIC-53 is unique among UBX family members. Deletional analysis has shown that unlike p97, the ERGIC-53-UBXD1 interaction takes place in the extreme amino terminus of UBXD1, (within the first 10 amino acids) which is predicted by computer modeling to form a hydrophobic binding pocket. Further site-directed mutagenesis work has clearly shown four amino acids (3 highly hydrophobic) are crucial for maintaining this interaction. They have been modeled to form a conserved alpha-helix. ßCOPI, a primary member of the COPI coatomer complex which is involved in protectively coating ERGIC-53 positive vesicles, is also thought to be involved with the ERGIC-53-UBXD1-p97 pathway. ßCOPI has been identified as a UBXD1-independent interactor with p97. Modest UBXD1 over- expression using a ponasterone inducible system has shown that UBXD1 modulates ERGIC-53 localization. Additionally, a functional link between UBXD1, p97 and ERGIC-53 in autophagy has been discovered through the use of a highly efficient, miR30-based, inducible knockdown system. Upon individual knockdown of UBXD1, p97 and ERGIC-53, autophagic markers p62 and LC3-II accumulate at relatively high levels in normal culture conditions, strongly suggesting a role in mediating basal autophagy. However, when placed under starvation conditions, autophagy progresses and p62 is degraded. It is speculated from these studies that a p97/UBXD1 complex plays a role in regulating the trafficking of ERGIC-53 positive vesicles and this activity plays an important role in autophagy.
Temple University--Theses
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Chan, Gabriel. "The role of Crk adaptor proteins in human breast cancer /." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81608.
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The family of Crk adaptor proteins is integral to the molecular signaling of cellular migration. Only limited research has demonstrated a role in human carcinogenesis. Through the analysis of human breast cancers with immunohistochemistry, there is a near significant association implicating Crk proteins over-expression and a worsened overall survival. There was no statistical difference in a relation to nodal status. The RNA interference of Crk in human cancer cell lines caused a significant decrease in cell migration. This in vitro suppression of a malignant characteristic could provide a new avenue towards the molecular targeting of breast cancer that may eventually benefit patient outcomes.
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Luke, Courtney. "Expression, molecular interactions and functions of Ruk, a novel adaptor protein." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/29855.
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In this work I confirmed the Ruk gene structure using phage library analysis, which showed that it encompasses 320 kb and contains 24 exons with introns that are relatively large (³l00kb). There are 5 promoters found in Ruk and differential usage of these promoters together with alternative splicing generate 12 types of Ruk mRNA, which are for the majority developmentally down-regulated; the exceptions are Rukt and Rukh2 which are upregulated in adult testis. The 12 transcripts encode 7 different proteins and the only domain that is common for all of them is the coiled-coil. Therefore, the coiled-coil was targeted for conditional inactivation using the Cre loxP system. The tissue specific inactivation of Ruk dimerisation will help to elucidate the function of the individual isoforms depending on which tissues are targeted. GST pulldown and co-immunoprecipitation studies have shown that isoforms are able to interact with one another through various domains. Similar techniques have been used to elucidate the fine mechanism of the interaction between Ruk proteins and the p85α regulatory subunit of PI3-kinase. Ruk isoforms that were expressed in mammalian cells were shown to localize to vesicular structures, probably late endosomes, suggesting that certain domains interacting with membrane bound proteins cause the entire protein to be translocated to the endosomal membrane.
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Abu-Thuraia, Afnan. "Characterization of the interaction between the adaptor protein Nck and the protein kinase PKR." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96908.
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Tight regulation of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is critical for the maintenance of cellular homeostasis due to its potent inhibitory role on general translation. Previously, we have identified the adaptor protein Nck-1 as a novel cellular regulator of PKR activation through its interaction with PKR. In this study, we further confirmed that Nck-1 limits PKR activation under normal conditions. However, we demonstrate that the control of PKR activation by Nck-1 is reversible, since significant levels of dsRNA override Nck-1's negative control of PKR activation and induce dissociation of Nck-1 from PKR. Our data show that Nck-1 needs to be in full length to interact and modulate PKR. In addition, we observed that the interaction of Nck-1 with PKR is independent of any functional Src-homology domains of Nck-1, but our findings showing that Nck-1 interacts with both the N- and C-terminus of PKR challenge this concept. Nonetheless, we uncovered that upon significant levels of dsRNA, dissociation of Nck-1 from PKR is due to the activation of the catalytic activity of PKR rather than to competition by dsRNA binding or change of PKR conformation during its activation. Finally, we provided further evidence supporting the occurrence of Nck-1 phosphorylation by PKR in vivo. Hence, Nck-1 not only buffers PKR activation but appears to be a substrate of PKR. Therefore, we propose that PKR-mediated phosphorylation is part of the mechanism that promotes Nck-1 dissociation from activated PKR. Taken together, our data confirm Nck-1 as a novel cellular modulator of PKR that limits PKR activation under physiological conditions.La protéine kinase PKR, activée par l'ARN double brins (ARNdb), est connue pour jouer un rôle inhibiteur de la traduction des protéines. La régulation de PKR est donc critique pour le maintien de l'homéostasie cellulaire. Nous avons précédemment identifié la protéine adaptatrice Nck 1 comme étant un potentiel régulateur de l'activation de PKR, suivant son interaction avec PKR. Dans la présente étude, nous avons été en mesure de confirmer que, dans des conditions physiologiques, Nck-1 peut limiter l'activation de PKR par l'ARNdb. Cependant, le contrôle qu'exerce Nck-1 sur PKR est réversible puisque, lorsque la quantité d'ARNdb dépasse une certaine concentration, PKR est activée et alors Nck-1 se dissocie de PKR, l'empêchant ainsi de limiter son activation. Nos données démontrent également que Nck-1 doit être dans sa forme native pour interagir et moduler l'activation de PKR. De plus, il semble que l'interaction entre Nck-1 et PKR ne nécessite pas que les différents domaines homologues de Src (SH2 et SH3) présents chez Nck-1 soient fonctionnels. De plus, nous avons observé que Nck-1 interagit à la fois avec les domaines N- et C-terminaux de PKR. Nous démontrons également que lorsque les niveaux d'ARNdb atteignent un niveau seuil, Nck-1 se dissocie de PKR non pas à cause d'une compétition avec l'ARNdb, ni à cause d'un changement de conformation de PKR ou son autophosphorylation, mais est plutôt dû à l'activation du domaine catalytique de PKR. De plus, il semble que Nck-1 puisse être phosphorylé par PKR in vivo. Nck-1 est donc non seulement un modulateur de l'activation de PKR mais peut également servir de substrat pour PKR. Ceci nous amène donc à proposer que la phosphorylation de Nck-1 par PKR activée soit responsable du mécanisme de dissociation entre Nck-1 et PKR. En conclusion, nos résultats confirment Nck-1 comme étant un nouveau modulateur cellulaire de PKR, en limitant son activation dans des conditions physiologiques.
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Nasertorabi, Fariborz. "Biochemical and Structural Studies on the Adaptor Protein p130Cas." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4777.
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Domiziana, De Tommaso. "Astrocytes contribute to neuroinflammation during EAE by shaping the CNS microenvironment via Rai signalling." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1105117.
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Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). One of the pathological hallmarks of MS is the T cells-mediated destruction of myelin sheath, which result in axonal damage and subsequent neurological dysfunction. Current MS therapies are focused on immunosuppression as they are aimed at limiting the entry of immune cells into CNS, thereby preventing neuroinflammation. Although these therapies have been shown to be potent disease-modifying agents they fail to prevent or reverse disease progression. Astrocytes, among CNS resident cells, has been recently suggested as alternative highly promising therapeutic targets because of the key role played by these cells in driving disease progression in MS. Indeed, thanks to a close contact between astrocyte end-feet and blood vessels, the crosstalk of astrocytes with encephalitogenic T cells is centrally implicated in MS pathogenesis (Ponath et al., 2018). In this view, we have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice accounts for a reduced demyelination and a milder experimental autoimmune encephalomyelitis (EAE), notwithstanding a higher frequency and pathogenicity of autoreactive T cells infiltrated within CNS highlighting the key role played by astrocytes in T cell modulation during EAE (Ulivieri et al., 2016).
In the first part of this project we have investigated the molecular mechanism underlying the ability of ShcC/Rai-deficient astrocytes to generate an efficient T cell suppressive microenvironment in the pathological setting of EAE.
At the beginning, we focused to study the ability of astrocytes to control the balance between extracellular ATP and adenosine in response to encephalitogenic T cells. We found that astrocytes respond to autoreactive T cells injury by enhancing the expression and activity of CD39 ectonucleotidase, responsible for the enzymatic hydrolysis of extracellular ATP into the immunosuppressive mediator adenosine, and that ShcC/Rai couples CD39 to its negative regulator RanBPM thereby limiting its activity. Accordingly, we measured high adenosine concentration in conditioned medium of Rai-/- astrocytes. As a result, T cells in the presence of microenvironment shaped by Rai knock-out astrocytes showed reduced proliferation and an up-regulation of inhibitory receptor CTLA-4, indicating that higher levels of adenosine are responsible to immunosuppression. We further characterized the impact of Rai on the protein composition of astrocytes-derived extracellular vesicles (EVs) in response to T cell-derived cytokines. Data obtained using a proteomic approach revealed that several proteins are differentially present in EVs released from Rai deficient or control astrocytes. Interestingly, enrichment analysis showed that these proteins participate in glutamate metabolism, in the control of protein folding and in the protection from oxidative stress suggesting that Rai controls pathways involved in brain homeostasis.
Additionally, we examined functional polarization of astrocytes towards a neuroprotective (A2) or a neurotoxic (A1) phenotype. We show that Rai-/- astrocytes skew towards the A2 neuroprotective phenotype in response to encephalitogenic T cells both in vitro and in the EAE mouse model of MS by enhancing the activation of STAT3 transcription factor.
In the second part of the project we have analyzed the role of ShcC/Rai in adenosine signaling in T cells. We identify a novel mechanism by which Rai in T cells dampens immunosuppressive effect of adenosine by inhibiting A2A receptor signalling interfering with the activation of transcription factor CREB. Characterization of molecular mechanism in a Jurkat T cell lines overexpressing Rai shows that Rai forms a complex with CREB upon A2AR triggering. In this respect, the phosphorylation/activation of CREB was significantly higher in Rai knock-out T cells compared with control following A2AR stimulation.
Collectively, these data identify Rai/ShcC adaptor protein as critical regulator of astrocytes responses to T cells mediated neuroinflammation and highlight a new molecular mechanism to which Rai prevents establishment of an immunosuppressive program in T cells by limiting the transcriptional activity of nuclear factor CREB.
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Demone, Jordan. "Characterizing the role of the transcriptional adaptor ADA2: an integrating node in the cold response mechanism in «Brachypodium distachyon»." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114517.
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Freezing stress limits crop productivity and generates substantial economic losses every year. While most temperate cereal crops exhibit some degree of cold tolerance, they require exposure to low, non-freezing temperatures in order to acclimate. This involves the induced expression of cold-regulated (COR) genes. COR gene promoters contain sequences that are recognized by C-repeat Binding Factor 1 (CBF1), a transcription factor that may mediate gene expression via the SAGA (SPT-ADA2-GCN5-acetyltransferase) complex in response to cold stress. The goal of this study was to characterize the function of the adaptor protein ADA2, a member of the SAGA complex that may link CBF1 to chromatin remodeling proteins. Expression analysis confirmed that Brachypodium CBF1 and ADA2 were expressed synchronously in response to cold treatment. Bimolecular fluorescence complementation (BiFC) analysis demonstrated that ADA2 and CBF1 interact directly in planta. These results further support the hypothesis that the SAGA complex exists in Brachypodium and that it may play an important role in mediating the cold response mechanism.Pour les pays nordiques, les épisodes de gel précoces et tardifs limitent considérablement le rendement des cultures et génèrent de lourdes pertes économiques. Bien que la plupart des plantes céréalières cultivées au Canada possèdent d'emblée un certain niveau de tolérance au gel, elles nécessitent toutes une période d'exposition à de basses températures (de 2 à 10°C) afin de maximiser leurs niveaux de tolérance. Ce processus d'acclimatation repose sur l'expression induite des gènes COR (Cold-Regulated Genes) contrôlés en grande partie par le régulateur CBF1 (C-repeat Binding Factor 1). Récemment, il a été proposé que CBF1 pourrait interagir avec le complexe chromatinien SAGA dans le but d'accomplir sa fonction. Cette interaction serait médiée par une sous-unité du complexe SAGA, la protéine adaptatrice ADA2. Cette dernière représenterait donc le lien moléculaire unissant les mécanismes de régulation génique traditionnels et chromatiniens impliqués dans le développement de la tolérance au gel des plantes. Le but de cette étude était de caractériser l'interaction physique entre CBF1 et ADA2 dans un contexte in planta. Des analyses d'expression en temps réel ont démontré que BradiCBF1 et BradiADA2 sont exprimés de façon similaire en réponse aux basses températures. De plus, des analyses d'interactions utilisant la technique de complémentation bimoléculaire de fluorescence (BiFC) ont démontré pour la première fois une interaction in planta entre les protéines BradiCBF1 and BradiADA2. Les résultats présentés ici suggèrent fortement qu'un complexe apparenté au complexe SAGA existe chez Brachypodium distachyon et que ce dernier pourrait jouer un rôle important lors du développement de la tolérance au gel chez les plantes céréalières.
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Kong, Mei 1972. "Epidermal growth factor-induced DNA synthesis : key roles for phosphatidylinositol 3-kinase and the adaptor protein Gab2." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82904.
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In primary rat hepatocytes, we found that activation of the Pl3-kinase pathway is both necessary and sufficient to account for EGF-induced DNA synthesis. To identify the mechanism of EGF-induced Pl3-kinase activation, we demonstrated that three distinct p85-associated complexes were formed following EGF: ErbB3-p85, Shc-p85 and a large complex Gab2-Grb2-SHP2-p85. The latter accounted for >80% of total Pl3-kinase activity. Further experiments showed that these complexes are differentially localized in rat liver following EGF treatment. ErbB3-p85 and Shc-p85 complexes were localized to PM and Endosomes; whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly and exclusively in cytosol. A central role for Gab2 in EGF-induced Pl3-kinase activation and DNA synthesis was established when we observed that over-expression of wild-type Gab2 augmented these EGF actions, whereas a Gab2 mutant lacking p85 binding sites did not effect such augmentation. Over-expression of the PH-domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, Pl3-kinase activation and DNA synthesis, whereas over-expressed Gab2 lacking the PH-domain was comparable to wild-type Gab2 in respect to these EGF-induced signals. These data demonstrated that Gab2 is phosphorylated and mediates EGF signaling in a PH-domain independent manner. We then explored the mechanism of Gab2 phosphorylation by EGF; our results demonstrated that PP1, a selective inhibitor of Src family kinases, blocked EGF-induced Gab2 tyrosine phosphorylation and downstream events. Moreover, Gab2 phosphorylation was increased in Csk knock-out cells in which Src family kinases are constitutively activated. A constitutive association between Gab2 and Src via proline rich sequences on Gab2 was demonstrated since deletion of proline rich sequences in Gab2 prevented EGF-induced association of Src with Gab2, Gab2 phosphorylation, Pl3-kinase/Akt activation, and DNA synthesis. The role of SHP2 was defin
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Tan, Meng Kwang Marcus. "Deciphering the biological functions of F-box proteins through the use of Parallel Adaptor Capture (PAC) proteomics." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11164.
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The timely and selective proteasomal degradation of proteins is important for the maintenance of proper cellular processes. Prior to proteasomal degradation, proteins destined for degradation are polyubiquitiylated by ubiquitin ligases (E3s). The SKP1-CUL1-F-box protein (SCF) complex is a member of the cullin-RING ligase (CRL) superfamily of modular, multi-protein E3s, in which the F-box protein (FBP) acts as a substrate specificity factor for the recruitment of substrates to the SCF complex. Many of the 69 human FBPs remain uncharacterized. From our current knowledge of FBPs, we know that they regulate a myriad and diverse set of cellular processes and their misregulation is associated with diseases, including cancer. Though many approaches exist for the identification of SCF substrates and/or interactors, most existing genetic and quantitative proteomic methods are not capable of adaptor identification, while interaction proteomic approaches are generally performed in a low throughput manner. To facilitate our characterization of FBPs, we have developed a novel, facile proteomic approach for the identification of interactors, including substrates, of FBPs. In this method, FBPs, which together with SKP1 form the adaptor subunit of the SCF complex, are individually expressed in cells. These cells are subsequently exposed to 3 conditions: left untreated, treated with MLN4924 (neddylation inhibitor) or treated with Bortezomib (proteasome inhibitor). After treatment, cells are lysed, and the lysates are affinity-purified and processed in parallel for proteomic analysis. This approach, which we have named Parallel Adaptor Capture (PAC) proteomics, was successfully applied for the characterization of three novel FBP/substrate interactions: FBXW11 (β-TrCP2)/DEPTOR (Appendix 1), FBXL17/BACH1 (Chapter 2) and FBXO22/KDM4A (Chapter 3). Besides the characterization of these FBPs, PAC proteomics was performed on 19 leucine-rich repeats containing FBPs, the FBXLs, identifying 230 high confidence interacting proteins, including known regulatory proteins and substrates. Deciphering the biological context and significance of these interactions will allow us to understand the importance of FBXLs in the cellular processes in which they regulate. Since CRLs require neddylation to efficiently ubiquitylate their substrates and most CRL substrates are degraded by the proteasome, PAC proteomics, in principle, can also be applied to other CRL adaptors.
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Davies, Alexandra Katherine. "An investigation of the function of adaptor protein complex 4 (AP-4)." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/289777.
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Vesicle trafficking provides the solution to the 'sorting problem' - how the eukaryotic cell maintains the distinct identities, and thus functional properties, of its membrane-bound organelles. During vesicle trafficking, proteins are selectively sorted into membrane bound transport intermediates by vesicle adaptors, which include those of the highly conserved adaptor protein (AP) complex family. Each AP complex has a distinct subcellular localisation and functions in the sorting of a specific subset of transmembrane cargo proteins. Adaptor protein complex 4 (AP-4) is one of the more recently identified AP complexes, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder, suggesting an important role in neuronal development and homeostasis. However, the pathomechanisms that underly the neuronal pathology in AP-4 deficiency are currently unknown. AP-4 is proposed to function in protein sorting at the trans-Golgi network (TGN), so AP-4 deficiency can be thought of as a disease of missorting. The aim of this study was to apply unbiased global proteomic approaches to define the composition of AP-4 vesicles and to identify physiological cargo proteins of the AP-4 pathway. Using 'Dynamic Organellar Maps' and comparative analysis of vesicle-enriched fractions from wild-type and AP-4-depleted cells, three ubiquitously expressed transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, were found to be mislocalised in AP-4-deficient cells. Two novel cytosolic AP-4 accessory proteins, RUSC1 and RUSC2, were also identified. Further proteomic analyses confirmed the interactions between these proteins. AP-4 deficiency was found to cause missorting of ATG9A in diverse cell types, including patient derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A and SERINC-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the 'ATG9 reservoir' required for autophagosome biogenesis. This study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.
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Fournier, Tanya M. "The role of signalling pathways downstream from the Grb2 adaptor protein in Met receptor and Tpr-Met oncoprotein biological activities /." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36925.
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Activation of the Met receptor tyrosine kinase by its ligand, Hepatocyte Growth Factor (HGF), leads to mitogenesis, cell motility, morphogenesis, and angiogenesis. Mutational analysis has demonstrated the requirement of a single tyrosine within the carboxy-terminus (Y1356) of the Met receptor for the recruitment and activation of all Met-dependent signalling pathways and for the transformation of fibroblasts by the Tpr-Met oncogene. The selective abolishment of Grb2 from the Tpr-Met oncoprotein, by generating an asparagine to histidine mutation two amino acids downstream from Y1356 (N1358H), led to a reduction in Tpr-Met-mediated transformation of fibroblasts. Moreover, Met receptor studies demonstrate that while a Grb2 binding site is not required for epithelial cell motility, it is critical for the formation of branching tubules when cells are suspended in a collagen matrix. This suggests that Grb2-dependent pathways are involved in the organization and polarization of epithelial cells following Met receptor stimulation.Grb2 associated molecules, Gab1 and Cbl, are highly phosphorylated following stimulation of the Met receptor. Moreover, signaling pathways associated with Gab1 are critical for branching tubulogenesis in epithelial cells. Expression of a constitutively active version of Cbl, 70z-Cbl, results in an epithelial-mesenchymal transition, leading to the breakdown of cellular junctions and reorganization of the actin cytoskeleton. The amino-terminal SH2 domain is the minimal region required to induce morphological changes, which may be mediated through its interaction with the Met receptor, and/or an unidentified protein of 150 kDa.
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Chen, Bin, and 陈斌. "Structural and functional characterization of human APPL2, a novel adaptor protein involved in insulin signaling." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B4552757X.
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Langel, Felicia D. "Molecular mechanisms of Bcl10-mediated NF-kappaB signal transduction /." Download the dissertation in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/langel2006.pdf.
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Zhu, Weidong, and 朱伟东. "APPL1 and APPL2: a pair of adaptor proteins as "yin-and-yang" regulators of insulin signaling in skeletalmuscle." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B45980470.
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Grazette, Affif. "Molecular characterisation of squamous cell carcinoma antigen recognised by T-cells 3, an adaptor protein of ubiquitin specific protease 15." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33081/.
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The deubiquitinating enzyme USP15, a member of the USP family, reverses the process of ubiquitination thereby altering the fate of a plethora of substrates. As such, USP15 has been implicated in a numerous important cellular pathways including cell cycle progression, transcriptional modification and DNA damage repair. The spliceosomal subunit recycling protein SART3 has been shown to bind to USP15 enhancing its deubiquitination of histone H2B thereby providing histone dimers for reassembly during subsequent rounds of transcription and splicing. Furthermore SART3 has also been shown to bind to USP4, a close homologue of USP15. In addition to this histone chaperone activity, SART3 primarily functions by mediating the re-annealing of the U4 and U6 snRNPs to facilitate consequent rounds of splicing in addition to its implication in several disease states including numerous cancer types and HIV through interactions with various cellular proteins. In this thesis ITC, ESI-MS, analytical size exclusion, X-ray crystallography, SAXS and pull-down assays are used to characterise the interaction between USP15 and SART3, solve the structure of the N-terminus of SART3 and identify novel binding partners of the USP15-SART3 complex. The structure of SART3’s N-terminus (residues 96-574) has been solved to 3.04Å, revealing that it is a homodimer comprised of a series of anti-parallel α-helices that form a shape reminiscent of a bowtie. This dimeric arrangement is retained in solution and can bind two molecules of USP15DU. The DU-finger of USP15 plays a pivotal role in co-ordinating the binding interaction with SART3, as small changes to this region can affect the nanomolar affinity interaction between USP15 and SART3. In addition the USP15-SART3 complex appears to interact with several cellular proteins which could have significant impact in several disease states.
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Lundmark, Richard. "Sorting nexin 9 in clathrin-mediated endocytosis." Doctoral thesis, Umeå : Department of Medical Biochemistry and Biophysics, Umeå University, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-197.
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Yuan, Ming. "Antiphagocytosis by Yersinia pseudotuberculosis : role of the YopH target proteins." Doctoral thesis, Umeå : Umeå University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-957.
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Kebache, Sem. "A role for the Nck adapter in protein translation /." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82899.
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In mammals, Nck, represented by two genes, is a 47kDa protein lacking intrinsic enzymatic function. It is composed solely of three N-terminal Src-homology 3 (SH3) domains and a single C-terminal Src-homology 2 (SH2) domain. Nck is classified as an adapter molecule that links cell surface receptors, via its SH2 domain, to downstream effectors, through its SH3 domains. Two cDNAs coding for the carboxy-terminal region of the beta subunit of the eukaryotic initiation factor 2 (eIF2beta) were isolated from a two-hybrid screen to identify new effector molecules interacting with the SH3 domains of Nck. eIF2beta is a component of the molecular complex eIF2 responsible for one of the early steps in the initiation of protein synthesis. In this thesis, I determined in vivo that the first and the third SH3 domains of Nck were both required for its interaction with eIF2beta. Furthermore, Nck and eIF2beta colocalized in an enriched ribosomal fraction. The localization of Nck in this compartment is enhanced upon insulin stimulation. I also established that Nck-1 overexpression is concomitant with an increase in protein translation. Interestingly, the effect of Nck on translation was dependent on its first and third SH3 domains originally found to mediate its interaction with eIF2beta. To elucidate how Nck modulates protein translation, I examined whether Nck maintains its positive effects under ER stress conditions where protein synthesis is normally inhibited. Interestingly, my data revealed that Nck-1 overexpression antagonized ER stress-induced inhibition of translation. Furthermore, I demonstrated that overexpression of Nck-1 prevented PERK activation, eIF2alpha phosphorylation, BiP induction and cell survival in response to ER stress treatments. This integrates Nck in the regulation of the ER unfolded protein response (UPR). I also provided evidence that the effects of Nck on the UPR involve its second SH3 domain and a phosphatase-dependent mechanis
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Podmore, Lauren. "The role of the p66-Shc adapter protein in mammary tumourigenesis." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119690.
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The ShcA adaptor protein is a key signaling molecule in the regulation of survival, angiogenesis, immune suppression and metastasis in breast cancer cells. The mammalian ShcA gene encodes three isoforms which are produced through alternative translation initiation (p46Shc, p52Shc) or different promoter usage (p66Shc). Although p46Shc and p52Shc are constitutively expressed and confer pro-tumorigenic signals, p66Shc levels vary in cancer cell lines. While activation of p66Shc through phosphorylation of its serine-36 (S36) is known to induce reactive oxygen species (ROS) formation, the role it plays in tumourigenesis is poorly understood. Our objective was to characterize the impact of p66Shc expression on mammary tumourigenesis both in vitro and in vivo. To this end, p66Shc-expressing vectors were stably transfected into two cell lines low in endogenous p66Shc; one driven by oncogenic ErbB2 activation (NIC-4360) and one lacking ErbB2 expression altogether (MDA-MB-231). These cells were characterized in vitro before being injected into the mammary fat pad (MFP) of immune-compromised mice for characterization in vivo. Our data revealed that while increased p66Shc expression promotes tumour initiation by way of increased angiogenesis, sustained p66Shc activation serves to decrease tumour cell proliferation. Our observation that p66Shc delayed tumour outgrowth in ErbB2-expressing cells also indicates a link between p66Shc activation and ErbB2 expression. The results of this study demonstrate that p66Shc confers both pro- and anti-tumourigenic properties, and that its role in mammary tumourigenesis is largely dependent on its ability to induce production of ROS.La protéine adaptatrice ShcA est une molécule clé dans la régulation de la survie, de l'angiogénèse, de la suppression tumorale et des métastases des cellules cancéreuses du sein. Le gène ShcA, chez les mammifères, encode trois isoformes qui sont produites par site d'initiation alternatif pour la transcription (p46Shc, p52Shc) ou l'utilisation d'un différent promoteur (p66Shc). Malgré le fait que p46Shc et p52Shc soient constitutivement exprimés et confèrent un signal bénéfique aux tumeurs, les niveaux de p66Shc varient dans les lignées cellulaires cancéreuses. L'activation de p66Shc via la phosphorylation de Sérine36 (S36) est connue pour induire la formation de dérivés réactifs de l'oxygène (ROS), toutefois le rôle que cela joue dans la tumorigénèse est mal compris. Notre objectif était de caractériser l'impact de l'expression de p66Shc sur la tumorigénèse mammaire, à la fois in vitro et in vivo. À cette fin, des vecteurs exprimant p66Shc ont été transfectés, de façon stable, dans deux lignées cellulaires ayant une expression endogène faible en p66Shc : une mû par l'activation de l'oncogène ErbB2 (NIC-4360), l'autre complètement exempte de ErbB2 (MDA-MB-231). Ces cellules ont été caractérisées in vitro avant leur injection dans les tissus graisseux mammaires de souris immunodéficientes pour la caractérisation in vivo. Nos données démontrent qu'une augmentation de l'expression de p66Shc promouvoit l'initiation tumorale via une augmentation de l'angiogénèse, toutefois une activation soutenue de p66Shc entraîne une diminution de la prolifération des cellules tumorales. Notre observation que p66Shc retarde la croissance tumorale dans les cellules exprimant ErbB2 dénote un lien entre l'activation de p66Shc et l'expression de ErbB2. Les résultats de cette étude démontrent que p66Shc confèrent des propriétés à la fois bénéfiques et néfastes pour les tumeurs, et que son rôle dans la tumorigénèse mammaire est largement dépendante de son habilité à induire la production de ROS.
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Battersby, Alysia. "Identification of molecules that interact with the adaptor protein Dof." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962733261.
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Wang, Ying. "Genetic dissection of adaptor molecules in lymphocyte development, homeostasis and function." Aix-Marseille 2, 2006. http://www.theses.fr/2006AIX22035.
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LAT (Linker for activation of T cells) assembles an essential platform for recruiting multiple proteins after TCR engagement. Mice homozygous for a single mutation in tyrosine residue 136 of the LAT adaptor showed impeded T cell development. However, later in life they accumulated polyclonal Th2 CD4+ effector T cells, which triggered tissue eosinophilia and massive maturation of B cells into plasma cells secreting high level IgG1 and IgE. To investigate the molecular and cellular pathways that might be involved in the triggering of this pathological condition, we used short-term and long-term adoptive transfer into hosts that were specifically deprived of T cells. We found that CD4 T cells from LATY136F mutant mice displayed similar homeostasis to their wild-type counterpart at the beginning of the reconstitution. However, 8 weeks after reconstitution, transfer of the LATY136F derived CD4 T cells gives rise to a condition that fully reproduce the lymphoproliferative disease originally observed in LATY136F mice. Accordingly, the reconstituted mice showed a large accumulation of CD4 T cells and B cells in secondary lymphoid organs, as well as hypergammaglobulinemia IgG1 and IgE in serum, and eosinophilia in multiple tissues. This demonstrated that the LATY136F disorder was T cell autonomous. Further results derived from comparison between CD3 epsilon-deprived hosts that are either MHCII-sufficient or-deficient showed that in the absence of MHCII molecules, LATY136F CD4 T cells were capable, however with a two-fold reduced efficiency, of proliferating and of helping B cells to mature into IgG1 and IgE plasma cells. These results suggested that the lymphopoliferation of CD4 T cells in LATY136F mutant mice and the ensuing T/B cooperation that leads to massive amount of IgE and IgG1 are largely independent on MHC Class II molecules. This almost TCR-independent and thus “autistic” behavior vis-à-vis MHC Class II molecule appears to be a unique property of CD4 T cells harboring the LATY136F mutation. NTAL (non-T cell activation linker, also called LAB), a newly identified transmembrane adaptor protein, shared structural similarity with LAT. It is highly expressed in B cells, NK cells and mast cells. NTAL/LAB is rapidly phosphorylated by 4 Src-family kinases (Lck or Lyn) and Syk- family kinases (ZAP-70 or Syk) after ligation of BCR, FcγRI and FcεRI receptors. The major proteins that associated with NATL/LAB after phosphorylation were identified as Grb2, SOS1, C-Cbl and Gab1 etc. The disruption of NTAL/LAB expression in B cell line can lead to decreased calcium mobilization and Erk activation, which suggested that NTAL/LAB is acting in BCR-mediated calcium flux and Ras-MAPK activation. To delineate the respective roles of NTAL/LAB and LAT in B cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed with same efficiency in Lat-/- Ntal-/- double-deficient mice and in mice lacking either of the two adaptors and in wild-type mice, which demonstrated that NATL/LAB is dispensable in B cell development. Upon B cell antigen receptor cross-linking, Ntal-/- B cells exhibited slightly increased Ca2+ mobilization and proliferation. Analysis of T-dependent and T-independent humoral responses showed that Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Specific serum immunoglobulins at normal titers were produced in response to T cell-independent antigens. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play in B cells a role symmetric to the role played by LAT in T cellsL’adaptateur transmembranaire LAT (Linker for Activation of T cells) constitue une plateforme moléculaire assurant le recrutement de nombreuses protéines impliquées dans la transduction des signaux médiés par le TCR. Les souris homozygotes pour une mutation ponctuelle de la tyrosine 136 du domaine intracytoplasmique de LAT présentent un défaut dans le développement des lymphocytes T. En outre, elles développent progressivement une lymphoprolifération T CD4+ polyclonale qui est associée à une éosinophilie tissulaire et à une maturation massive des cellules B en plasmocytes sécrétant des niveaux élevés d’IgG1 et d’IgE. Pour disséquer les mécanismes cellulaires et moléculaires associés au développement de cette pathologie, nous avons mis en oeuvre un système de transfert adoptif à court terme et à long terme dans des hôtes présentant une immunodéficience sélective en lymphocytes T. Nous avons montré que les cellules T CD4+ des souris mutantes LATY136F présentent une homéostasie similaire à celle des cellules wt en début de reconstitution. En revanche, 8 semaines après reconstitution, les cellules T CD4+ desLATY136F induisent une pathologie lymphoproliférative qui reproduit en tout point celle observée initialement dans les souris LATY136F. Ainsi, les souris reconstituées présentent-elles une accumulation massive de cellules T CD4+ et de lymphocytes B dans les organes lymphoïdes secondaires, une ypergammaglobulinémie IgG1 et IgE et une éosinophilie marquée dans de nombreux tissus. Ceci démontre que les lymphocytes T sont nécessaires et suffisants pour induire la pathologie LATY136F. Par comparaison des reconstitutions réalisées dans des hôtes déficients en CD3 et exprimant ou non les molécules d’histocompatibilité de classe II, nous avons établi que les lymphocytes T CD4+ LATY136F conservent, en l’absence de molécules d’histocompatibilité de classe II, leur capacité à proliférer et à stimuler la maturation des lymphocytes B en plasmocytes sécrétant des IgG1 et des IgE -avec une efficacité néanmoins deux fois moindre-. Ces résultats suggèrent que la lymphoprolifération des cellules T CD4+ dans les souris mutantes LATY136F et que la coopération T/B associée qui conduit à des concentrations sériques considérables d’IgE et IgG1 sont, pour une large part, indépendants des moléculesd’histocompatibilité de classe II. Ce comportement largement indépendant du TCR et pouvant à ce titre être qualifié de comportement autistique vis-à-vis des molécules de classe II apparaît comme une propriété unique des cellules T CD4+ portant la mutation LATY136F. NTAL (Non T cell Activation Linker, également appelé LAB) est un adaptateur transmembranaire récemment identifié qui possède des similarités structurales avec LAT. NTAL est fortement exprimé dans les cellules B, les cellules NK et les mastocytes. NTAL est rapidement phosphorylé par les kinases de la famille Src (Lck ou Fyn) et de la famille Syk (Zap-70 ou Syk) après engagement du récepteur des cellules B (BCR) ou des récepteurs au fragment Fc des immunoglobulines de type FcγRI et FcεRI. Les principales protéines identifiées comme associées à la molécule NTAL phosphorylée sont Grb2, SOS1, C-Cbl, Gab1. La suppression de l’expression de NTAL dans une lignée B conduit à une diminution des flux calciques et de l’activation des molécules Erk, ce qui suggère que NTAL participe à l’induction des flux calciques et à l’activation de la voie Ras-MAPKassociées à la stimulation du BCR. Pour identifier les rôles respectifs de NTAL et de LAT dans le développement et la fonction des lymphocytes B, des souris déficientes pour Ntal ont été générées et croisées avec des souris déficientes pour LAT. L’ analyse de ces souris a montré que les cellules B se développent avec la même efficacité dans les souris doublement déficientes Lat-/-Ntal-/-, dans les souris déficientes dans l’un de ces deux gènes et dans les souris wild-type, ce qui démontre que NTAL n’est pas indispensable pour le développement des lymphocytes B. L’agrégation des récepteurs des cellules B induit, par ailleurs, des niveaux de prolifération et des niveaux de flux calciques légèrement augmentés dans les souris déficientes pour Ntal. L’analyse des réponses humorales T dépendantes et T indépendantes a montré que les souris déficientes pour Ntal possèdent, en outre, des niveaux augmentés d’anticorps naturels et des réponses humorales légèrement amplifiées en réponse à des antigènes T dépendants. Des titres normaux d’immunoglobulines sériques spécifiques sont observés en réponse à des antigènes T indépendants. Enfin, bien que NTAL soit également exprimé dans les plasmocytes, son absence n’affecte pas l’hypergammaglobulinémie E et G1 sedéveloppant dans les souris porteuses de la mutation LATY136F. NTAL ne constitue donc pas dans les lymphocytes B l’équivalent fonctionnel de LAT dans les lymphocytes T
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Aghaei, Amin. "Symmetry-Adapted Molecular Modeling of Nanostructures and Biomembranes." Research Showcase @ CMU, 2013. http://repository.cmu.edu/dissertations/295.
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Tremendous advances in nanoscience during the past decades have drawn a new horizon for the future of science. Many biological and structural elements such as DNA, bio-membranes, nanotubes, nanowires and thin films have been studied carefully in the past decades. In this work we target to speed up the computational methods by incorporating the structural symmetries that nanostructures have. In particular, we use the Objective Structures (OS) framework to speed up molecular dynamics (MD), lattice dynamics (phonon analysis) and multiscale methods. OS framework is a generalization of the standard idea for crystal lattices of assuming periodicity of atomic positions with a large supercell. OS not only considers the translational periodicity of the structure, but also other symmetries such as rotational and screw symmetries. In addition to the computational efficiency afforded by Objective Structures, OS provides us with more flexibility in the shape of the unit cell and the form of the external deformation and loading, comparing to using the translational periodicity. This is because the deformation and loading should be consistent in all cells and not all deformations keep the periodicity of the structures. For instance, bending and twisting cannot be modeled with methods using the structure's periodicity. Using OS framework we then carefully studied carbon nanotubes under non-equilibrium deformations. We also studied the failure mechanism of pristine and twisted nanotubes under tensile loading. We found a range of failure mechanisms, including the formation of Stone-Wales defects, the opening of voids, and the motion of atoms out of the cross-section. We also used the OS framework to make concrete analogies between crystalline phonons and normal modes of vibration in non-crystalline but highly symmetric nanostructures.
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Tufan, Hale Ann. "Cellular and molecular defence responses of wheat to adapted and non-adapted magnaporthe species." Thesis, University of East Anglia, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522402.
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Blast disease of wheat has emerged as a new and serious field disease affecting wheat production in South America. The causal agent of wheat blast, Magnaporthe oryzae, infects cultivated crops including rice, wheat and barley, while M grisea is adapted to infect wild grass species. Durable disease resistance of crops against fungal pathogens may be achieved through non-host resistance; which is defined as resistance of a plant species to a non-adapted pathogen. Cellular defence responses and transcriptional changes of wheat cv. Renan, infected with adapted and non-adapted isolates of Magnaporthe spp., were investigated. Appositions formed beneath attempted penetration sites appeared to prevent colonisation by the nonadapted M grisea isolate, but were breached by the adapted M oryzae isolates. Microarray analysis indicated that wheat undergoes extensive transcriptome reprogramming following inoculation with both adapted and non-adapted isolates of Magnaporthe spp. A distinct set of transcripts were induced exclusively in response to the non-adapted M grisea isolate, while others were induced in response to both adapted and non-adapted isolates. Defence-related transcripts induced in common by adapted and non-adapted isolates were differentially regulated in response to M oryzae and M grisea isolates over time. Establishment of a virus induced gene silencing (VIGS) system in wheat cv. Renan enabled functional analysis of differentially expressed transcripts. Silencing the gene WIRT increased cell-to-cell spread of M oryzae hyphae, but not penetration efficiency, suggesting a postpenetration resistance role for this gene. Another gene, TaNIN was induced at early time points in response to both adapted and non-adapted Magnaporthe isolates. A partial sequence of this gene was cloned and shown to be the first nodule inception (NM-like gene characterised in wheat. This study provides an insight into the development of Magnaporthe spp. on wheat, together with functional characterisation of transcripts differentially expressed during adapted and nonadapted Magnaporthe spp. -wheat interactions.
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Talbot, Suzanne. "The role of the TLR4 adaptor molecules Mal and TRIF during Salmonella infections." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611823.
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Haghdoust, Rouja. "Genetic and molecular analysis of resistance to adapted and non-adapted (heterologous) rust pathogens in barley." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/24387.
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Rust diseases caused by Puccinia spp. are specialised to different economically important crops such as wheat and barley and can cause significant crop failure when severe. For many decades, genetic resistance has proven effective in protecting crops against rust pathogens. However, the ability of rust pathogens to evolve rapidly can lead to the emergence of new virulent races that can overcome genetic resistance. Therefore, the identification of new durable sources of resistance is essential for crop production and food security. Barley is a diploid crop with a reference genome sequence that is a near non-host to some non-adapted rust pathogens. Thus, the barley-Puccinia pathosystem is amenable for studies of the genetic architecture of resistance to rust pathogens. This thesis aims to determine the genetic basis of specificity of resistance in different barley-Puccinia pathosystems and to identify valuable sources of resistance in barley that can be used for genetic improvement of cereal crops. Chapter one contains a detailed review of the literature, addressing aspects of biology of barley and rust pathogens as well as mechanisms of host and non-host resistance. Chapter two investigates the genetic architecture of non-host resistance in barley using diverse Puccinia isolates and identifies non-host resistance (NHR) QTL that are suitable for either map-based or rapid cloning approaches. Chapter three examines the isolate specificity and polygenic inheritance of resistance in barley to different P. striiformis isolates. Chapter four generates a high-density linkage map using DArT-Seq markers with the aim of mapping loci for seedling and adult plant resistance to adapted stripe, leaf and stem rust pathogens. Chapter five delivers an overall conclusion based on the findings of the thesis and includes future paths and perspectives.
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Hachet, Virginie. "The role of the adaptor molecule Importin α in nuclear envelope assembly in vitro." Paris 11, 2004. http://www.theses.fr/2004PA112091.
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Chez les eucaryotes, le noyau, contenant l'information génétique est délimité par l'enveloppe nucléaire. Au cours du cycle cellulaire, l'enveloppe nucléaire se désintègre puis se reforme autour des chromosomes. Les mécanismes responsables de la reformation de l'enveloppe nucléaire peuvent être étudiés en utilisant des extraits d'œufs de Xénope. Nous avons étudié le rôle de l'Importine alpha, molécule impliquée dans l'import de protéines contenant un signal de localisation nucléaire (NLS), dans ce processus. L'addition en excès de cette protéine ou de son substrat BSA-NLS inhibe la formation d'une enveloppe nucléaire autour de la chromatine. Ces résultats suggèrent un rôle de l'Importine alpha dans ce processus. De plus, le retrait de la protéine endogène des extraits d'œufs de Xénope inhibe l'assemblage de l'enveloppe nucléaire. Trop ou trop peu d'Importin alpha dans le système inhibe la reformation de l'enveloppe nucléaire in vitro. L'équilibre entre la quantité d'Importine alpha et de ses substrats NLS semble crucial pour ce processus. Nous avons démontré que l'Importine alpha pouvait s'associer aux membranes et que cette association était régulée par phosphorylation. L'Importine alpha cytosolique est plus phosphorylée que l'Importine alpha liée aux membranes. La phosphorylation de l'Importine alpha liée aux membranes induit la dissociation de celle-ci des membranes. L'utilisation de formes mutantes de l'Importine alpha qui soit ne lient pas les membranes, soit ne sont pas relarguées des membranes par phosphorylation, a permis de démontrer un rôle direct de la forme membranaire de l'Importine alpha dans la reformation de l'enveloppe nucléaire in vitroIn eukaryotes, the nucleus, containing the genetic information, is delimited by the nuclear envelope (NE). During the cell cycle, the NE disassembles and then re-assembles around the chromosomes. The molecular mechanisms underlying NE formation can be studied using a nuclear assembly reconstitution assay based on Xenopus laevis egg extract. We analysed the role of Importin alpha, adaptor molecule involved in the import of NLS-containing proteins. Addition of excess Importin alpha or BSA-NLS substrate to a nuclear assembly reaction inhibits the formation of a NE around chromatin, suggesting a direct or indirect role for the protein in this process. Moreover, addition of affinity purified antibodies directed against Importin alpha or depletion of the endogenous protein from Xenopus egg cytosol inhibits NE formation. Thus, too much or too little Importin alpha negatively affects nuclear assembly in vitro. The balance of Importin apha and NLS proteins in the system is of critical importance. An unexpected finding of our study was that Importin alpha can associate with membranes and that this association is regulated by phosphorylation. We could show that cytosolic Importin alpha is highly phosphorylated as compared to membrane-bound Importin alpha. Phosphorylation of membrane-associated Importin alpha leads to its release from membranes. Phosphopeptide mapping and mutagenesis analysis allowed us to identify some of the phospho-residues within Importin alpha. Using mutant forms of Importin alpha that either do not bind membranes or are not released from them by phosphorylation, we provide evidence that membrane-bound Importin alpha functions in NE formation in vitro
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Hohenstein, Edward G. "Implementation and applications of density-fitted symmetry-adapted perturbation theory." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/42699.
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Noncovalent interactions play a vital role throughout much of chemistry. The understanding and characterization of these interactions is an area where theoretical chemistry can provide unique insight. While many methods have been developed to study noncovalent interactions, symmetry-adapted perturbation theory (SAPT) stands out as one of the most robust. In addition to providing energetic information about an interaction, it provides insight into the underlying physics of the interaction by decomposing the energy into electrostatics, exchange, induction and dispersion. Therefore, SAPT is capable of not only answering questions about how strongly a complex is bound, but also why it is bound. This proves to be an invaluable tool for the understanding of noncovalent interactions in complex systems.
The wavefunction-based formulation of SAPT can provide qualitative results for large systems as well as quantitative results for smaller systems. In order to extend the applicability of this method, approximations to the two-electron integrals must be introduced. At low-order, the introduction of density fitting approximations allows SAPT computations to be performed on systems with up to 220 atoms and 2850 basis functions. Higher-orders of SAPT, which boasts accuracy rivaling the best theoretical methods, can be applied to systems with over 40 atoms. Higher-order SAPT also benefits from approximations that attempt to truncate unneccesary unoccupied orbitals.
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Alqadri, N. A. "Molecular genetic analysis of the Drosophila MRL adapter protein in hyperplastic growth and experimental metastasis." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3003392/.
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Members of the Mig-10/RIAM/Lamellipodin (MRL) family of adapter proteins transduce signals derived from growth factor receptors, via interactions with Ras GTPases and/or phospholipids, to changes in the actin cytoskeleton, increased cell motility, and altered cell adhesion properties. MRL proteins have roles in normal development and their overexpression is implicated in tumour development and cancer progression. The focus of this thesis has been to explore signalling upstream and downstream of the Drosophila MRL protein, encoded by pico, to better understand the effects of manipulating its function. This has been done both in the developing wing, where overexpressed pico is capable of driving hyperplastic overgrowth, and in the larval CNS, where pico cooperates with oncogenic Ras to promote both hyperproliferation and cell invasion. An important downstream consequence of pico overexpression is thought to be activation of the Serum Response Factor (SRF), which responds to changes in actin dynamics through the action of its cofactor Mal. An SRF-responsive reporter gene, whose expression recapitulates the distribution of SRF protein in the developing wing, has been used to provide the first in vivo evidence that pico, and its associated actin regulatory proteins, promote SRF signalling. This work has also helped to identify deterin, which encodes Drosophila Survivin, as an SRF target that is necessary for pico-mediated overgrowth of the wing by suppressing proliferation-associated cell death. SRF is also likely to be a key effector of pico in the brain, based on genetic evidence and the observation that cooperation between pico and oncogenic Ras is restricted to two populations of Repo-positive glial cells, which are enriched for SRF expression. This work has also explored the involvement of a potential cascade of protein phosphorylation upstream of pico using a panel of site-directed mutants in pico designed to abolish binding to specific MRL-associated proteins and/or mimic its phosphorylation state. Importantly, these findings are consistent with the known roles of MRL proteins in promoting growth via changes in the actin cytoskeleton, and support the potential involvement of MRL gain-of-function in tumour cell invasion and metastasis.
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Trusselle, Melanie Nancy. "Molecular dynamics simulations of citrate synthase from organisms adapted to different temperatures." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413659.
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Lorenz-Baath, Katrin. "Comparative analysis of the functions of integrin adaptor molecules ILK and PINCH1 in the skin epithelium." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-125025.
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Smeenk, Cecilia. "Identification of molecular changes and virulence determinants in a mouse adapted influenza virus A/FM/1/47." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/9963.
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The human influenza virus A/FM/1/47 (FM) was mouse-adapted by serial lung passage to produce the more virulent variant A/FM/1/47-MA (FM-MA). Previous genetic analysis identified four genome segments, 4, 5, 7 and 8 that are statistically associated with virulence. The aim of this investigation was first, to find the mutations on these four genome segments and then to determine a role for these changes in disease production. Upon sequencing segments 4 and 7, single amino acid replacements were found at amino acid 47 of the HA2 subunit of the hemagglutinin (HA) and at amino acid 139 of the matrix protein (M1). Segments 5 and 8 had not mutated on mouse-adaption. Viral growth kinetics were studied both in the mouse lung and in cell culture with MDCK cells. For viral pathology, standard hematoxylin, phloxine, saffron staining of formalin fixed sections, fluorescent antibody labelling of frozen sections and flow cytometric analysis for infected cells and immune cell recruitment was used to detect differences between the viruses. The matrix protein has a role in both replication and pathology and its coding change can be observed as a shift on SDS-PAGE. It was shown that viruses containing the matrix protein of FM-MA could replicate as quickly and to as high a titer as FM-MA itself. In pathology, it was apparent that segment 7 contributes to the early onset of interstitial pneumonia and that these reassortants can infect a greater number of cells than FM or the segment 4 reassortants. The parental FM-MA strain infects more cells in the lung and is more virulent than FM X FM-MA reassortants containing segments 4 and 7. (Abstract shortened by UMI.)
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Bailly, Jane E. "Determination of the mechanism of viral interference manifested by a mouse-adapted strain of influenza A virus." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq20989.pdf.
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Donnelly, Kevin. "Molecular and adaptive variation in the Caledonian Pine, Pinus sylvestris (L.)." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/10574.
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The remnants of the Caledonian Pine Forest represent the north western boundary of the Eurasian Pinus sylvestris (L.) distribution. Remnant populations occupy a diverse range of environments within Scotland, subject to a steep rainfall gradient, and previous investigations have found evidence of local adaptation. Additionally, studies of biochemical and molecular markers have indicated that Scotland’s native pinewoods originated from more than one glacial refugium. Whole-genome-shotgun (WGS) sequencing was employed for the discovery of mitochondrial (mt) variants that may provide further insight into the origins of P. sylvestris populations both in Scotland and mainland Europe. DNA extractions were performed on megagametophyte tissue from Scottish, Finnish, and Spanish populations. Three members of the closely related P. mugo species complex were also sequenced. Using similarity-based approach, 160kbp of putative mitochondrial sequence was recovered by comparison of de novo assembled contigs with the mtgenome of the gymnosperm Cycas taitungensis. In total, 16 novel variants were identified among samples, which may be used in future phylogeographic studies. A study of needle characters was performed for eight native populations of P. sylvestris in an outdoor provenance/progeny trial of 192 saplings. A negative correlation was detected between longitude and the number of stomatal rows present on needle surfaces. It was posited that this may be an adaptive response to lower water availability in eastern pinewoods, possibly in conjunction with increasing altitude. The west coast of Scotland is one of the wettest regions in Europe: western pinewoods may receive in excess of 3,000mm of rainfall in a year, compared with an average of 800mm eastern sites. To determine whether native pinewoods are differentially adapted to waterlogging, a glasshouse based provenance/progeny trial of 432 saplings from nine native populations was undertaken, in which 50% were subject to a long-term waterlogging treatment, and the remainder used as a control. Two studies were then conducted. In the first, responses to the treatment were assessed in terms of phenological and growth traits. Bud flush was delayed in response to waterlogging, and growth was impeded relative to the control. Although population differences were observed, treatment × population interactions were not detected. In the second study physiological traits known to be sensitive to plant stress and water balance were measured at intervals throughout the experiment. Prior to the commencement of the treatment needle δ13C was found to exhibit interpopulation differentiation, and was positively correlated with longitude. This seems likely to represent differential selection for water use efficiency between eastern and western pinewoods. Photochemical efficiency and stomatal conductance were found to be reduced by waterlogging, and needle δ13C was increased. After generalising populations into ‘high’ and ‘low’ rainfall groups (monthly averages of 214.9mm and 72.8mm, respectively), high rainfall populations were observed to maintain consistently higher photochemical efficiency under waterlogging the low rainfall populations. In addition, the low rainfall group exhibited greater variability in response to flooding (in terms of phenotypic and additive genetic variance) which may be indicative of a lack of past selection pressure.
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Tran, Van Chuoi Myriam. "Molecular cloning of an angiotensin II receptor isoform in the European eel, Anguilla anguilla." Thesis, Queen Mary, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368077.
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Bennett, Rhona Marie. "A molecular study on the adaption and response of plants to windy habitats." Thesis, Bangor University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446543.
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Abubaker, Nagah Suleman. "Molecular identification and physiological characterisation of bacteria adapted to grow at high salinity." Thesis, University of Sheffield, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555713.
Full textAbstract:
Three strains (AM7, JC33 and NW53) were isolated from an oil pipeline in Kuwait using rich medium and aerobic incubation. 16S rDNA sequencing was used to identify the strains as Bacillus licheniformis (AM7, 98.9% identity), Staphylococcus pasteuri (Je33, 98.8%) and Brevibacterium stationis (97.9%). Due to their origin from oil pipeline samples, the strains were tested for their ability to grow as biofilms on filter paper on agar plates and also by using the microtitre assay. All three strains were able to form biofilms on filter paper and in microtitre plates. Biofilm and planktonic growth of the three strains was determined at different temperatures, pH values and salinities J and optimum values for biofilm and planktonic growth were determined. The response of the three strains to salinity was carried out, because it was known that seawater and groundwater were used to propel the oil to the refinery. Using rich LB medium, all three strains grew well at 1 M NaCI and after adaptation, good growth was found at salinities up to 3 M NaCl for B. licheniformis and S. pasteuri. Growth was also found for Br. stationis at 3 M NaCl, but it was significantly less than at the lower salinities. Further studies on salinity tolerance were carried out on Br. stationis, since this was the least characterised of the three strains isolated. NMR analyses were used to determine the compatible solutes used by Br. stationis when grown on M9 minimal medium. It was found that glycine betaine (betaine) was the major compatible solute, but that proline and an unknown sugar were also present at high salinities, when glucose was the carbon source. When betaine was the carbon source, only betaine was accumulated as a compatible solute. When the compatible solute ectoine was used as the sole source of carbon in M9 medium, ectoine was the dominant compatible solute at low salinities, but betaine was still accumulated at high salinities. These results suggest that betaine is the major compatible solute used by Br. station is. Further characterisation of Br. stationis involved measuring NADH oxidase activity in crude cell-free extracts to examine the effects oftemperature and salinity on intracellular enzyme activity.
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ElDoliefy, Ahmed ElFatih Amin. "Molecular Mapping of Fusarium Head Blight Resistance in Two Adapted Spring Wheat Cultivars." Diss., North Dakota State University, 2015. https://hdl.handle.net/10365/27412.
Full textAbstract:
Releasing bread wheat (Triticum aestivum) cultivars with resistance to Fusarium head blight (FHB) disease can be endangered by narrowing the variation in genetic sources. However, resistance to FHB rely on five different types. FHB resistance types I, II, III, IV and V were assessed in field and greenhouse under multiple locations and years experiment’s combination. In our study, the genetic of FHB resistance in two widely cultivated hard red spring wheat varieties (‘Glenn’ and ‘Parshall’) were dissected. The specific objectives of the study were to generate recombinant inbred lines (RIL) populations, phenotypic assessment for FHB resistance, different informative genotypic marker data, genetic map, and finally QTL analysis. For Glenn/MN00216-4 (GM) population, 112 RIL were developed; while for Parshall/Reeder (PR) population, 110 RIL were developed. The RIL, checks and the two parents were evaluated for five FHBrelated and one agronomic-related traits over two to six environments in North Dakota, Minnesota, and South Dakota. Two genetic maps were developed covering 2,229 cM of length using 645 DArT markers for GM population, and 470.4 cM length using 154 DArT/SNP combined markers for PR population. Composite interval mapping identified 37 QTL for the GM population, and 10 QTL for the PR population. Results showed that Glenn lacks the major consistent (Fhb1 and Fhb5A) QTL from the Chinese source Sumai3, while acquired (Fhb2). Parshall proved to be domestic with no exotic resistance background, though it acquired similar genomic regions to Fhb2 of Sumai3. PR genome contains five major QTL including three novel QTL with multiple FHB resistance and two with stable effect (1AS and 4BL) across at least two environments. Along with these previously identified QTL for FHB resistance, in both populations, new QTL were also identified such as Fhb-1B1L.c and 7D1S.b in Glenn, and Fhb.5AL, 7AS and 4BL in Parshall. In conclusion, our study added to the wheat genome, two genetic maps, new QTL for FHB resistance and two germplasms with new recombination for QTL and/or resistance sources. Finally, Glenn and Parshall can be of great importance if implemented in wheat enhancement and molecular assisted breeding programs nationally and internationally.
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Nystedt, Björn. "Evolutionary Processes and Genome Dynamics in Host-Adapted Bacteria." Doctoral thesis, Uppsala universitet, Molekylär evolution, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-107720.
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Many bacteria live in close association with other organisms such as plants and animals, with important implications for both health and disease. This thesis investigates bacteria that are well adapted to live inside an animal host, and describes the molecular evolutionary processes underlying host-adaptation, based on bacterial genome comparisons. Insect-transmitted bacteria of the genus Bartonella infect the red blood cells of mammals, and we investigate host adaptation and genome evolution in this genus. In Bartonella, many host-interaction systems are encoded in a highly variable chromosomal segment previously shown to be amplified and packaged into bacteriophage particles. Among all genes imported into the Bartonella ancestor, we identify the short gene cluster encoding these phage particles as the most evolutionary conserved, indicating a strong selective advantage and a role in niche adaptation. We also provide an overview of the remarkable evolutionary dynamics of type IV and type V secretion systems, including a detailed analysis of the type IV secretion system trw. Our results highlight the importance of recombination and gene conversion in the evolution of host-adaptation systems, and reveal how these mutational mechanisms result in strikingly different outcomes depending on the selective constraints. In the insect endosymbionts Buchnera and Blochmannia, we show that genes frameshifted at poly(A) tracts can remain functional due to transcriptional slippage. Selection against poly(A) tracts is very inefficient in these genomes compared to other bacteria, and we discuss why this can lead to increased rates of gene loss. Using the human pathogen Helicobacter pylori as a model, we provide a deeper understanding of why highly expressed genes evolve slowly. This thesis emphasizes the power of using complete genome sequences to study evolutionary processes. In particular, we argue that knowledge about the complex evolution of duplicated gene segments is crucial to understand host adaptation in bacteria.
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Randall, T. S. "A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1370581/.
Full textAbstract:
Kinesins are motor proteins that have roles in cell division as well as the transport of various organelles and protein complex cargoes within cells. Kinesin-1 is considered the conventional motor protein and consists of three main isoforms, KIF5A, KIF5B and KIF5C. The family of Trafficking Kinesin proteins (TRAKs) bind to the cargo binding domain of kinesin-1 forming a link between the motor protein and their cargoes. The best characterised cargo transported by TRAKs is mitochondria. To understand further the mode of association between kinesin and TRAKs, the aim of this study was to determine the structure of the C-terminal cargo binding domain of kinesin-1. Due to the known binding of TRAK2 with the kinesin-1 family it was hypothesized that TRAKs may stabilize the KIF5A cargo binding domain. Hence, the cDNA encoding the KIF5A cargo binding domain, KIF5A800-1032, and the cDNA encoding the kinesin interacting domain of TRAK2, i.e. TRAK2100-380 were cloned into a bicistronic expression vector to result in epitope-tagged constructs. The expression of both proteins was characterized with respect to yield and solubilisation efficiency. TRAK2100-380 was stabilized by KIF5A800-1032. Affinity chromatography of soluble extracts found that KIF5A800-1032 and TRAK2100-380 did co-purify indicating that they do associate. However, the yield was insufficient for further characterisation. To determine the structure of the cargo binding domain of KIF5A several C-terminal constructs which varied in size and epitope tag location were generated. Bacterial growth and solubilisation conditions were optimized to maximize the yield of the various KIF5A cargo domain recombinant proteins. Each was purified by an appropriate affinity chromatography resin. Conditions were established to optimize the purity, yield, stability and aggregation state. One construct, KIF5A800-951 was isolated to a sufficient yield and did not aggregate. Therefore, KIF5A800-951 is a good candidate for further structural analysis. To refine further the TRAK binding site within the cargo domain of kinesin-1 a series of rationally designed C-terminal truncations of KIF5A and KIF5C were generated and co-expressed with either TRAK1 or TRAK2 in mammalian cells. The binding of kinesin-1 truncations to the TRAKs was determined by co-immunoprecipitation assays followed by quantitative immunoblotting. Deletion of the distal 71 amino acids of KIF5A resulted in an increased co-immunoprecipitation of KIF5A with TRAK2. Three possible TRAK2 binding sites within the KIF5A and KIF5C cargo binding domains were found. Furthermore, differences were found between TRAK1 and TRAK2 in terms of their association sites with KIF5A. Overall these studies yield new insights into kinesin/kinesin adaptor interactions which may impact in the future on a better understanding of neurodegenerative diseases such as hereditary spastic paraplegia and Charcot–Marie–Tooth disease which have both been linked to deficiencies in neuronal transport mechanisms of mitochondria.
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Garousi, Javad. "Development of ADAPT-based tracers for radionuclide molecular imaging of cancer." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-327419.
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ABD-Derived Affinity Proteins (ADAPTs) is a novel class of small engineered scaffold proteins based on albumin-binding domain (ABD) of streptococcal protein G. High affinity ADAPT binders against various therapeutic targets can be selected. In this thesis, we report a development of ADAPT-based radionuclide imaging agents providing high sensitivity and specificity of molecular imaging of HER2 expression in disseminated cancers. We investigated the feasibility of the use of ADAPTs as imaging agents and influence of molecular design and radiolabeling chemistry on in vivo targeting and biodistribution properties of the tracers. In Paper I we demonstrated the feasibility of the use of anti-HER2 ADAPT6 molecule as a high contrast imaging agent; In Paper II we evaluated the influence of composition of histidine-containing tag on in vivo biodistribution of ADAPT-based tracers labeled with 99mTc using 99mTc(CO)3 binding to histidine-containing tags and 111In using DOTA chelator at N-terminus; In Paper III we evaluated the influence of different aspects of N-terminus leading sequence on targeting including effect of sequence size on clearance rate and effect of the composition of the sequence on biodistribution profile; In Paper IV, we evaluated the influence of residualizing properties and positioning of the label on biodistribution and targeting; and In Paper V, we compared tumor-targeting properties of the ADAPT6 labeled at C-terminus with 99mTc using N3S chelator and 111In using DOTA chelator. In conclusion, ADAPTs constitute a very promising class of targeting probes for molecular imaging providing high contrast. Molecular design of the ADAPT proteins and chelators/linkers for labeling has an appreciable effect on their imaging properties.
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Chen, Chang. "Genetical and molecular systematic study on the genus Montagnea Fr., a desert adapted Gasteromycete." Thesis, Virginia Tech, 1999. http://hdl.handle.net/10919/10103.
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Montagnea arenaria [Hymenogastrales, Basidiomycota] , adapted to desert and xeric habitats, is morphologically and phenotypically variable. Species have been described on the basis of macromorphology and spore shape and size. This study was initiated to investigate populations of M. arenaria from Namibia in Africa and the Southwestern United States. It was hypothesized that biological species would exist in the widely separated populations. Spores from single sporocarps were germinated, single spore isolates were obtained and selfed to obtain mating types. On transfer, clamp connections were not maintained and mating patterns could not be achieved. Nuclear staining revealed multinuclei in the hyphae of both single spore isolates and compatible crosses. Spores were stained and found to have either 1 or 2 nuclei, but only four sterigmate basidia were observed. Limited partial compatibility was achieved and in some cases clamp connections formed within and between crosses from the two continents. Genomic DNA was extracted from old herbarium specimens. The ITS1, 5.8S, and ITS4 regions of nuclear ribosomal DNA were amplified and sequenced directly. Phylogenetic analysis using PAUP was performed. The hypothesis that Montagnea would form different biological species based on continental separation was rejected. In fact, the complex of isolates from widely varying locations not only had partial compatibility, but the variation in ITS sequences among widely distributed collections was relatively low. Lastly, no correlation between sporocarp size and gene flow among specimens from a wide variety of habitats was found. It appears that M. arenaria is a single, highly variable, widely distributed species.Master of Science
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Hjorth, Ingrid. "Molecular genetic analysis of the microbial community structure in nitrifying biofilms adapted to different salinities." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-14053.
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Nitrification, the microbial oxidation of ammonia to nitrate, is a key step inthe biological cycling of nitrogen, and is often used in wastewater treatmentfor removal of ammonium. Fish-farming in flow-through systems such asopen net pens is the largest contributor to human discharge of inorganicnutrients along the Norwegian coast. It is important to limit these emissionsby converting to recirculating systems for nitrogen removal. However, thenitrification process is known to be sensitive to high concentrations of salts,a factor of importance when establishing technology for treatment of salinewastewaters.The aim of this thesis was to investigate and compare the microbialcommunities of nitrifying biofilms adapted to different salinities. Two con-tinuous biofilm reactor systems were operated, one supplied with seawater-based cultivation medium, while the other was supplied with tapwater-basedcultivation medium. The microbial communities in the two reactors wereinvestigated by denaturing gradient gel electrophoresis (DGGE) and fluo-rescence in situ hybridization (FISH). A batch culture salinity response testwas carried out to investigate the acute effect of different salinities on thenitrification activity in the seawater-adapted culture.DGGE analysis based on 16S rRNA and amoA sequences showed thatthe seawater-based reactor had lower microbial diversity than the tapwater-based reactor, and that different nitrifiers seemed to dominate in the tworeactors. Ammonia- and nitrite-oxidizers affiliated with the nitrosomonadsand with Nitrospira were identified in both reactors. Ammonia-oxidizersrelated to Nitrosomonas oligotropha seemed to dominate in the tapwater-based reactor, while Nitrosomonas halophila seemed to dominate in theseawater-based reactor. The batch culture salinity respons test, comparedto a similar experiment by (Kristoffersen 2004), indicated that the nitrify-ing culture adapted to high salinity was more halotolerant than a cultureadapted to low salinity.Nitrifikasjon er en mikrobiell prosess der ammonium oksideres til nitrat.Nitrifikasjon er en viktig prosess i den biologiske nitrogensyklus, og er oftebrukt innen vannrensing for ̊ fjerne ammonium. Fiskeoppdrett i ̊aapnemerder er den største bidragsyteren til menneskaskapte utslipp av uorgan-iske næringssalter langs norskekysten, og det er viktig begrense disse ut-slippene ved ̊ g ̊ over til resirkulerte systemer for nitrogenfjerning. Nitri-a afikasjonsprosessen er kjent for ̊ være følsom for høye saltkonsentrasjoner,aog det er viktig ̊ ta hensyn til dette n ̊ teknologi for rensing av avløpsvannaarmed høyt saltinnhold etableres.M ̊ med denne oppgaven var ̊ undersøke og sammenlikne mikro-aletabielle samfunn i nitrifiserende biofilmer adaptert til ulike saliniteter. Tokontinuerlige reaktorsystemer ble drevet, ́n ble forsynt med sjøvannsbasertekultiveringsmedium, den andre ble forsynt med springvannsbasert kultiver-ingsmedium. De mikrobielle samfunnene i de to reaktorene ble undersøktved hjelp av denaturerende gradient gelelektroforese (DGGE) og fluorescensin situ hybridisering (FISH). En salinitetsresponstest ble utført for ̊ un-adersøke den akutte effekten av ulike saliniteter p ̊ nitrifikasjonsaktiviteten iaden sjøvanns-adapterte kulturen.DGGE-analyse basert p ̊ sekvenser av 16S rRNA og amoA indikerteaat den sjøvannsbaserte reaktoren hadde lavere mikrobiell diversitet enn denspringvannsbaserte reaktoren, og at ulike nitrifiserende bakterier dominerte ide to reaktorene. Ammonium- og nitrittoksiderende bakterier beslektet medNitrosomonas og Nitrospira ble funnet i begge reaktorene. Ammonium-oksiderende bakterier beslektet med Nitrosomonas oligotropha s ̊ ut til ̊aadominere i den springvannsbaserte reaktoren, mens Nitrosomonas halophilavar mer dominerende i den sjøvannsbaserte reaktoren. Salinitetsrespons-testen, sammenliknet med et liknende eksperiment utført av Kristoffersen(2004), indikerte at den nitrifiserende kulturen adaptert til høy salinitet varmer halotolerant enn en kultur adaptert til lav salinitet.
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au, kryan@ccg murdoch edu, and Karon Magdalene Leanne Ryan. "Variation of flour colour in Western Australia adapted wheat: comparative genomics, molecular markers and QTL analysis." Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20061019.130337.
Full textAbstract:
The yellowness of flour colour ranges is an important quality trait in wheat for end-use products and is determined by the accumulation of carotenoids in the endosperm. The aims of this study were to develop EST-based molecular markers for genes encoding enzymes of the carotenoid biosynthetic pathway leading to xanthophyll accumulation and identify quantitative trait loci for flour colour (b*) and xanthophyll content in Western Australian adapted germplasm.
A novel bioinformatic strategy was developed to identify rice genes encoding key enzymes of the carotenoid biosynthetic pathway and to predict wheat orthologues on the short arm of chromosome 3 or long arm of chromosome 7. The bioinformatic strategy involved the identification of rice carotenoid genes on BAC/PAC contigs aligned to wheat mapped ESTs. Rice genes predicted to have wheat orthologues were selected based on ESTs mapping to regions on wheat homoeologous chromosomes 3 and 7 known to be involved in flour colour. The rice genes predicted to have wheat orthologues were Geranylgeranyltransferase I Ò¡Vsubunit (GGT-Ibeta) and Rab geranylgeranyltransferase component A (RGGT-A) on the short arm of chromosome 3, Lycopene Ò¡Vcylcase (LBC) on the long arm of chromosome 3 and Lycopene £`¡Vcylcase (LEC) on the long arm of chromosome 7.
The prediction of these wheat orthologues provided the basis for development of EST-based molecular markers for detecting variation in xanthophyll content. Wheat ESTs with unknown chromosomal locations and having the highest similarity to GGT-Ibeta, RGGT-A and LBC were selected for the development of molecular markers. No EST homologues were identified for LEC and therefore this gene was not further considered. Orthology was confirmed by sequencing and deletion lines were used to confirm chromosomal locations. Two partial orthologues of GGT-Ibeta were identified on the short arms of chromosomes 3B and 3D. A partial orthologue of RGGT-A was mapped to the proximal regions of the short and long arms of chromosome 3B. At least two or more orthologues of LBC were identified from nullisomic-tetrasomic lines. An EST-based molecular marker for GGT-Ibeta was found to be involved in minor variation of xanthophyll content in a Westonia*2/Janz doubled haploid population.
QTL analysis from three doubled haploid populations indicated variation in WA-adapted germplasm may be due to different alleles controlling flour colour. QTLs for b* and xanthophyll content were found to coincide on the short arms of chromosomes 3A, 4D, and 7B and the long arm of chromosomes 7A and 7B in WA-adapted germplasm. Homoeologous expression of regions controlling variation in b* and xanthophyll content on the long arm of chromosomes 7A and 7B suggests the shut-down of genes in the same region on chromosome 7D. The main outcome of this study is flour colour and identification of gene orthologues in wheat controlling variation in xanthophyll content is complex most likely because of the interaction of the carotenoid biosynthetic pathway with other pathways.
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Ryan, Karon Magadalene Leanne. "Variation of flour colour in Western Australia adapted wheat : comparative genomics, molecular markers and QTL analysis /." Access via Murdoch University Digital Theses Project, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20061019.130337.
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Allcock, David. "Investigating the molecular basis of cold temperature and high pressure adapted growth in Photobacterium profundum SS9." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/3859.
Full textAbstract:
Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.
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Ryan, Karon Magdalene Leanne. "Variation of flour colour in Western Australia adapted wheat: comparative genomics, molecular markers and QTL analysis." Thesis, Ryan, Karon Magdalene Leanne (2005) Variation of flour colour in Western Australia adapted wheat: comparative genomics, molecular markers and QTL analysis. PhD thesis, Murdoch University, 2005. https://researchrepository.murdoch.edu.au/id/eprint/285/.
Full textAbstract:
The yellowness of flour colour ranges is an important quality trait in wheat for end-use products and is determined by the accumulation of carotenoids in the endosperm. The aims of this study were to develop EST-based molecular markers for genes encoding enzymes of the carotenoid biosynthetic pathway leading to xanthophyll accumulation and identify quantitative trait loci for flour colour (b*) and xanthophyll content in Western Australian adapted germplasm.
A novel bioinformatic strategy was developed to identify rice genes encoding key enzymes of the carotenoid biosynthetic pathway and to predict wheat orthologues on the short arm of chromosome 3 or long arm of chromosome 7. The bioinformatic strategy involved the identification of rice carotenoid genes on BAC/PAC contigs aligned to wheat mapped ESTs. Rice genes predicted to have wheat orthologues were selected based on ESTs mapping to regions on wheat homoeologous chromosomes 3 and 7 known to be involved in flour colour. The rice genes predicted to have wheat orthologues were Geranylgeranyltransferase I beta-subunit (GGT-Ibeta) and Rab geranylgeranyltransferase component A (RGGT-A) on the short arm of chromosome 3, Lycopene beta-cylcase (LBC) on the long arm of chromosome 3 and Lycopene epsilon-cylcase (LEC) on the long arm of chromosome 7.
The prediction of these wheat orthologues provided the basis for development of EST-based molecular markers for detecting variation in xanthophyll content. Wheat ESTs with unknown chromosomal locations and having the highest similarity to GGT-Ibeta, RGGT-A and LBC were selected for the development of molecular markers. No EST homologues were identified for LEC and therefore this gene was not further considered. Orthology was confirmed by sequencing and deletion lines were used to confirm chromosomal locations. Two partial orthologues of GGT-Ibeta were identified on the short arms of chromosomes 3B and 3D. A partial orthologue of RGGT-A was mapped to the proximal regions of the short and long arms of chromosome 3B. At least two or more orthologues of LBC were identified from nullisomic-tetrasomic lines. An EST-based molecular marker for GGT-Ibeta was found to be involved in minor variation of xanthophyll content in a Westonia*2/Janz doubled haploid population.
QTL analysis from three doubled haploid populations indicated variation in WA-adapted germplasm may be due to different alleles controlling flour colour. QTLs for b* and xanthophyll content were found to coincide on the short arms of chromosomes 3A, 4D, and 7B and the long arm of chromosomes 7A and 7B in WA-adapted germplasm. Homoeologous expression of regions controlling variation in b* and xanthophyll content on the long arm of chromosomes 7A and 7B suggests the shut-down of genes in the same region on chromosome 7D. The main outcome of this study is flour colour and identification of gene orthologues in wheat controlling variation in xanthophyll content is complex most likely because of the interaction of the carotenoid biosynthetic pathway with other pathways.
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Ryan, Karon Magdalene Leanne. "Variation of flour colour in Western Australia adapted wheat: comparative genomics, molecular markers and QTL analysis." Ryan, Karon Magdalene Leanne (2005) Variation of flour colour in Western Australia adapted wheat: comparative genomics, molecular markers and QTL analysis. PhD thesis, Murdoch University, 2005. http://researchrepository.murdoch.edu.au/285/.
Full textAbstract:
The yellowness of flour colour ranges is an important quality trait in wheat for end-use products and is determined by the accumulation of carotenoids in the endosperm. The aims of this study were to develop EST-based molecular markers for genes encoding enzymes of the carotenoid biosynthetic pathway leading to xanthophyll accumulation and identify quantitative trait loci for flour colour (b*) and xanthophyll content in Western Australian adapted germplasm.
A novel bioinformatic strategy was developed to identify rice genes encoding key enzymes of the carotenoid biosynthetic pathway and to predict wheat orthologues on the short arm of chromosome 3 or long arm of chromosome 7. The bioinformatic strategy involved the identification of rice carotenoid genes on BAC/PAC contigs aligned to wheat mapped ESTs. Rice genes predicted to have wheat orthologues were selected based on ESTs mapping to regions on wheat homoeologous chromosomes 3 and 7 known to be involved in flour colour. The rice genes predicted to have wheat orthologues were Geranylgeranyltransferase I beta-subunit (GGT-Ibeta) and Rab geranylgeranyltransferase component A (RGGT-A) on the short arm of chromosome 3, Lycopene beta-cylcase (LBC) on the long arm of chromosome 3 and Lycopene epsilon-cylcase (LEC) on the long arm of chromosome 7.
The prediction of these wheat orthologues provided the basis for development of EST-based molecular markers for detecting variation in xanthophyll content. Wheat ESTs with unknown chromosomal locations and having the highest similarity to GGT-Ibeta, RGGT-A and LBC were selected for the development of molecular markers. No EST homologues were identified for LEC and therefore this gene was not further considered. Orthology was confirmed by sequencing and deletion lines were used to confirm chromosomal locations. Two partial orthologues of GGT-Ibeta were identified on the short arms of chromosomes 3B and 3D. A partial orthologue of RGGT-A was mapped to the proximal regions of the short and long arms of chromosome 3B. At least two or more orthologues of LBC were identified from nullisomic-tetrasomic lines. An EST-based molecular marker for GGT-Ibeta was found to be involved in minor variation of xanthophyll content in a Westonia*2/Janz doubled haploid population.
QTL analysis from three doubled haploid populations indicated variation in WA-adapted germplasm may be due to different alleles controlling flour colour. QTLs for b* and xanthophyll content were found to coincide on the short arms of chromosomes 3A, 4D, and 7B and the long arm of chromosomes 7A and 7B in WA-adapted germplasm. Homoeologous expression of regions controlling variation in b* and xanthophyll content on the long arm of chromosomes 7A and 7B suggests the shut-down of genes in the same region on chromosome 7D. The main outcome of this study is flour colour and identification of gene orthologues in wheat controlling variation in xanthophyll content is complex most likely because of the interaction of the carotenoid biosynthetic pathway with other pathways.
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Tauer, Anthony Philip. "Theoretical Investigations of Pi-Pi and Sulfur-Pi Interactions and their Roles in Biomolecular Systems." Thesis, Georgia Institute of Technology, 2005. http://hdl.handle.net/1853/7573.
Full textAbstract:
The study of noncovalent interactions between aromatic rings and various functional groups is a very popular topic in current computational chemistry. The research presented in this thesis takes steps to bridge the gap between theoretical prototypes and real-world systems.
The non-additive contributions to the interaction energy in stacked aromatic systems are measured by expanding the prototype benzene dimer into trimeric and tetrameric systems. We show that the three- and four-body interaction terms generally do not contribute significantly to the overall interaction energy, and that the two-body terms are essentially the same as in the isolated dimer.
The sulfur-pi interaction is then studied by using the hydrogen sufide-benzene dimer as a prototype system for theoretical predictions. We obtain higly-accurate potential energy curves, as well as an interaction energy extrapolated to the complete basis set limit. Energy decomposition analysis using symmetry-adapted perturbation theory shows that the sulfur-pi interaction is primarily electrostatic in nature.
These theoretical results are then compared to an analysis of real sulfur-pi contacts found by searching protein structures in the Brookhaven Protein DataBank. We find that the most frequently seen configuration does not correspond to the theoretically predicted equilibrium for sydrogen sulfide-benzene, but instead to a configuration that suggests an alkyl-pi interaction involving the carbon adjacent to the sulfur atom. We believe our findings indicate that environmental effects within proteins are altering the energetics of the sulfur-pi interaction so that other functional groups are preferred for interacting with the aromatic ring.