Dissertations / Theses on the topic 'Proline-rich'
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1
Dikic, Inga. "Signal Transduction by Proline-Rich Tyrosine Kinase Pyk2." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5316-3/.
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Bess, Kirstin. "Transcriptional repression by the proline rich homeodomain protein." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390994.
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Murray, Nicola Jane. "NMR studies of salivary proline-rich proteins and tannins." Thesis, University of Sheffield, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284595.
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Swingler, Tracey. "Transcriptional repression by the proline rich homeodomain protein (PRH)." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419215.
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Drzymala, Luke. "Phosphorylation of human salivary proline-rich proteins in cultured cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0001/MQ40692.pdf.
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Soufi, Abdenour. "Oligomerisation and phosphorylation of the proline-rich homeodomain protein (PRH)." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432944.
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Chan, Maggie Tin Lai. "Proteolytic processing of recombinant human salivary proline-rich protein precursors (PRPs)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0025/MQ50333.pdf.
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Kofler, Michael. "GYF domains a class of proline rich ligand binding adaptor domains /." kostenfrei, 2007. http://www.diss.fu-berlin.de/2007/261/index.html.
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Lu, Ying. "Characterization of the interaction of human salivary proline-rich proteins with tannins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29245.pdf.
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Charlton, Adrian Jon. "Study of the interaction between salivary proline-rich proteins and plant polyphenols." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327666.
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Noy, Peter John. "Coordinate regulation of Vegf signalling genes by the proline rich homeodomain protein." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1527/.
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PRH is a transcriptional repressor that regulates development of the haematopoietic and vascular systems. VEGF is a mitogen that stimulates cell survival via cell surface receptors including VEGFR1 and VEGFR2. This thesis identifies Vegf, Vegfr1, and Vegfr2 as bona fide PRH target genes and shows PRH can control cell survival through the modulation of VEGF and VEGF receptor signalling. CK2 is a stress-activated protein kinase with pleiotropic activity and CK2 phosphorylation of PRH inhibits its DNA binding activity. Here I show that CK2 can antagonise PRH induced cell death and transcriptional regulation. Furthermore I show that CK2 reduces PRH stability and decreases nuclear retention of PRH. The oncogenic BCR-ABL fusion protein increases CK2 activity. I show that Inhibition of BCR-ABL in CML cells results in decreased PRH phosphorylation and the down-regulation of PRH target genes. Reestablishment of gene control by PRH is partly responsible for the therapeutic effects of BCR-ABL inhibition in CML and that the reestablishment of PRH function could be valuable for the treatment of leukaemias with elevated CK2 activity. These data show that PRH is a key regulator of the VEGF family and loss of PRH transcriptional activity, through elevated CK2, has a role in leukaemogenesis.
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Kan, Su-Yin. "The proline-rich repeat and thioester domains of streptococcal fibronectin-binding proteins." Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/6134.
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Streptococcus pyogenes is an important human pathogen. One of the most prominent virulence factors produced by S. pyogenes is SfbI, a surface adhesin composed of three domains: thioester domain (TED), proline-rich repeat domain (PRR) and fibronectin-binding repeat domain (FnBD). The structures and functions of TED and PRR and their contributions to the pathogenesis of streptococcal diseases are unknown. The interaction between PRR and its putative target, the intracellular actin cytoskeleton regulator Arp2/3, was investigated by both in vitro and in vivo studies. PRR was shown to inhibit Arp2/3-dependent actin polymerisation. The expression of PRR in HeLa cells caused disruption to the cytoskeleton of the cells. All data point towards a role of PRR in inhibiting the Arp2/3 complex but more evidence is needed to support this. The N-terminal domain of SfbI (TED) and four homologous domains from S. pyogenes, group G streptococci and Streptococcus pneumoniae were characterised by mass spectrometry, NMR spectroscopy and biochemical assays. All were shown to possess intramolecular thioester bonds, spontaneously formed between sides chains of Cys and Gln residues. Fibrinogen (Fg) was identified as the first binding target of bacterial TEDs with direct evidence that the thioester bond was involved in the interaction with Fg. A pull-down experiment using human plasma showed Fg is a specific binding partner of SfbI-TED. The binding sites were narrowed down to the thioester-forming Gln of SfbI-TED and Lys residues in the Fg-Aα chain, and binding potentially occurred via covalent isopeptide linkage. The data presented here suggest two new roles for SfbI, previously unknown in bacterial pathogenesis. The PRR may be the first bacterial inhibitor of the actin cytoskeleton acting by inhibiting the Arp2/3 complex. Thioester domains appear to be a shared common feature of surface proteins of many Gram-positive pathogens. They may form covalent crosslinks between bacteria and host tissue.
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Shiban, Ehab [Verfasser]. "Characterization of the Proline-rich Synapse-associated Protein 1 Knockout Mouse / Ehab Shiban." Ulm : Universität Ulm. Medizinische Fakultät, 2013. http://d-nb.info/1035802562/34.
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Greenwood, Michael T. "Isolation and characterization of Dictyostelium discoideum genes encoding a common proline-rich region." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39517.
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CABP1 is a cAMP-binding protein found in Dictyostelium discoideum. Anti-CABP1 monoclonal antibody cross-reacts with several polypeptides. The gene encoding CABP1, capA, cross-hybridizes to seven genomic fragments on a genomic DNA Southern blot. Clones representing four different capA-related sequences were obtained by screening both cDNA and genomic libraries with the anti-CABP1 monoclonal antibody and with a capA cDNA. Sequence analysis revealed that the structural similarities between CABP1 and two CABP1-related polypeptides are restricted to a region highly enriched for proline, glycine, glutamine, and tyrosine residues. One of these capA-related sequences encodes the p24 actin-binding protein and the other encodes a homologue of human annexin VII. The levels of the p24 transcript were found to decrease in response to the addition of extracellular cAMP and to increase in the presence of the protein synthesis inhibitor cycloheximide. As a preliminary step towards understanding the function of annexin VII, specific antibodies were generated. Immunoblot analysis revealed the existence of two polypeptides with sizes expected for the two products of the alternatively spliced annexin VII encoding gene.
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Ferraz, Junior Jose Candido de Souza. "Prime-boost vaccination strategies using the Mycobacterium tuberculosis APA (alanine-proline rich antigen)." Thesis, University College London (University of London), 2004. http://discovery.ucl.ac.uk/1446909/.
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Alaoui-Ismaili, Moulay Hicham. "Roles of three proline-rich proteins at late stages of entomopoxvirus and baculovirus morphogenesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/NQ55295.pdf.
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Williams, Hannah. "Investigating the DNA binding properties of the proline rich homeodomain, an oligomeric transcription factor." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503856.
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Genes are the basic units of heredity, containing the DNA code for RNA molecules. Transcription is the process that converts the genetic code of genes into RNA molecules, which then perform a range of functions, including encoding proteins in the case of mRNA. Transcription is commonly regulated by proteins known as transcription factors, which can act in either a positive or negative manner.
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Geng, Wei, and 耿瑋. "The role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43224106.
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Geng, Wei. "The role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43224106.
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BIGI, ALESSANDRA. "Characterization of human sialidase NEU4: role of the proline-rich region in signal transduction." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/19197.
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Sialidases or neuraminidases are glycohydrolytic enzymes removing sialic acid residues from glycoproteins and glycolipids. They are widely distributed in nature, from microorganisms to vertebrates. In mammals, four sialidases with different subcellular localization and biochemical features have been described: a lysosomal sialidase (NEU1), a cytosolic sialidase (NEU2), and two membrane-associated sialidases (NEU3 and NEU4). NEU4, the most recently identified member of the human sialidase family, is found in two forms, long and short, differing in the presence of a 12 amino acids sequence at the N-terminus of the protein. Contradictory data are present in the literature about the subcellular distribution of these enzymes, and their membrane anchoring mechanism is still unclear.
First of all, in this work we investigated NEU4 long and NEU4 short membrane anchoring mechanism and their subcellular localization in COS-7 and HeLa cells. As observed by solubilization and cross-linking experiments, NEU4 long and short are extrinsic membrane proteins, probably associated to the lipid bilayer through protein-protein interactions. These results are in accordance with primary structure analysis that did not evidence any transmembrane sequence, nor the presence of any membrane binding motifs. Subcellular localization studies, performed through confocal immunofluorescence and subcellular fractionation, showed that human NEU4 is a membrane-bound enzyme; in particular, the long form of NEU4 localizes in mitochondria, while the short one is mainly associated with the endoplasmic reticulum. In addition, a finer submitochondrial fractionation and a protease treatment of intact mitochondria and mitoplasts provided evidence for NEU4 long location in the outer mitochondrial membrane.
Moreover, primary structure analysis showed the presence of a proline-rich region which is unique to NEU4, having no counterpart in any other human sialidase. Deletion mutants lacking this loop showed subcellular distributions similar to those of wild-type proteins in COS-7 cells, suggesting that this region does not directly affect the association of NEU4 to the membranes. We subsequently hypothesized an involvement in the interaction with signaling pathway components.
Studies in collaboration with the Department of Medical Chemistry, Biochemistry and Biotechnology (L.I.T.A.) of the University of Milano evaluated the effect of NEU4 long transfection in human neuroblastoma SK-N-BE cell line. Similarly, we produced stable SK-N-BE clones transfected with the mutated form of NEU4 long (N4LnoP). The mRNA level of both NEU4 and the other human sialidases were checked in both wild-type and mutated NEU4 clones by RT-PCR and real-time PCR. In addition, only the wild-type form of NEU4 long was able to alterate the sialoglycoprotein profile and significantly enhance the proliferative ability of SK-N-BE cells, suggesting that the Pro-rich region of NEU4 is involved in both these phenomena. Subsequently, in order to study the function of NEU4 in SK-N-BE cell line, we analyzed the effect of NEU4 expression also under retinoic acid induced differentiating conditions. Our results show that retinoic acid treatment increases the expression of NEU4, either wild-type or mutated, due to the presence of RA response elements (RARE) in the CMV promoter.
In addition, both morphological change analysis and neurite outgrowth quantification were consistent with acetylcholinesterase activity data, indicating a role for NEU4 long and its Pro-rich region in the early phases of retinoic acid induced neuronal differentiation process.
Since potential Akt and Erk1 kinase motifs were found in NEU4 proline-rich region, activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathways were studied in stable SK-N-BE clones. Our results demonstrate that the expression of NEU4, both wild-type and mutated, does not significantly affect retinoic acid induced activation of both Akt and Erk1/2 pathways in SK-N-BE cells, suggesting that NEU4 could be located in a downstream place in these signaling pathways. As confirmed by immunoprecipitation experiments, NEU4 long interacts with Akt kinase in SK-N-BE cells. Conversely, the lacking of the proline-rich region impaired interaction between NEU4 and Akt, indicating that the formation of NEU4-Akt complex occurs through the proline-rich region. On the contrary, no interactions between NEU4 and Erk1/2 kinase were observed, suggesting that NEU4 is not a substrate of this kinase. Finally, treatment with LY294002 PI3K inhibitor demonstrates that PI3K/Akt signaling pathway is required for neuronal differentiation induced by retinoic acid in this neuroblastoma cell line. On the whole, these data suggest that NEU4 long is a downstream component of Akt signaling pathway required for RA induced neuronal differentiation in SK-N-BE cells.
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Wu, Yih-Yiing. "Response of skin to noxious stimuli : studies using in situ hybridisation." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263124.
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実里, 岩田, and Minori Iwata. "Study of mechanisms for the axonal localization of the tau protein in neurons." Thesis, https://doors.doshisha.ac.jp/opac/opac_link/bibid/BB13142995/?lang=0, 2020. https://doors.doshisha.ac.jp/opac/opac_link/bibid/BB13142995/?lang=0.
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微小管結合タンパク質の1つであるタウは、神経細胞の軸索に特異的に局在している。タウの軸索局在化分子機序を解明するために、外因性タウを神経細胞の発達初期に一時的に発現させ、軸索特異的に局在させる方法を構築した。この方法を用い、proline rich region 2 (PRR2)がタウの軸索局在化に重要であること、PRR2のリン酸化が軸索への移動に関与することを示唆した。またこの系の確立は局在や細胞内動態などの検討を行うことを可能にした。Microtubule-associated protein tau localizes specifically to neuronal axons. In order to elucidate the molecular mechanism of the axon localization of tau, we constructed an expression system for axon specific localization of exogenous tau in immature neurons in culture. Using this system, it suggested that the proline rich region 2 (PRR2) and phosphorylation of PRR2, which contains important phosphorylation sites, is critical for the localization. In the future, this experimental system will contribute greatly to the study of tau in normal and in the pathology of Alzheimer's disease.
博士(理学)
Doctor of Philosophy in Science
同志社大学
Doshisha University
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Sun, Kin-wai, and 孫建維. "The significance of proline rich tyrosine kinase 2 (PYK2) in proliferation and invasiveness of hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40687375.
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Sun, Kin-wai. "The significance of proline rich tyrosine kinase 2 (PYK2) in proliferation and invasiveness of hepatocellular carcinoma." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40687375.
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Sawasdichai, Anyaporn. "The regulation of cell growth by the proline rich homeodomain protein and its importance in human diseases." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546220.
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Faure, Camille. "Régulation de la localisation et de l'activation de la tyrosine kinase Pyk2 (Proline-rich tyrosine kinase 2)." Paris 6, 2010. http://www.theses.fr/2009PA066736.
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Proline-rich tyrosine kinase 2 (Pyk2) est une tyrosine kinase non récépteur de 110-kDa deappartenant à la famille de focal adhesion kinase (FAK). Elle a été initialement caractérisée comme une enzyme cytoplasmique. Elle est, régulée par autophosphorylation (sur la tyrosine 402) en réponse à l’des augmentations de la concentration de Ca2+calcium libre cytosolique. L’autophosphorylation de Pyk2 est cruciale pour son activation puisqu’elle permet le recrutement des membres de la famille de Src, conduisant à l’ac’tivation de nombreuses voies de signalisation. Pyk2 existe sous deux isoformes produites par épissage alternatif, une isoforme enrichie dans le système nerveux contenant l’exon alternatif et une forme spécifique des cellules hématopoïétiques, dans laquelle cet exon est absent. Le laboratoire s’intéresse à l’isoforme de Pyk2 abondante dans les neurones qui joue un rôle important dans l’induction de la plasticité synaptique dans l’hippocampe. Au cours de ma thèseN, nous nous sommes intéressés à la régulation de l’activation et de la localisation de Pyk2 en réponse à la dépolarisation membranaire. Nous avons montré observé dans différents modèles, que des augmentations de Ca2+ induisent indépendamment l’activation et la relocalisation de Pyk2 dans le noyau en impliquant la calcineurine, probablement en amont de ces évènements. Nous avons la dépolarisation de la membrane induisait une accumulation nucléaire rapide et transitoire de Pyk2. Nous avons montré que cette relocalisation de Pyk2 dans le noyau était calcium-dépendante, mais indépendante de son autophosphorylation, de son activité kinase et du recrutement des kinases de la famille de Src. En revanche, nous avons démontré qu’en réponse à la dépolarisation, l’autophosphorylation et la localisation subcellulaire de Pyk2 sont régulées par une protéine sérine/thréonine phosphatase, la calcineurine. Afin de caractériser le mécanisme moléculaire permettant la régulation de la localisation de Pyk2, nous avons recherché les séquences responsables de son trafic nucléo/cytoplasmique. Nous avons utilisé une stratégie de délétion qui nous a permis d’isoléer une région de Pyk2, localisée entre les domaines kinase et FAT, nécessaire à lsa localisation cytoplasmique de Pyk2 contenantdans les cellules non stimulées. Nous avons démontré la présence d’ une séquence d’export nucléaire qui dans cette région, comprenant les résidus hydrophobes Leu-735, Phe-739, Val-741. Au cours de ces expériences, nous avons observé que cette région mimait en partie la régulation de la localisation de Pyk2 induite par la dépolarisation membranaire suggérant que l’accessibilité de ces séquences d’export et/ou d’import nucléaire est régulée au cours de la stimulation pour permettre la translocation nucléaire de Pyk2. De manière intéressante, la séquence d’export nucléaire que nous avons caractérisée appartient en partie à l’exon alternatif et n’est pas fonctionnelle dans l’isoforme spécifique des cellules hématopoïétiques révélant. Ces données suggèrent que cet épissage alternatif traduirait une spécificité tissulaire de lala régulation de sla localisation de Pyk2 mise en évidence par la dépolarisation membranaire. Nous avons également pu observéer une accumulation nucléaire de Pyk2 dans d’autres modèles, ainsi qu’ in vivo, suggérant l’existence des fonctions nucléaires de Pyk2 différentes de ses fonctions cytoplasmiques connues. L’ensemble de ces résultats permet de révéler que des augmentations de calcium induites par la dépolarisation membranaire induisent indépendamment l’activation de Pyk2 et sa re-localisation dans le noyau. Ces deux phénomènes impliquent la calcineurine, qui agit probablement en amont de ces évènements. D’autre part, la localisation cytoplasmique de l’isoforme neuronale de Pyk2 est assurée par une séquence d’export nucléaire située au niveau du site d’épissage alternatif, permettant d’appréhender les bases d’une spécificité neuronale Tyrosine kinase
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Yamasaki, Masahide. "Monocyte chemoattractant protein-1 causes differential proline-rich tyrosine kinase 2-mediated signaling in THP-1 cells." Kyoto University, 2001. http://hdl.handle.net/2433/150583.
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Mok, Ka Wai. "Proline-rich membrane anchor (PRiMA) of acetylcholinesterase (AChE) : characterization of its splicing variants and their expression profiles in different chicken tissues /." View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202009%20MOK.
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Charlton, Bernard. "Cloning and characterization of Drosophila Nedd4 as a putative binding partner of the proline-rich region of Inscuteable." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0004/MQ45499.pdf.
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Roberts, Daniel Stephen. "The role of the proline rich homeodomain in the regulation of proliferation, survival and migration of breast cells." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/4873/.
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This thesis demonstrates that the Proline Rich Homeodomain transcription factor (PRH/HHEX) plays an important role in regulating the proliferation and migratory behaviour of breast cells. In tumourigenic MCF-7 breast cells, shRNA knockdown of PRH results in a pro-invasive and pro-proliferative phenotype. Key genes regulated by PRH in MCF-7 cells include TP53, endoglin (ENG) and e-cadherin (CDH1), which regulate migration/invasion in breast cells. Significantly, exogenous PRH functions as an inhibitor of cell proliferation/survival and migration/invasion in all breast cell types examined. Furthermore, the effects of exogenous PRH on cell proliferation/survival are dependent on the DNA binding activity of PRH. This work provides an explanation for the finding that PRH expression is associated with increased overall survival in breast cancer patients. In contrast with this work, MCF-7 xenograft experiments reveal that expression of exogenous PRH in MCF-7 cells is oncogenic. Furthermore, shRNA knockdown experiments in MDA-MB-231 cells show that endogenous PRH increases proliferation of these cells. This thesis therefore demonstrates that the role of PRH can differ dramatically between breast cell types and between ex vivo and in vivo conditions.
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Donnelly, Sean Frederick Hilton. "Isolation and characterisation of a gene encoding verprolin, a novel proline-rich protein in the yeast Saccharomyces cerevisiae." Thesis, University of Leicester, 1993. http://hdl.handle.net/2381/34405.
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A gene (VRPl) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridisation to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src Homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature-sensitive. The vrpl mutants are impaired in both cell shape and size and display aberrant chitin and actin localisation. These phenotypic characteristics resemble those observed in yeast cytoskeletal mutants. Using the data generated in this study, together with the rapidly accumulating data on SH3 domains, the cytoskeleton and signal- transduction, it is possible to speculate as to the in vivo role of verprolin.
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Fowler, Thomas James. "Characterization of four proline-rich protein genes in Arabidopsis and identificaiton of a defective kernel mutant in maize /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487847761307916.
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Wang, Shu-Zhen. "Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm." Diss., Virginia Polytechnic Institute and State University, 1985. http://hdl.handle.net/10919/49959.
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Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)⁺ mRNA were found to direct in vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)⁺ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with ³²P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not.
Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single U gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons. There are eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln in the N-terminal one-half of the polypeptide. The codon specifying the third proline in the hexapeptide repeating unit is identical, CCG, in all eight repeats. It is likely that these highly conserved tandem repeats are of critical importance to the function of gamma-zein which is presently unknown. Alternatively, it is conceivable that selective pressures responsible for conserving these tandem repeats may be operating at the nucleic acid level.Ph. D.
incomplete_metadata
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Mack, Birgit H. "Identification of proline-rich motifs in human WIP that mediate a conserved interaction with the yeast Hof1p SH3 domain." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/391078.
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Wiskott-Aldrich Syndrome protein (WASP) is defective in Wiskott-Aldrich Syndrome, an inherited immunodeficiency disorder. WASP promotes assembly of branched actin filaments and functions in a complex with WASP-interacting protein (WIP). Both proteins are proline-rich and bind the Src-homology 3 (SH3) domains of several other cytoskeletal proteins to facilitate signaling to the actin cytoskeleton. The yeast Saccharomyces cerevisiae has an actin cytoskeleton similar to mammalian cells and the homologues of WASP and WIP in S. cerevisiae are Las17p and Vrp1p. In yeast, Vrp1p binds the SH3 domain of Hof1p, a key regulator of the yeast cytoskeleton, and this binding promotes cytokinesis while a lack of Vrp1p inhibits cytokinesis.
This study investigates the possibility that the interaction between Vrp1p and the Hof1p SH3 domain has been conserved in humans. This study proposes that human WIP, which is known to function in yeast cells lacking Vrp1p, binds the yeast Hof1p SH3 domain through proline-rich motifs and thereby counteracts an inhibitory effect of the unbound yeast Hof1p SH3 domain on cytokinesis.
It was found that the yeast Hof1p SH3 domain interacts with two proline-rich motifs (PRMs) in the C-terminus of human WIP. Yeast two-hybrid interaction tests and in vitro pull-down assays both confirmed the motif PRM1 (PATPQLPSRS) as one of the interaction sites. More yeast two-hybrid interaction tests revealed the second PRM (PRM2) mediating interaction to be PPPPPSTS, a PRM that has been previously reported to be required for the rescue of cytoskeletal defects in yeast cells lacking Vrp1p. However, this result could not be confirmed in in vitro pull-down assays. The other PRM essential for interaction between the C-terminus of human WIP and the Hof1p SH3 domain was found to be PRM3 (PPLPPIPR), which is located at the extreme C-terminus of human WIP.
Unexpectedly, yeast cells lacking Vrp1p but expressing mutated full-length human WIP lacking PRM1 and PRM2 or PRM1 and PRM3 did not exhibit any cytoskeletal defects at elevated temperatures (a stress condition). A yeast twohybrid interaction test with these mutated full-length human WIP constructs revealed that, despite the deletion of the PRMs in the C-terminus of human WIP mediating Hof1p SH3 domain interaction, the full-length WIP protein was still able to interact with the Hof1p SH3 domain.
This study provides evidence that the interaction between Vrp1p and the Hof1p SH3 domain in yeast has been conserved in humans. Two PRMs were found in the C-terminus of human WIP that mediate interaction with the yeast Hof1p SH3 domain. Further, the human homologue of Hof1p, PSTPIP1, interacted with the same C-terminal fragment of human WIP in the yeast two-hybrid interaction test demonstrating that a similar mechanism exists in humans. In the absence of WIP, the unbound SH3 domain of Hof1p (or PSTPIP1 in humans) might be the cause cytoskeletal defects possibly contributing to Wiskott-Aldrich Syndrome. The results of this study help in revealing the mechanisms of cytoskeletal rearrangement in the yeast model allowing us to unravel similar mechanisms in humans in the future.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Xie, Qunhui. "Transcriptional regulation and function of PRiMA (proline-rich membrane anchor), a membrane anchor of globular acetylcholinesterase, in muscle and neuron /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202006%20XIE.
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Savastano, Adriana [Verfasser]. "Investigation on the Physiological and Pathological Aspects of the Proline-Rich Region of the Microtubule-Associated Protein Tau / Adriana Savastano." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1223706311/34.
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Hosono, Yuji. "Splicing factor proline/glutamine-rich is a novel autoantigen of dermatomyositis and associated with anti-melanoma differentiation-associated gene 5 antibody." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225498.
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BOROUMAND, MOZHGAN. "Extensive proteomics characterization of basic proline-rich proteins in human saliva and investigation on their properties as substrates of epithelial transglutaminase 2." Doctoral thesis, Università degli Studi di Cagliari, 2019. http://hdl.handle.net/11584/260381.
Full textAbstract:
This PhD thesis describes the work carried out during three years upon the Department of “Scienze della Vita e dell’Ambiente” of the Cagliari University, work that was also partly performed upon the “Istituto di Biochimica e Biochimica Clinica” of the Faculty of Medicine of the Catholic University of Rome.
Topics of the thesis were centered on the structural and functional characterization of some human salivary proteins.
In particular, the topics investigated were the following:
a) Extensive identification of the components of the family of basic (and glycosylated basic) proline-rich proteins (bPRPs and gPRPs), the most complex and heterogeneous family of the human salivary proteins, by a proteomic top-down platform with the aim to achieve the most complete knowledge possible of the main parent proteins present in human saliva and of their post-translational modifications. This study allowed the characterization of 55 new components of the family bringing the total number of naturally occurring components to 110.
b) Exploration of the reactivity in vitro of some bPRPs (P-C, P-H, P-D, P-D P32⟶A, II-2, P-F and P-J) as substrate of the epithelial transglutaminase-2 (TG-2). The aim of this study was to establish whether these bPRPs can potentially contribute in vivo to the formation of the “oral mucosal protein pellicle”, a protein network covering the oral mucosal epithelia devoted to the protection of the oral cavity. This study allowed establish that almost all the bPRPs are substrates of TG-2 and therefore are potential components of the “oral mucosal protein pellicle” and that, despite the great sequence similarity, their reactivity is significantly different.
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Faurie, Benoit. "Les sucres et l'astringence : effet des polysaccharides présents dans le vin sur les interactions tanins-protéines." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0191/document.
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Les tanins jouent un rôle clé dans les qualités organoleptiques du vin rouge. Ils sont à l’origine de l'Astringence, une sensation de sècheresse perçue en bouche lors de la dégustation. Cette sensation est la conséquence d'une interaction spécifique entre des tanins et des protéines salivaire, principalement les Protéines Riches en Prolines (PRPs). Une première partie de ce travail a consisté à étudier l'influence de divers sucres sur le processus d'auto-association des tanins ainsi que sur les interactions tanins-protéines. Le comportement colloïdal d’un tanin (l’Epigallocatechine Gallate – EGCG), ainsi que son interaction avec un peptide modèle IB9-14 représentatif des PRPs, ont été étudiés en présence de différents sucres simples et polysaccharides. Les paramètres de l’interaction ont été déterminé pour l’ensemble des systèmes, mettant en évidence l’existence d’une interaction entre EGCG et sucres dont l’affinité semble dépendre du degré de polymérisation du sucre. Cette interaction ne perturbe pas, dans les conditions expérimentales testées, l’association entre les tanins et le peptide. Une deuxième partie de ce travail correspond à la synthèse complète de la protéine IB9 incluant la séquence peptidique d’IB9-14, et à l’étude de son interaction avec deux procyanidines : l’EGCG et le dimère B3. Cette étude a permis d’observer et de confirmer une influence de la longueur de la chaîne peptidique sur les interactions avec les taninsTannins play a key role in the organoleptic qualities of red wine. They are responsible for wine astringency, a dry, rought and puker sensation perceived in the mouth while tasting. This sensation is the consequence of a specific interaction between tannins and saliva proteins, mainly Proline Rich Protein (PRPs). The first part of this work was to study the influence of various sugars on the self-association of tannins process as well as tannins - proteins interactions. The colloidal behavior of a tannin (the epigallocatechin gallate - EGCG), as well as its interaction with a representative peptide IB9-14 of PRPs was studied in the presence of various simple sugars and polysaccharides. The parameters of the interaction were determined for all systems, highlighting the existence of an interaction between EGCG and sugars whose affinity seems to depend on the sugar polymerization degree. This interaction does not interfere, under the experimental conditions tested, on the association between tannins and peptide. The second part of this work was to realize the full synthesis of the protein IB9 including the peptidic sequence of IB9-14, and to study its interaction with two procyanidins: EGCG and dimer B3. The results show and confirm the influence of the length of the peptide chain interactions with tannins
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ORRU', ROBERTO. "Sequenziamento e Analisi Molecolare di Varianti Alleliche del Gene PRB1." Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266626.
Full textAbstract:
The aim of this work was the molecular characterization of gene PRB1 in three subjects with different salivary proteome profile. The PRB1 gene encodes a member of the heterogeneous family of Basic Proline rich Proteins (PRB), produced by the parotid gland and secreted in human saliva. The protein polymorphism resulting from post-translational modifications of the pre-protein PRB1, are further complicated by the gene allelic variants. Those variants involves the third exon of the gene, and are related to the presence or absence of a tandem repeat sequence 183 bp long. That tandem repeat may originate three different allelic forms, defined small, medium or large depending on the number of repeats inside the third exon.
The information reported in the literature about the class of PRP genes, and in particular PRB1 are, from the molecular point of view, fragmentary and difficult to reconstruct. In fact, the literature on this topic covers a time span ranging from the seventies (Azen & Oppenheim, 1973), when the associated peptides have been characterized for the first time, up to the end of the nineties (Stubbs et al., 1998). Since the eighties, there have been various attempts to characterize the PRB1 allelic variants, based on the resulting peptides. This approach has greatly complicated the classification of the gene, but also of his allelic variants and the deriving proteins (Maeda et al., 1985 ; Lyons et al., 1988a, b; Azen et al., 1993). Today we know that from PRB1, considering its allelic and splicing variants, are expressed six different proteins (Marconi et al., 2015). However, amongst the three allelic variants of this gene, the only of which we have the complete nucleotide sequence is the medium. Conversely, for the small and large we have so far only partial sequences, referring only to the third exon. Among the objectives of this study, there was the reconstruction of the complete sequences of the small and large variants, of which there have been previously characterized two putative bearers from the proteomic point of view. At the same time, we also sequenced the PRB1 gene from a putative homozygotic bearer of the medium variant.
While for the subject bearer of the small variant, we were able to identify and sequence the entire gene, the same was not possible for the subject bearer of the large variant. In fact, from our analysis this last subject has shown to be homozygous carrier of the medium variant. This result is incompatible with the presence in his saliva of Ps2 protein, which is characteristic of the large variant. In the absence of further mass spectrometric analysis, we cannot explain the reason for this discrepancy between genomic and proteomic data. The analysis of the complete sequences did not allow us to understand why it has not been identified to the mass spectrometer the peptide relative to the splice variant classified by Maeda with the acronym cP5 (Maeda et al., 1985), while in all the subject has been identified the peptide and the relative cP4 splice variant. Pk-o protein has so far been reported to be expressed from the large variant only, although the first classification of splice variants had been made from an individual carrying the medium variant. Since in none of the three sequenced subjects the splice acceptor site in 3' of the cP5 variant results to be altered, it is possible that the relative peptide has not been identified to the mass spectrometer. Otherwise, specific splicing factors, that recognize Splicing Regulatory Elements (SRES; Hernandez-Imaz et al., 2015) in the neighborhood of the two molecular acceptor sites, prevented the transcription of the cP5 in favor of cP4. In order to shed light on this aspect, there are some possible strategies in perspective. Recently, in fact, studies have been published in which extensive in vivo screening for the detection of SRES is coupled with RNA affinity purification and mass spectrometry, which allow also the identification of the splicing factors which bound to the SRES (Wang Y & Z Wang, 2014; Wang et al., 2013). This strategy could be effectively integrated with the analysis of minigenes (Hernandez-Imaz et al., 2015), specifically designed on the model of the third exon of PRB1, where alternative splicing takes place and where are probably placed the SRES.
Molecular analysis has also enabled us to identify a set of Single Nucleotide Polymorphims (SNPs), most of which have never been described so far, localized specially inside introns and some even within exons. In particular, for two of these, we have detected a non-synonymous substitution at the amino acid level, the role of which can only be clarified by means of appropriate functional studies. Although translationally silent, even synonymous SNPs may have an important function, especially in alternative splicing. In fact, these substitutions can determine the genesis or destruction of SRES, or strengthen cryptic donor /acceptor sites. Furthermore, they can result in the alteration of the secondary structure of the mRNA, important for the exons definition and the pause sites of RNA II. This could, in turn, alter the processivity of the polymerase, with possible consequences on the choice of splicing sites.
The reconstruction of the complete sequences and the comparison with the data available in the literature (Lyons et al., 1988) allowed us to produce an updated model, which allows to explain the generation of allelic variants small and large from the medium variant. Hopefully, the reconstruction also of the large allelic variation, and possibly the very large, will lay the basis for the validation of our model and possibly also its extension to other allelic variants of the PRB gene class.
The genotypic characterization of allelic variants of PRB1 may be, in the future, an important predictive tool about the subjective susceptibility towards a series of oral pathogens. In fact, polymorphic variations in the PRB1 peptides can build the basis for the prediction of individual differences at the level of the oral microflora, with repercussions on the susceptibility to infections and diseases in this body part (Newman et al., 1993 and 1996; O'Sullivan et al., 2000).
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Pelillo, Chiara. "Therapeutic potential of BAC7(I-35), a Proline-rich Antimicrobial Peptide: in vitro and in vivo studies and Pegylation strategy to improve its bioavailability." Doctoral thesis, Università degli studi di Trieste, 2011. http://hdl.handle.net/10077/5978.
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2009/2010The antimicrobial peptides (AMPs) are an important component of the innate defense against invading microorganisms, are widespread in nature and may have multiple and diversified mechanisms of bactericidal action. In addition to their direct antimicrobial activity the are also involved in other biological processes. The aim of this project was to investigate the in vivo activity of Bac7(1-35), a bovine proline-rich antimicrobial peptide, having in mind its possible use as a lead compound for the development of novel anti-infective agents. Before moving to animal models of infection, the in vitro stability of the peptide in the presence of murine and human serum or plasma as well as its biodistribution in mouse were investigated. Antibacterial activity assays against Salmonella enterica showed that the presence of murine blood components largely inhibits the antibacterial activity of the peptide. On the contrary, in human serum and plasma Bac7(1-35) maintains its efficacy. This is due to the more rapid degradation by proteases of murine blood. The in vivo biodistribution of Bac7(1-35) was investigated by using a time-domain optical imaging apparatus and a fluorescently-labeled Bac7(1-35) derivative. The compound reaches the kidney and the bladder respectively 1 and 3 hours after i.p. injection. The in vivo and ex vivo analyses performed after 24 h confirm that the compound has been totally excreted. A mouse model of S. typhimurium infection was set up and used to test the therapeutic efficacy of Bac7(1-35). Treatment of infected mice with the peptide injected i.p. immediately after a lethal, intraperithoneal bacterial challenge, increased the mean survival time and reduced significantly the number of viable bacterial cells in liver and spleen of treated mice at 3 days post-inoculum. In 1/3 of the organ homogenates, the bacterial presence was undetectable and this result matches the percentage of cured animals (35%). In an attempt to improve its pharmacokinetic profile, the peptide was conjugated with polyethylene glycol (PEG), a non-toxic, non-immunogenic and FDA-approved polymer. Different strategies of pegylation have been considered to find the best method in terms of chemical yield and of maintenance of biological activity. Pegylation via a thioether ligation resulted the best strategy to obtain a slow active peptide release in human blood components with a reduced renal clearance and an increased bioavailability of Bac7(1-35), as biodistribution analyses demonstrated. Several important pathogens, such as S. enterica, cause disease by surviving and replicating within host cells. Since many AMPs have also immunomodulatory activities, we investigated the effect of Bac7(1-35) on the interaction between macrophages and Salmonella. We carried out phagocytosis assays with macrophages and the results suggest that Bac7(1-35) plays a positive modulatory effect on this function. Phagocytosis assays were also performed to determine if Bac7(1-35) could inhibit survival and replication of intracellular Salmonella. The results show that the peptide inhibits the replication of intracellular Salmonella, suggesting that it can exert its antibacterial activity within eukaryotic cells. Further studies are required to fully understand the details of the Bac7(1-35) biological activities. The results obtained provide encouraging evidence for future investigations on Bac7(1-35) and on the pegylated form Bac7(1-35)CAM-PEG20k also in other models of infection and with different intracellular pathogens.
XXIII Ciclo
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Liu, Xiao. "Bioinformatic Identification and Analysis of Hydroxyproline-rich Glycoproteins in Plants." Ohio University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou150037001088989.
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Canon, Francis. "Contribution de la spectrométrie de masse à l'étude des interactions entre les protéines salivaires riche en proline et les tanins." Thesis, Montpellier, SupAgro, 2010. http://www.theses.fr/2010NSAM0015/document.
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L'astringence résulte de l'interaction des tanins, polyphénols abondants dans les végétaux, avec les protéines salivaires et plus particuliÈrement les protéines salivaires riches en proline (PRP), appartenant à la famille des protéines peu structurées. Les tanins participent aux mécanismes de défense des végétaux et présentent des effets antinutritionnels dus à leur capacitè à inhiber les enzymes digestives. La synthÈse de PRP salivaires constitue un mècanisme d'adaptation à la consommation d'aliments riches en tanins. Ce travail vise à caractèriser les complexes ètablis en solution entre les PRP et les tanins, par une approche basèe sur la spectromètrie de masse (MS). Pour cela, les protèines salivaires humaines IB5, PRP basique, et II-1, PRP glycosylèe, ont ètè produites par voie hètèrologue. AprÈs purification, les deux protèines ont ètè caractèrisèes par MS avec une source d'electronèbullisation (ESI-MS) et avec une source MALDI (Matrix-Assisted Laser Desorption/Ionisation). L'ètude des interactions par ESI-MS a confirmè la prèsence en solution de complexes non-covalents IB5tanin et permis de prèciser leurs stchiomètries. Des expèriences de compètition entre diffèrents tanins et de dissociation des complexes IB5tanin ont mis en èvidence l'influence des principales caractèristiques structurales des tanins sur cette interaction. L'ètude structurale des èdifices IB5tanin, par diffèrentes techniques de MS/MS (Collision Induced Dissociation, Electron Capture Dissociation et photodissociation) et par mobilitè ionique couplèe à la MS, a mis en èvidence la prèsence de plusieurs sites d'interaction sur IB5 ainsi que des changements conformationnels liès à l'interactionAstringency is an important organoleptic property of plant-based food. It is attributed to interactions of tannins, which are polyphenolic compounds, with salivary proteins and especially proline rich proteins (PRPs), which belong to the group of intrinsically unstructured protein (IUP). Tannins play an important part in plant defence mechanisms. Indeed, they have an antinutritional effect as they inhibit digestive enzymes. Production of salivary PRP is thus an adaptation process to tannin-rich diets. The purpose of this work is to provide a closer look at PRPtannin supramolecular edifices in solution, using a mass spectrometry (MS) approach. The human salivary proteins IB5, a basic PRP, and II-1, a glycosylated PRP, have been produced by heterologous expression. After purification, both proteins have been characterized by MS using electrospray (ESI) and Matrix-Assisted Laser Desorption/Ionisation (MALDI) sources. The study of the interaction between IB5 and model tannins by ESI-MS confirmed the presence of IB5tannin non covalent complexes in solution and provided new information on their stoichiometries. Competitive interaction experiments between IB5 and two tannins, along with IB5tannin complexes dissociation studies revealed the impact of the main tannin chemical features on this interaction. Structural studies performed on IB5tanin edifices by Collision induced dissociation (CID), Electron Capture Dissociation (ECD) and photodissociation MS/MS experiments and by ion mobility coupled with MS showed the presence of several interaction sites on IB5 and conformational changes arising from the interaction
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Weiler, Mary Claire. "Identification of the SH3 binding domains and the in vitro map kinase phosphorylation sites of CR16, a novel proline-rich protein expressed in Rat Brain neurons /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487951214938305.
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Fleau, Charlotte. "Maladie d'Alzheimer et polyphénols : effet des tanins sur la phosphorylation de la protéine Tau." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0776/document.
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L’hyperphosphorylation de la protéine Tau, à l’origine de la formation de dégénérescences neurofibrillaires et de la mort cellulaire observée, chez les patients atteints de la maladie d’Alzheimer, est causée par un déséquilibre entre l’activité des kinases et des phosphatases. Cette modification post traductionnelle touche de nombreux sites dans la Région Riche en Proline (RRP) de Tau. Or, des études RMN, réalisées au sein du laboratoire, ont montré que les polyphénols sont capables d’interagir avec des fragments de la protéine issues de la RRP avec des affinités de l’ordre de grandeur de celles décrites pour les kinases. Dans ce contexte, il a été envisagé de synthétiser une bibliothèque de polyphénols et de mettre au point une réaction de phosphorylation sur des peptides modèles afin d’étudier, par spectrométrie de masse, la capacité de ces composés à protéger Tau contre l’attaque des kinasesHyperphosphorylation of Tau protein, which leads to their abnormal aggregation into neurofibrillary tangles and to neuronal loss observed in patients with Alzheimer disease, is regulated by a disequilibrium between kinases and phosphatases activities. This post traductional modification affects several residues, in particular in the Proline Rich Region of Tau (PRR). But, NMR studies, realized in our laboratory, have shown that polyphenols are able to interact with peptide Tau models issued from the PRR and the affinity are in the same range that those described for the kinases. In this context, we have envisaged to synthetize several polyphenols and to develop a phosphorylation reaction of peptide models in order to study, by mass spectrometry, the ability of these compounds to protect Tau against kinase attack
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Palm-Forster, Mieder Anthony Thomas [Verfasser], Dierk [Akademischer Betreuer] Scheel, Sacha [Akademischer Betreuer] Baginsky, and Wolfgang [Akademischer Betreuer] Dröge-Laser. "Identification and functional characterisation of three novel Proline Rich Proteins that are Mitogen Activated Protein Kinase substrates / Mieder Anthony Thomas Palm-Forster. Betreuer: Dierk Scheel ; Sacha Baginsky ; Wolfgang Dröge-Laser." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2013. http://d-nb.info/1031915028/34.
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Gehrke, Janin. "Untersuchungen zu tanninbindenden Speichelproteinen des Rehs und anderer Wiederkäuer." Phd thesis, Universität Potsdam, 2002. http://opus.kobv.de/ubp/volltexte/2005/42/.
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Am Beispiel der Wiederkäuer wurde unter Zuhilfenahme von biochemischen und molekularbiologischen Methoden die Adaptation von Pflanzenfressern (Herbivoren) an pflanzliche Sekundärmetabolite wie z.B. Tannine untersucht. Tannine können in nicht an ihren Verzehr adaptierten Spezies durch ihr Proteinbindungsvermögen die Nahrungsverwertung und damit Wachstum und Gesundheit des Pflanzenfressers beeinträchtigen (antinutritive Wirkung). Einige Wiederkäuerarten wie z.B. das Reh (Capreolus capreolus) haben in ihrem Nahrungsspektrum viele stark tanninhaltige Pflanzen, leiden aber nicht unter den erwähnten postdigestiven Konsequenzen. Eine Möglichkeit, die antinutritive Wirkung von Tanninen zu neutralisieren, besteht in der Produktion tanninbindender Speichelproteine.
Der Speichel verschiedener Wiederkäuerarten wurde auf das Vorhandensein tanninbindender Proteine untersucht. Diese Arten wurden so ausgewählt, dass alle drei Ernährungstypen (Konzentratselektierer, Intermediärtyp, Gras- und Rauhfutterfresser) in den Vergleich eingeschlossen werden konnten. Als Referenzspezies wurde der Konzentratselektierer Reh herangezogen.
Die Speichelproteine des Rehs und die der Intermediärtypen (Rentier, Rangifer tarandus; Damhirsch, Cervus dama; Moschusochse, Ovibos moschatus) banden ungefähr doppelt so effektiv an hydrolysierbare Tannine (Tanninsäure), wie die der untersuchten Gras- und Rauhfutterfresser (Rind, Bos taurus; und Mufflon, Ovis orientalis musimon). Diese Abstufung zeigte sich auch bei der Untersuchung der Bindung an kondensierte Tannine (Quebracho). Eine Ausnahme stellte Mufflonspeichel dar, dieser band ebenso gut an Quebracho wie die Speichelproteine der anderen Ernährungstypen.
Über eine Aminosäuretotalanalyse konnte festgestellt werden, dass der Speichel einiger untersuchter Wiederkäuerarten prolinreiche Proteine (PRPs) enthielt. Unter Ausnutzung ihrer Trichloressigsäure (TCA)-Löslichkeit wurden diese angereichert und genauer untersucht. Die Analyse der TCA-löslichen Speichelproteine der Konzentratselektierer (Reh, Elch) ergab einen relativen Prolingehalt von über 35 %, während beim Moschusochsen noch 29 % gemessen wurden. In Damhirsch- und Rinderspeichel wurden keine prolinreichen Proteine gefunden.
Für die TCA-löslichen Speichelproteine des Rehs konnte eine hohe Tanninbindungskapazität nachgewiesen werden. Diese banden 24 - 30 x effektiver an Tannine als die TCA-löslichen Speichelproteine des Rindes. Die Tanninbindungskapazitäten der TCA-löslichen Speichelproteine von Moschusochse und Damhirsch waren ebenfalls höher als die des Rindes, aber niedriger als die des Rehs.
Die Kohlenhydrat-Analyse der TCA-löslichen Speichelproteine des Rehs erbrachte, dass es sich bei ihnen um Glykoproteine handelt. Mittels Gelfiltration und zweidimensionaler Polyacrylamidgelektrophorese konnten fünf Proteingruppen mit Molekulargewichten zwischen 15 und 50 kd sowie isoelektrischen Punkten zwischen 4,0 und 8,2 detektiert werden.
Von 15 dieser Proteine konnten die N-terminalen Aminosäuresequenzen ermittelt werden. Ausgehend von diesen Informationen wurden Reh-PRP spezifische mRNAs isoliert und partiell sequenziert. Die meisten dieser Fragmente hatten eine gemeinsame 18 Aminosäuren lange C-terminale Sequenz PPPEEQPEE/QSPDEE/DSPSE.
Die Suche nach Übereinstimmungen der analysierten Sequenzen mit anderen Säugetier-PRPs in der Genbank ergab keine sinnvollen Ähnlichkeiten. Die Ergebnisse können zu Informationen über tanninbindende Proteine anderer Wiederkäuer führen. Die Sequenzinformationen stellen einen Ausgangspunkt bei der Analyse der evolutiven Zusammenhänge der Cerviden dar.
Investigation of tannin binding salivary proteins of roe deer and other ruminants:
In this work the adaptation of herbivores to plant secondary metabolites was investigated with help of biochemical and molecular biological methods. In unadapted species plant secondary metabolites as tannins can reduce food digestibility and thus diminish growth rate and health status (antinutritive action). Tannins act through its astringency, that means the high capacity to bind proteins, other macromolecules and metal ions. Some ruminant species feed on tannin containing plant but do not suffer from the mentioned nutritive consequences. The production of tannin binding proteins is one possible adaptation mechanism to neutralize the effects of the tannins.
Saliva of six different ruminant species was investigated for the presence of tannin binding proteins. All three feeding types (concentrate selector, intermediate type and grass and roughage eater) were included in the comparison.
Salivary proteins from roe deer (Capreolus capreolus, concentrate selector) and from the intermediate feeding types (rein deer, Rangifer tarandus; fallow deer, Cervus dama; musk ox, Ovibos moschatus) bound twice as effective to hydrolysable tannins (tannic acid) as those from the investigated grass and roughage eaters (cattle, Bos taurus; moufflon, Ovis orientalis). This differentiation could also be observed investigating the binding capacities to condensed tannins (quebracho) except for moufflon. Moufflon salivary proteins bound with the same intensity to quebracho as the salivary proteins from the other feeding types.
Proline rich proteins (PRPs) could be accumulated from roe deer, moose and musk ox saliva by use of its solubility properties in 5 % trichloro acetic acid (TCA). Roe deer and moose TCA soluble salivary proteins contained more than 35 %, musk ox proteins 29 % proline. In fallow deer and cattle saliva PRPs could not be detected.
A tannin binding assay demonstrated for the TCA soluble salivary proteins from roe deer, musk ox and fallow deer but not from cattle, that they are able to bind tannins. Roe deer salivary proteins bound 24 to 30 more effective to tannins as cattle proteins. Tannin binding capacity of the proteins from musk ox and fallow deer saliva was higher as those from cattle but lower as those from roe deer.
For further analysis of ruminant tannin binding proteins we chose roe deer as reference species. Carbohydrate analysis of TCA soluble proteins from roe deer saliva showed that they were glycoproteins. With help of gel filtration and two dimensional polyacrylamid gel electrophoresis five proteins groups with molecular weights from 15 to 50 kd and isoelectric points from 4.0 to 8.2 could be detected.
N-terminal amino acid sequences of 15 of the roe deer salivary TCA soluble proteins were determined by Edmann degradation. This information led to partially sequenced roe deer PRP specific cDNA. An 18 amino acid long C-terminal sequence was common in most of the clones. The obtained roe deer PRP sequences did not match with known mammalian PRP sequences from data banks.
The finding in this work can lead to information about salivary tannin binding proteins in other ruminants. The sequence information represent a starting-point for the investigation of cervid evolution.
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Paulhe, Frédérique. "Régulation différentielle du métabolisme des phosphoinositides par les intégrines αvβ3 et αvβ5 lors de la migration des cellules musculaires lisses." Paris 7, 2002. http://www.theses.fr/2002PA077140.
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Mardirossian, Mario. "Internal targets and killing mechanism of the cathelicidin Bac7 in Gram-negative bacteria." Doctoral thesis, Università degli studi di Trieste, 2013. http://hdl.handle.net/10077/8640.
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2011/2012Bac7(1-35) is the smallest fragment of the proline-rich cathelicidin Bac7 that shows the same antibacterial activity as the whole natural peptide of 60 residues. In this work, we remarked that the unique gene whose deletion can confer resistance to E. coli against Bac7(1-35) is sbmA, coding for an inner membrane protein involved in the penetration of this peptide into bacterial cells. Moreover, we provided evidence that SbmA is also involved in the transmembrane transport of a fragment of another proline-rich antimicrobial peptide, arasin1(1-23), isolated from the spider crab. These findings suggest a general role of this membrane protein in the uptake of proline-rich antimicrobial peptides (PR-AMPs) into Gram-negative bacteria. We then measured the intrabacterial concentration reached by Bac7(1-35) in E. coli, and observed that this increases from micromolar in the medium to millimolar within the bacterial cell, suggesting that it may bind to cytosolic structures. For this reason, we looked for possible interactions between Bac7(1-35) and macromolecules involved in viable processes of bacteria. These studies showed that Bac7(1-35) completely inhibits in vitro the transcription/translation process starting from a concentration of 50 μM. Then we demonstrated that inhibition is: i) specific for Bac7(1-35), since it is not exerted by other cathelicidin-derived AMPs not belonging to the Prorich group, and ii) not stereo-specific, since it is exerted at the same level by the all-D isomer of Bac7(1-35). We also demonstrated the ability of Bac7(1-35) to bind DNA in vitro, but we excluded that this binding may represent the primary mechanism of bactericidal action. We also showed that the peptide does not significantly affect in vitro the transcription process, deducing that the inhibition of the transcription/translation targets primarily the translation process. We verified these data in vivo on E. coli cells measuring the incorporation of radioactive precursors of bacterial macromolecules. We observed that the exposure of bacteria to Bac7(1-35) inhibited the incorporation of radioactive leucine, but not of radioactive thymidine and uridine, indicating a specific block at the protein synthesis level and not of DNA and RNA synthesis. In the near future, a clearer definition of the intrabacterial target(s) of Bac7(1-35) would hopefully lead to the experimentation of this molecule or of its derivatives as a new generation antibiotic drug.
Bac7(1-35) è il più breve frammento del peptide Bac7, una catelicidina ricca in prolina di 60 residui, dotato della stessa attività battericida del peptide intero. In questo lavoro di tesi, abbiamo rimarcato che sbmA è l’unico gene la cui delezione conferisce ad E. coli una resistenza a Bac7(1- 35). Tale gene codifica per una proteina della membrana interna coinvolta nell’ingresso del peptide nel citoplasma della cellula batterica. Inoltre, abbiamo dimostrato che SbmA è coinvolta anche nel trasporto transmembrana di un frammento di un altro peptide antimicrobico, l’arasina1(1-23). Tali risultati suggeriscono che questa proteina giochi un ruolo generale nell’internalizzazione di peptidi antimicrobici ricchi in prolina nei batteri Gram-negativi. Abbiamo quindi misurato la concentrazione intrabatterica raggiunta da Bac7(1-35) in E. coli e abbiamo osservato che questa aumenta da valori micromolari nel terreno di coltura a millimolari nel citosol batterico, suggerendo un suo legame a strutture interne della cellula. Per questo abbiamo cercato possibili interazioni tra il peptide e macromolecole coinvolte in processi vitali del batterio. Con questi studi abbiamo appurato che Bac7(1-35) in vitro inibisce completamente il processo di trascrizione/traduzione a partire da una concentrazione di 50 μM. Successivamente abbiamo dimostrato che questa inibizione è una peculiarità di Bac7(1-35), in quanto altri AMP derivati da catelicidine ma non ricchi in prolina non hanno dimostrato un’attività comparabile. Inoltre, questa inibizione non è stereo-specifica, in quanto anche l’isomero D di Bac7(1-35) blocca tale processo esattamente come il suo isomero L. Abbiamo inoltre dimostrato la capacità di Bac7(1-35) di legare in vitro il DNA, ma abbiamo escluso che tale legame rappresenti il meccanismo primario della sua attività battericida. Abbiamo anche dimostrato che il peptide non interferisce in vitro in maniera significativa con il processo di trascrizione, deducendo che l’effetto osservato sul processo di trascrizione/traduzione fosse da attribuirsi prevalentemente all’inibizione della traduzione. Abbiamo verificato tali dati in vivo su cellule di E. coli misurando l’incorporazione di precursori radioattivi delle macromolecole batteriche. Abbiamo osservato che l’esposizione di batteri a Bac7(1-35) bloccava l’incorporazione di leucina radioattiva ma non di timidina ed uridina, indicando un blocco specifico della sintesi proteica ma non di quelle di DNA e RNA. In futuro, una definizione ancora più chiara del target intrabatterico di Bac7(1-35) potrebbe portare alla sperimentazione di tale molecola o di suoi analoghi come farmaci antibiotici di nuova generazione.
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Wiza, Claudia [Verfasser], and Michael [Akademischer Betreuer] Feldbrügge. "Proline-rich Akt substrate of 40 kDa (PRAS40): A new modulator and target of insulin action. Characterization of the function of PRAS40 in Akt- and mammalian target of rapamycin complex 1 (mTORC1)-signaling pathway. / Claudia Wiza. Gutachter: Michael Feldbrügge." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1046670646/34.
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