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New Insights Into the Role of Equine Infectious Anemia Virus S2 Protein in Disease ExpressionCovaleda Salas, Lina M. 2010 May 1900 (has links)
Equine infectious anemia virus (EIAV) is an important animal model to study the
contribution of macrophages in viral persistence during lentiviral infections. EIAV is
unique amongst the lentiviruses in that it causes a rapid, rather than the very slow
disease progression, characteristic of other lentiviral infections. The accessory gene, S2,
unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene
is required to achieve high-titer viremia and the development of disease in infected
horses. Despite its essential role, the mechanisms by which S2 influences EIAV
pathogenesis remain elusive. The goal of this research was to gain insight into the role of
S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and
its S2 protein in the regulation of the cytokine and chemokine responses in macrophages,
(ii) Assessed the influence of EIAV infection and the effect of S2 on global gene
expression in macrophages and (iii) Identified host cellular proteins that interact with S2
as a starting point for the identification of host factors implicated in S2 function.
The results from this study provide evidence for a role of S2 in enhancing a proinflammatory
cytokine and chemokine response in infected macrophages. Specifically,
S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly
discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation
may contribute to disease development by optimizing the host cell environment to
promote viral dissemination and replication. Microarray analyses revealed an interesting
set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were
involved in the immune response, transcription, translation, cell cycle and cell survival.
Finally, we used the yeast two-hybrid system to identify S2 host cellular
interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S
ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is
needed to demonstrate the physiological relevance of these interactions in vivo.
In summary, the results from this study contribute towards our understanding of
the role S2 in disease expression and allow the formulation of new hypotheses as to the
potential mechanisms of action of S2 during EIAV infection.
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Identification of TEF cofactor(s) in skeletal muscles utilizing yeast two hybrid system /Zhang, Aijing. January 2004 (has links)
Thesis (M.S.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references (leaves 70-75). Also issued on the Internet.
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Identification of the Na,K-ATP interacting proteinsJing, Yonghua. January 2006 (has links)
Thesis (M.S.)--Medical University of Ohio, 2006. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Major advisor: Zijian Xie. Includes abstract. Document formatted into pages: iv, 50 p. Title from title page of PDF document. Bibliography: pages 40-49.
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Etablierung eines Zwei-Hybrid-Screening-Systems zur Suche und Charakterisierung von Ras-Raf-EffektorenFriese, Anke. January 2002 (has links)
Frankfurt (Main), Univ., Diss., 2002.
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Entwicklung modifizierter Zweihybrid-Systeme zur effizienten Untersuchung multipler Protein-Protein-Interaktionen unter Verwendung fluoreszenzaktivierter Zellsortierung am Beispiel des humanen CytomegalovirusFahr, Kristina. Unknown Date (has links) (PDF)
Universiẗat, Diss., 2002--Jena.
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Untersuchungen zur funktionellen Charakterisierung von regulatory-protein T-lymphocyte-1 (rpt-1, Trim 30)Späth, Kerstin. Unknown Date (has links)
Universiẗat, Diss., 2005--Düsseldorf. / Erscheinungsjahr an der Haupttitelstelle : 2004.
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Estudos estruturais e funcionais de septinas humanas: a ligação e hidrólise de GTP por SEPT3 e a busca de parceiros funcionais de SEPT1, SEPT5 e SEPT7 / Functional and structural studies of human septins: GTP hydrolyse and binding, and screening of functional partners to SEPT1, SEPT5 e SEPT7Joci Neuby Alves Macêdo 24 September 2010 (has links)
Septinas são proteínas que pertencem a super família das GTPases e que foram inicialmente identificadas em Saccharomyces cerevisae, mas logo em seguida, também em eucariotos superiores, exceto em plantas. Estas proteínas estão envolvidas em uma variedade de processos celulares tais como segregação de cromossomos, polaridade celular, dinâmica da membrana, tráfego de vesículas, exocitose, apoptose, entre outros. Mutações ou alterações no padrão de expressão de septinas são associadas com vários cânceres e doenças neurológicas. Objetivando contribuir com informações funcionais sobre tais proteínas, as septinas humanas 1, 5 e 7 foram usadas como iscas em ensaios de duplo híbrido em leveduras visando à identificação de seus parceiros protéicos. Após a varredura de bibliotecas de cDNA de leucócitos e cérebro fetal humano, os parceiros protéicos predominantemente encontrados foram outras septinas de grupos diferentes aos das iscas. As interações septina-septina envolveram o domínio de ligação a GTP. Ainda, outros parceiros, diferentes de septinas, foram também identificados nas bibliotecas e estes se mostraram funcionalmente relacionados à endocitose, à regulação da atividade de GTPases, ao tráfego intracelular, aos ciclos de sumoilação, à manutenção da placa metafásica e à maturação do centrossomo. Algumas destas funções são inéditas e foram pela primeira vez relacionadas às septinas. Este trabalho também esteve voltado à caracterização biofísica das septinas 3 e 5 (SEPT3 e SEPT5). Muitos protocolos diferentes foram desenvolvidos na tentativa de obter amostras homogêneas de SEPT5, mas não foram bem sucedidos. Por outro lado, SEPT3 recombinante, destituída do domínio amino-terminal (SEPT3GC) foi eficientemente produzida em E. coli. O estado monomérico de SEPT3GC em solução foi confirmado por cromatografia de exclusão molecular e espalhamento de raios-X a baixos ângulos (SAXS). SEPT3GC mostrou-se ativa e capaz de hidrolizar GTP in vitro. A afinidade de SEPT3GC por GTPγS e GDP foi avaliada por calorimetria de titulação isotérmica (ITC), sendo que o KD de SEPT3GC para GTPγS foi de 5,43 μM e a ligação para este nucleotídeo foi dependente de Mg2+. A ligação para GDP não foi detectável. Agregados de SEPT3GC induzidos por temperatura foram capazes de ligar a sonda fluorescente tioflavina-T, sugerindo uma natureza amilóide para tais estruturas. / Septins are proteins that belong to the superfamily of GTPases, which were initially identified in Saccharomyces cerevisiae, and then in higher eukaryotes, except plants. These proteins are involved in a variety of cellular processes such as chromosome segregation, cell polarity, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, among others. Mutations or changes in the expression of septins have been associated with various cancers and neurological diseases. Aiming to provide functional information about these proteins, the human septins 1, 5 and 7 were used as baits in yeast two-hybrid assay in order to identify their protein partners. After screening cDNA libraries from human leukocytes and fetal brain, the protein partners predominantly found were septins from others groups. The septin-septin interactions involved the GTP binding domain. Others non-septins interactors have also been identified in the libraries and were functionally related to endocytosis, the regulation of the GTPase activity, intracellular trafficking, sumoylation, maintenance of metaphase plate and centrosome maturation. Some of these functions are new and were related to the septins for the first time. This work also focused on the biophysical characterization of the septins 3 and 5 (SEPT3 and SEPT5). Many different protocols were developed aiming to obtain homogeneous samples of SEPT5, but were not successful. On the other hand, recombinant SEPT3, without the amino-terminal domain (SEPT3GC), was produced in a homogeneous form in E. coli. SEPT3GC is monomeric in solution as confirmed by size exclusion chromatography and small-angle X-ray scattering (SAXS). Also, SEPT3GC has shown to be active and able to hydrolyze GTP in vitro. The SEPT3GC affinity by GTPγS and GDP were evaluated by isothermal titration calorimetry (ITC). The KD for GTPγS was about 5,43 μM and it was observed to be Mg2+ dependent. The binding to GDP was not detectable. SEPT3GC aggregates induced by temperature were able to bind the thioflavin-T fluorescent probe, suggesting an amyloid nature for such structures.
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Translationsaktivatoren der mitochondrialen Cytochrom b-Synthese in Saccaromyces cerevisiae: Membranassoziation, Mutagenese und Protein-Wechselwirkungen von Cbs1pKrause-Buchholz, Udo 10 September 2000 (has links) (PDF)
Die vorliegende Arbeit beschäftigt sich mit Cbs1p und Cbs2p, zwei spezifische Translationsaktivatoren der COB-mRNA. Im Mittelpunkt standen sowohl die weitere molekularbiologische und biochemische Charakterisierung von Cbs1p als auch ein Screening von Interaktionskandidaten, die mit Cbs1p und/oder Cbs2p physikalisch wechselwirken könnten. Cbs1p liegt als peripheres Membranprotein fest mit der inneren Mitochondrienmembran matrixseitig assoziiert vor. Dabei spielen möglicherweise hydrophobe und/oder Protein-Protein-Wechselwirkungen mit integralen Membranproteinen eine essentielle Rolle bei der Membranverankerung von Cbs1p. Durch die Identifizierug von atmungsdefekten Cbs1p-Mutanten, deren Mutationen in Bereichen mit Homologie zu RNA-bindenden Proteinen liegt, verstärken sich die Hinweise zur Beteiligung von Cbs1p an der direkten physikalischen Wechselwirkung mit dem 5´-leader der COB-mRNA. Darüber hinaus konnte gezeigt werden,, dass die abspaltbare Präsequenz nicht notwendig für einen mitochondrialen Import ist. Die Ergebnisse präzisieren und erweitern das vorliegende Modell der Wirkungsweise der Translationsaktivatoren Cbs1p und Cbs2p (Michaelis, 1991). Aufgrund der Membranverankerung von Cbs1p wird auch die gebundene COB-mRNA in räumlicher Nähe zur Membran gebracht. Darüber hinaus definiert Cbs1p damit möglicherweise auch den Ort der Insertion des nascierenden Apocytochrom b in die Membran. Cbs2p vermittelt die Bindung zur kleinen Untereinheit der mitochondrialen Ribosomen und könnte seinerseits ebenfalls in Interaktionen mit Untereinheiten des bc1-Komplexes involviert sein.
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Ανίχνευση νέων πρωτεϊνικών αλληλεπιδράσεων της μυοειδικής πρωτεΐνης δεσμίνης στα καρδιακά μυϊκά κύτταρα και προτάσεις νέων μηχανισμών δράσης της. / Novel protein-proteinΚυριακόπουλος, Ανδρέας 28 June 2007 (has links)
Η μυο-ειδική πρωτεΐνη δεσμίνη, αποτελεί μέλος των πρωτεϊνών του κυτταροσκελετού των ενδιαμέσων ινιδίων και εκφράζεται στους λείους και τους γραμμωτούς μυς. Στους συσταλτούς μύες, το πλέγμα του κυτταροσκελετού της Δεσμίνης περιβάλλει τους Ζ-δίσκους διασυνδέοντάς τους, ενώ παράλληλα συνδέει μεταξύ τους τις συσταλτές περιοχές της μυικής ίνας με την σαρκοπλασματική μεμβράνη, με διάφορα οργανίδια και με τον πυρήνα. Για να προσδιορίσουμε τους ακριβείς μηχανισμούς δράσης της δεσμίνης χρησιμοποιήσαμε το σύστημα υβριδισμού των ζυμών – yeast two hybrid screen system – προκειμένου να ανιχνεύσουμε πρωτεΐνες που αλληλεπιδρούν με τη δεσμίνη. Χρησιμοποιήσαμε ως «δόλωμα» αλληλουχίες των άκρων του μορίου της δεσμίνης του αμινο-τελικού και το καρβόξυ-τελικού. Μελετώντας τις πρωτεΐνες που προέκυψαν, διαπιστώσαμε ότι το αμινο-τελικό άκρο της δεσμίνης αλληλεπιδρά με διάφορες μιτοχονδριακές πρωτεΐνες. Με το ίδιο σύστημα αποκαλύψαμε αλληλεπιδράσεις της δεσμίνης με λυοσωματικές πρωτεΐνες όπως η καθεψίνη D και η προσαποσίνη οι οποίες αλληλεπιδρούν με το αμινοτελικό άκρο της δεσμίνης. Η καθεψίνη D είναι μια λυοσωματική πρωτεάση, που οδηγείται και ωριμάζει πλήρως στα λυοσώματα ενώ η προσαποσίνη είναι ένα πρόδρομο λυοσωματικό μόριο με πρωτεόλυση του οποίου, εντός του λυοσώματος, προκύπτουν οι σαποσίνες Α έως D. Η καθεψίνη D αποτελεί δείκτη καταστάσεων αυτοφαγία και τελευταία φαίνεται ότι επεμβαίνει σε φαινόμενα απόπτωσης επάγωντάς την κατά περίπτωση. Η αλληλεπίδραση της δεσμίνης με την καθεψίνη D επιβεβαιώθηκε και με βιοχημικές τεχνικές (in vitro) όπως η συνεργιστική ανοσοκαθίζηση /ανοσοκατακρήμνιση (co-immuno-precipitation) και η τεχνική GST pull-down. Μετά και από αυτές τις in vitro αποδείξεις, φαίνεται πως μάλλον συμβαίνει ευθεία αλληλεπίδραση μεταξύ της δεσμίνης και της καθεψίνης D. Γι’ αυτό, και με βάση όσα είναι γνωστά για την καθεψίνη D, προτείνουμε μια νέα λειτουργία του κυτταροσκελετού της δεσμίνης πιθανόν στην μετακίνησης και τη δημιουργία των λυοσωμάτων αλλά και έναν νέο ρυθμιστικό ίσως ρόλο της, σε διαδικασίες αυτοφαγίας και απόπτωσης, μέσω της πρόσδεσής της με σημαντικά μόρια ρυθμιστές τέτοιων διαδικασιών.................... / Desmin is the muscle - specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In order to identify the exact mechanisms of desmin’s action, we performed a yeast two-hybrid screen for desmin-interacting proteins. For this purpose, we used as baits the two non helical terminal regions of the desmin molecule, the amino (head)- and the carboxy (tail)- terminal domain. We have found that the head domain of desmin potentially interacts with two new groups of proteins, mitochondria and lysosome related. Specifically, in the second category, we have revealed an association of the head domain of desmin with Cathepsin D (one of the lysosomal proteinases) and prosaposin (a single precursor which gives rise to Saposins A-D by proteolytic cleavage in lysosomes and is also referred to as sphigolipid activator proteins). In addition to its targeting to lysosomes, Cathepsin D is also involved in apoptosis and autophagy processes. This protein interaction result has been retested. The interaction between cathepsin D and desmin has also been further confirmed both with reverse yeast transformation as well as biochemical assays such as co-immunoprecipitation and GST pull down assay. The above described strong evidence of direct interaction between desmin and cathepsin D, has allowed us to propose a novel function of desmin IFs in lysosomal trafficking and/or as a new regulator of autophagy and apoptotic cell death.
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Modified yeast two-hybrid screening identifies SKAP-HOM as a novel substrate of PTP-PESTScott, Adam Matthew. January 1900 (has links)
Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/12/09). Includes bibliographical references.
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