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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Prostasome ELISA - a potential marker for prostate cancer diagnosis

Thermaenius, Elisabeth January 2012 (has links)
Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.

Autorégulation de l'expression de PABPN1, le gène muté dans la dystrophie musculaire oculopharyngée

Pal, Gheorghe January 2015 (has links)
Les PABPs sont des protéines liant les répétitions d’adénosines se trouvant à l’extrémité 3’ des transcrits d’acides ribonucléiques messagers (ARNm) et par conséquent pouvant interagir avec la majorité d’entres eux. Cette classe de protéine est retrouvée chez la majorité des eucaryotes, mais elle semble absente chez les procaryotes. Il existe deux classes de PABPs : cytoplasmique et nucléaire. Mis à part les PABPs cytoplasmiques, il existe une PABP avec une localisation majoritairement nucléaire appelée PABPN1. Chez l’humain, PABPN1 est impliquée dans plusieurs processus cellulaires : la polyadénylation nucléaire des ARNms, le clivage et la polyadénylation alternative, l’expression de certains longs ARNs non-codant (lncARN) et l’export des ARNms du noyau vers le cytoplasme. La protéine contient plusieurs domaines dont une région riche en alanine. Cette région est sujette à des expansions trinucléotidiques qui ont pour conséquence l’ajout d’alanines supplémentaires. Ces ajouts entrainent, à long terme, l’apparition de la dystrophie musculaire oculopharyngée (DMOP). L’influence des expansions trinucléotidiques chez les patients atteints de la maladie est encore mal connue. En utilisant des cellules exprimant le transgène GFP-PABPN1, nous avons observé une régulation de l’expression de la protéine endogène, ce qui suggère une autorégulation du gène PABPN1. De plus, nous avons observé la présence de deux populations de transcrits du gène : un premier transcrit mature (ARNm) et le deuxième contenant l’intron terminal de PABPN1 (pre-ARNm). Expérimentalement, nous avons observé que l’expression du transgène module le ratio pre-ARNm/ARNm de l’endogène. Aussi, en utilisant un gène rapporteur (GFP-6/7) qui se comporte comme l’endogène, nous avons démontré que l’autorégulation de PABPN1 nécessite la présence d’un intron terminal ayant une séquence non optimale pour l’épissage. En plus, nos expériences suggèrent que l’autorégulation est indépendante des répétitions d’adénosines présentes à la fin du transcrit (queue poly-A). Par la suite, nous avons remarqué la présence d’une séquence riche en adénosine (A-riche) à l’intérieur de l’exon terminal de PABPN1. Nous avons démontré que cette séquence est liée par PABPN1 in vitro et in cellulo qu’elle influence l’autorégulation en inhibant l’épissage du transcrit. De plus, nous avons mis en évidence que le mécanisme de l’autorégulation passe par une compétition entre des facteurs qui influencent l’épissage et la dégradation du pre-ARNm par l’exosome nucléaire.

The cellular functions of the microprocessor complex

Cordiner, Ross Andrew Alex January 2016 (has links)
DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.

Structural and Functional Characterization of the Essential RNA Helicase Mtr4

Jackson, Ryan N. 01 May 2012 (has links)
The essential protein Mtr4 is a conserved Ski2-like RNA helicase that maintains the integrity of nuclear RNA by promoting the 3' end decay of a wide variety of RNA substrates. Mtr4 activates the multi-protein exosome in RNA processing, surveillance, and turnover pathways by unwinding secondary structure and/or displacing associated proteins from RNA substrates. While Mtr4 may be able to promote decay independently, it is often associated with large multi-protein assemblies. Specifically, Mtr4 is the largest member of the TRAMP (Trf4/Air2/Mtr4 polyadenylation) complex which targets a plethora of RNA substrates for degradation by appending them with small (~5nt) poly(A) tails via the polymerase activity of Trf4. Mtr4 preferentially binds and unwinds RNAs with short poly(A) tails. Notably, the mechanism by which Mtr4 recognizes the length and identity of the RNA 3' end is coupled to the modulation of poly(A) polymerase activity of Trf4. The lack of structural data for Mtr4 and associated complexes severely limits the understanding of Mtr4 function. Particularly, it is unclear how Mtr4 senses RNA features, acts on RNA substrates, delivers RNA substrates to the exosome, and assembles into larger protein complexes. Presented here is the x-ray crystal structure of Mtr4 combined with detailed structural and biochemical analysis of the enzyme. The structure reveals that Mtr4 contains a four domain helicase core that is conserved in other RNA helicases and a unique arch-like RNA binding domain that is required for the in vivo processing of 5.8S rRNA. Furthermore, kinetic and in vivo analysis of conserved residues implicated in the poly(A) sensing mechanism demonstrates that ratchet helix residues regulate RNA unwinding and impact RNA sequence specificity. A comparison of the apo Mtr4 structure with the RNA/ADP bound structure (determined elsewhere) provides a view of the range of motion that individual domains of Mtr4 adopt upon substrate binding as well as the possible conformations that occur during RNA translocation. These studies provide an important framework for understanding the fundamental role of Mtr4 in exosome-mediated RNA decay, and more broadly describe common themes in architecture and function of the Ski2-like helicase family.

Exploration of a novel non-lytic viral transmission mechanism utilized by a non-enveloped positive-sense RNA virus

Yang, Jie Eune 12 June 2018 (has links)
While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection. / 2019-06-12T00:00:00Z

Augmented liver tageting of exosomes by surface modification with cationized pullulan / カチオン化プルランを用いたエクソソームの表面修飾はエクソソームの肝指向性を増強する

Tamura, Ryo 25 September 2017 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20672号 / 医博第4282号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Endothelial Cell Derived MVs and Exosomes: Release and Functional Study

Liu, Langni 01 September 2015 (has links)
No description available.

Synergy Between the Exoribonucleases Rrp6p and Rrp44p in the Nuclear Exosome Complex

Axhemi, Armend 29 May 2020 (has links)
No description available.

Transfert du CFTR par vecteurs de gènes dérivés des adénovirus ou par trogocytose de microparticules membranaires : mécanismes moléculaires et applications à la mucoviscidose / Transfer of CFTR by gene tranfer vectors derived from adenoviruses or by trogocytosis of membrane-derived microparticle : molecular mechanisms and applications in cystic fibrosis

Gonzalez, Gaëlle 14 December 2011 (has links)
La mucoviscidose est une maladie génétique due à des mutations du gène CFTR, conduisant à une altération de la fonction de canal à ions chlorure de la glycoprotéine transmembranaire CFTR associée à une atteinte pulmonaire sévère. Plusieurs études récentes ont amené à reconsidérer l’utilisation des vecteurs adénoviraux (Ad) de sérotype 5 (Ad5) dans la mucoviscidose, lesquels induisent non seulement des réactions immunes anti-adénovirales mais aussi des effets cytopathiques indésirables. (1) Dans une première partie de notre étude, nous avons étudié l’entrée et le transit intracellulaire de l’Ad5/F35, vecteur chimérique portant les fibres de l’Ad sérotype 35 sur une capside de sérotype 5. Nous avons montré que la protéine fibre est déterminante dans l’internalisation et le trafic intracellulaire de ce vecteur. Le vecteur Ad5/F35 exprimant la fusion GFP-CFTR s’est révélé (i) être dépourvu de cytotoxicité, (ii) transduire efficacement les cellules épithéliales pulmonaires par voie apicale, et (iii) restaurer l’activité de canal à chlorure dans les cellules CFTR(-). Il constitue donc un vecteur de transfert du gène CFTR potentiellement utilisable en thérapie génique de la mucoviscidose. (2) Dans une seconde partie, nous avons exploré une stratégie alternative de transfert de la protéine CFTR par trogocytose. Nous avons fait l’hypothèse que le canal CFTR pouvait être véhiculé par des microvésicules ou microparticules membranaires (MP) émanant de la membrane cellulaire et libérées dans le milieu de culture. En utilisant un système d’expression stable de la protéine CFTR étiquetée par la protéine fluorescente GFP (GFP-CFTR) dans des cellules donneuses, nous avons pu démontrer que les MP sont capables de prendre en charge et délivrer la protéine GFP-CFTR à des cellules réceptrices, mais ce transfert n’est assuré que par une population réduite de MP (≤ 8 %), et la durée de vie du GFP-CFTR n’est que transitoire (≤ 24h). En fait, la majorité des MP transfèrent des molécules d’ARN messager ou polysomal GFP-CFTR. La protéine GFP-CFTR néosynthétisée à partir de ces ARNm est exprimée plus tardivement (> 48h) mais de façon prolongée (≥ 10 jours). La fonctionnalité du canal CFTR ainsi néosynthétisé est en cours d’évaluation. Les MP constituent donc un nouveau type de vecteurs de transfert non génique du CFTR qui pourraient être employés en thérapie de la mucoviscidose. / The cystic fibrosis (CF) is a genetic disease due to mutations of the CFTR gene, resulting in the alteration of the Cl channel function carried by the transmembranal glycoprotein CFTR, and associated with severe pulmonary complications. Several recent studies led the medical and scientific community to reconsider the use of adenovirus serotype 5 (Ad5)-based vectors as CFTR gene transfer vectors in CF gene therapy. Not only immune response against the vector itself and the ansduced cells have been observed, but also Ad5-induced nondesired cytopathic effects. (1) In the first part of our study, we analyzed the cell entry and traficking of Ad5/F35, a chimeric vector consisting of serotype 5 capsid carrying serotype 35 fibers. We showed that the fibre protein is the major, if not only, determinant of the internalisation and entry pathway of Ad5/F35. Ad5/F35-GFP-CFTR, expressing the fusin protein GF-CFTR, was found (i) to be devoid of detectable cytopathic effect, (ii) efficiently transduced airway epithélial cells via the apical pole, and (iii) restore the Cl channel function in CF cells. Ad5/F35 therefore represents a CFTR gene transfer vector with a great potential for gene therapy of CF. (2) In the second part of our study, we have investigated an alternative strategy based on the transfer of the mature CFTR protein via trogocytosis. We hypothesized that microvesicles or microparticules (MP) issued from the cell membranes and released into the culture medium could transport and achieve the cell-to-cell transfer of CFTR channel cargo. We engineered donor cells for stable expression of GFP-tagged CFTR protein (GFP-CFTR), and showed that donor cell-issued MP were capable of delivering GFP-CFTR protein to recipient cell. However, the GFP-CFTR protein was only transferred by a limited population of MP (≤ 8 %), and was only transient (≤ 24h). In fact, the major population of MP transferred mRNAGFP-CFTR or polysomal thereof. Interestingly, the GFPCFTR protein newly synthesized from this mRNAGFP-CFTR was expressed at late times after transfer (≥ 48 h) but in a prolonged manner (≥ 10 jours). The Cl canal function after MP-mediated CFTR transfer is being evaluated. MP represent a novel type of CFTR vectors which can be produced by specifically designed autologous donor cells, and which would overcome most of the inconveniences of gene therapy using viral or nonviral vectors.

CBC bound proteins and RNA fate / Titre non traduit

Giacometti, Simone 11 April 2016 (has links)
Le complexe de liaison de la coiffe des ARN (CBC) joue un rôle essentiel dans leur maturation et déclenche une variété de réactions biochimiques, via son interaction avec différents partenaires. Deux complexes, CBC-ARS2-PHAX (CBCAP), et CBC-ARS2-ZC3H18-NEXT (CBCN), ont récemment été montré comme important pour cibler les ARN vers l'export (CBCAP) ou la dégradation (CBCN). Cependant, les mécanismes par lesquels la sélection se fait pour l'une voie ou l'autre reste mystérieuse. Ainsi, une question majeure qui reste à résoudre est de savoir quand et comment ces complexes sont recrutés sur les ARN. Dans ce travail, j'ai utilisé la procédure du iCLIP (Cross-Linking and Immuno-Precipitation), afin d'identifier les cibles de ces complexes sur l'ensemble du transcriptome humain. J'ai réalisé un iCLIP sur cinq composants de CBCAP et CBCN, et j'ai comparé les résultats à ceux obtenus avec RBM7, un composant de NEXT précédemment étudié par iCLIP. Mes résultats indiquent que: (i) CBP20, ARS2, PHAX et ZC3H18 se lient près de la coiffe des ARN, tandis que RBM7 et MTR4 se lient partout; (ii) CBP20, ARS2, PHAX et ZC3H18 s'associent à un large ensemble d'ARN transcrits par l'ARN polymérase II et montrent une faible sélectivité; (iii) la liaison de ces protéines varie avec l'état de maturation des ARN, avec le CBC enrichi sur les ARN matures, tout comme ARS2/PHAX/ZC3H18 et MTR4 (bien que dans une moindre mesure), tandis que RBM7 est préférentiellement lié sur les pre-mRNAs non épissés; (iv) une liaison différentielle de RBM7 et MTR4 sur les ARN, avec RBM7 enrichi sur les introns et les PROMPTs, et MTR4 plus présent sur les ARN mature. Bien que des expériences additionnelles soient requises, nous proposons que le CBCAP et le CBCN se lient à un même ensemble d'ARN, ce qui indique à la fois une compétition entre ZC3H18 et PHAX pour la liaison à ces ARN, et l'absence de voies de routage bien déterminées qui ciblerait les ARN vers l'une ou l'autre de ces protéines. Le devenir des ARN pourrait ainsi être déterminé par d'autres caractéristiques des ARN, ou encore par des protéines additionnelles. Ces facteurs pourraient s'allier aux protéines liées à la coiffe afin de favoriser la formation du CBCAP ou du CBCN. Dans le but d’identifier des facteurs additionnels, j'ai réalisé un screen d'interaction par spectrométrie de masse après purification de ARS2 ou CBP80. Ceci a été fait dans des conditions natives ou après un cross-link des complexes à la formaldéhyde, afin de stabiliser les interactions transitoires. Ceci a permis d'identifier de nouveaux partenaires de ARS2 et de CBP80, dont la majorité sont impliqués dans l'épissage des ARN. Des expériences additionnelles seront nécessaires pour valider ces interactions. / The cap-binding complex (CBC) plays a pivotal role in post-transcriptional processing events and orchestrates a variety of metabolic pathways, through association with different interaction partners. Two CBC sub-complexes, the CBC-ARS2-PHAX (CBCAP) and the CBC-nuclear exosome targeting (NEXT) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii) binding varies with the RNA maturation stage, with the CBC being highly enriched on mature mRNA, ARS2/PHAX/ZC3H18/MTR4 less so, and RMB7 preferentially bound to pre-mRNAs; (iv) MTR4 and RBM7 show different specificities, with RBM7 being highly enriched on introns and promoter upstream transcripts (PROMPTs), while MTR4 is additionally present on mature RNAs. Although more experimental work is needed to fully support our model, we propose that CBCAP and CBCN bind overlapping sets of RNAs, indicating a competition between the proteins ZC3H18 and PHAX, and the lack of a strict RNA sorting mechanism. RNA fate may therefore be determined by additional RNA features and/or by other RNA-binding proteins, which may synergize with the cap and drive the formation of one specific CBC subcomplex instead of another. In an attempt to identify yet unknown factors that may interact with cap-bound CBCAP and CBCN, we performed a protein interaction screen leveraging affinity capture-mass spectrometry (ACMS), using ARS2 and CBP80 as bait proteins. As a complementary approach, we also employed a formaldehyde-based chemical cross-linking strategy, aimed at stabilizing weak/transient interactions. Although we failed to detect any transient interactions involving the CBC, we identified several potential CBC80 and ARS2 interactors, the majority of which are involved in pre-mRNA splicing. Additional quantitative experiments are required to validate our ACMS results and confirm the existence of such protein interactions in vivo.

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