Spelling suggestions: "subject:"rnas"" "subject:"rins""
121 |
Entwicklung eines Sleeping Beauty Transposon Systems zum simultanen und induzierbaren shRNA-Knock-down verschiedener Zielstrukturen in Zelllinien des Multiplen Myeloms / Development of a system for multi-targeted inducible shRNA-knock-downs with the Sleeping Beauty transposon system and establishment in Multiple Myeloma cell linesFink, Severin Lion Julian January 2021 (has links) (PDF)
Das Multiple Myelom (MM) ist ungeachtet neuer medikamentöser Therapien weiterhin eine für die allermeisten Patienten unheilbare Erkrankung. Aktuelle Whole-Genome-Sequenzierungen konnten die Annahme, dass hierfür die genetische Heterogenität des MM verantwortlich ist, bestätigen. Um dieses onkogene Signalnetzwerk auf effiziente Weise zu untersuchen, wurde ein induzierbares Knock-down-System basierend auf der weitverbreiteten RNA-Interferenz und dem neuen, innovativen Sleeping Beauty Transposon System (SBTS) entwickelt. Die Etablierung des SBTS im MM zeigte, dass die CMV-Promotor-vermittelte EGFP-Expression über 231 Tage stabil ist. Die Verwendung des SBTS ermöglicht es, Zellen bei niedrigster biologischer Schutzstufe (S1) stabil zu transfizieren. Am Beispiel des MAPK-Signalweges konnten stabile shRNA-vermittelte Knock-downs in verschiedenen MM-Zelllinien erfolgreich durchgeführt werden. Um ein induzierbares Tet-On-System zur shRNA-Expression zu erstellen, wurden zum einen dem verwendeten H1-Promotor zwei TetO-Sequenzen hinzugefügt. Zum anderen wurde durch die Verwendung eines zweiten, neu konstruierten SBTS Vektors mit anderer Selektionsresistenz in den verwendeten Zellen das Tet-Repressor-Protein (TetR) stabil exprimiert. Die notwendige Expression von TetR konnte nur mittels CAG-Promotors erreicht werden. Am Beispiel von ERK2 konnte gezeigt werden, dass es durch die stabile Transfektion und die Induktion des Knock-downs mit Doxycyclin möglich ist, den Knock-down Effekt zu erzielen. Das etablierte Plasmid-basierte System stellt somit ein versatiles, einfach anwendbares, kostengünstiges und zeitsparendes Werkzeug dar, induzierbare Knock-downs durch RNA-Interferenz für einzelne oder mehrere Proteine gleichzeitig zu erzielen. Es ist davon auszugehen, dass die Anwendung aufgrund der Verwendung etablierter Techniken und der leichten Modifizierbarkeit nicht nur auf das MM begrenzt sein wird. / Multiple Myeloma (MM) is regardless of new drug therapies still an incurable disease for the majority of patients. Large-scale whole-genome-sequencing studies strongly support the assumption that the reason for this is the large genetic heterogeneity of MM.
To gain knowledge about the complex oncogenetic signalling efficiently, in this work, a system for inducible knock-downs based on the wide-spread RNA-interference and the new innovative Sleeping Beauty Transposon System (SBTS) was developed. The successful establishment of the SBTS in MM revealed that EGFP-expression by a CMV-promotor is stable for at least 231 days without any further selection by antibiotics. The use of SBTS makes it possible to transfect cells stably at the lowest biological safety level (S1). Utilizing the MAPK-signal path as an example it was possible to successfully demonstrate shRNA-provided knock-downs for up to four targets at once in several different MM-cell lines. To create an inducible Tet-on-system for shRNA-Expression, two TetO-sequences were added to the used H1-Promotor. Furthermore, the required Tet-repressor-protein (TetR) was expressed by another newly constructed SBTS vector, which contains another selection resistance. After trying different approaches, it was possible to express the necessary amount of TetR by using the CAG-Promotor, which was verified by Western Blot. Using ERK2 as a sample target, it was possible to show that with stable transfection and induction of the knock-down with doxycycline it is possible to measure knock-down results without the toxic side effects of the electroporation necessary for transfection. The established plasmid-based system serves as an easily applicable, cost-efficient and time-saving tool for inducible knock-downs by RNAi for single or multiple proteins. Due to the fact that yet established techniques were used and thanks to the simplicity of the customization it is assumed that the application is not only limited to MM.
|
122 |
Identifizierung und Untersuchung TOP-mRNA - bindender Faktoren / Identification and examination of TOP mRNA binding factorsAmelingmeier, Florian January 2022 (has links) (PDF)
Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie benötigt und daher mittels vielfältiger Mechanismen streng reguliert wird. Ein Beispiel hierfür stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche hauptsächlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5’-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv ermöglicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. Nährstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird.
Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation ermöglicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erwägung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP – Sequenzen identifiziert werden, welche sich für Strukturanalysen eignen würden. Als weiterer TOP-interagierender Faktor wurde über verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert.
Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. Während gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7–Konstrukt sowie 7SK-Konstrukte für Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK – Interaktion ausüben, welcher in weiteren Studien berücksichtigt werden sollte. / In the nucleus of eucaryotic cells, genes are transcribed into mRNA which are heavily
processed and exported into the cytoplasm. There they are translated into proteins, a process
requiring large amounts of energy so this process is strongly regulated. One example is the
class of TOP RNAs consisting mainly of transcripts encoding for proteins involved in
translation. Some well-known examples include the proteins of the large and small ribosomal
subunits. TOP RNAs share a common motif at the start of their 5’ UTR comprising a sequence
entirely made of Pyrimidines immediately following the cap. This motif common to all TOP
RNAs enables translational control of this class of RNAs in a timely coordinated manner. In this
way, during conditions of stress like nutrient starvation, translation of TOP RNAs can be
inhibited to save energy.
The search for a regulator which binds to the TOP motif and enables coordinated regulation
started long ago. In principle the regulator could activate or inhibit translation. Different
proteins have been considered to be the regulator so far.
In this thesis the protein TIAR was identified as TOP interacting factor using mass
spectrometry. Its binding properties regarding the TOP motif have been evaluated using
EMSA. RNA and proteins of different organisms were evaluated to identify minimal binding
partner constructs for further structural analysis. Using different sequential purification
approaches, the 14-3-3ε protein was also identified as TOP binding factor.
Furthermore, the TOP binding proteins LARP1 and LARP7 and their target RNA sequences have
been evaluated in regard to their binding properties. It could be shown that LARP1 has an
inhibiting effect on translation of TOP RNAs. Using EMSA, minimal binding constructs of LARP7
and 7SK could be established which can be considered for further structural analysis. Also, it
could be shown that certain substances like tRNA and Arginine influence the interaction of
LARP7 and 7SK, an observation which should be considered in further experiments.
|
123 |
Contribution aux opérateurs arithmétiques GF(2m) et leurs applications à la cryptographie sur courbes elliptiques / Contributions to GF(2m) Operators for Cryptographic PurposesMétairie, Jérémy 19 May 2016 (has links)
La cryptographie et la problématique de la sécurité informatique deviennent des sujets de plus en plus prépondérants dans un monde hyper connecté et souvent embarqué. La cryptographie est un domaine dont l'objectif principal est de ''protéger'' l'information, de la rendre inintelligible à ceux ou à celles à qui elle n'est pas destinée. La cryptographie repose sur des algorithmes solides qui s'appuient eux-mêmes sur des problèmes mathématiques réputés difficiles (logarithme discret, factorisation des grands nombres etc). Bien qu'il soit complexe, sur papier, d'attaquer ces systèmes de protection, l'implantation matérielle ou logicielle, si elle est négligée (non protégée contre les attaques physiques), peut apporter à des entités malveillantes des renseignements complémentaires (temps d’exécution, consommation d'énergie etc) : on parle de canaux cachés ou de canaux auxiliaires. Nous avons, dans cette thèse, étudié deux aspects. Le premier est l'apport de nouvelles idées algorithmiques pour le calcul dans les corps finis binaires GF(2^m) utilisés dans le cadre de la cryptographie sur courbes elliptiques. Nous avons proposé deux nouvelles représentations des éléments du corps : la base normale permutée et le Phi-RNS. Ces deux nouveautés algorithmiques ont fait l'objet d'implémentations matérielles en FPGA dans laquelle nous montrons que ces premières, sous certaines conditions, apportent un meilleur compromis temps-surface. Le deuxième aspect est la protection d'un crypto-processeur face à une attaque par canaux cachés (dite attaque par «templates»). Nous avons implémenté, en VHDL, un crypto-processeur complet et nous y avons exécuté, en parallèle, des algorithmes de «double-and-add» et «halve-and-add» afin d'accélérer le calcul de la multiplication scalaire et de rendre, de par ce même parallélisme, notre crypto-processeur moins vulnérable face à certaines attaques par canaux auxiliaires. Nous montrons que le parallélisme seul des calculs ne suffira pas et qu'il faudra marier le parallélisme à des méthodes plus conventionnelles pour assurer, à l'implémentation, une sécurité raisonnable. / Cryptography and security market is growing up at an annual rate of 17 % according to some recent studies. Cryptography is known to be the science of secret. It is based on mathematical hard problems as integers factorization, the well-known discrete logarithm problem. Although those problems are trusted, software or hardware implementations of cryptographic algorithms can suffer from inherent weaknesses. Execution time, power consumption (...) can differ depending on secret informations such as the secret key. Because of that, some malicious attacks could be used to exploit these weak points and therefore can be used to break the whole crypto-system. In this thesis, we are interested in protecting our physical device from the so called side channel attacks as well as interested in proposing new GF(2^m) multiplication algorithms used over elliptic curves cryptography. As a protection, we first thought that parallel scalar multiplication (using halve-and-add and double-and-add algorithms both executed at the same time) would be a great countermeasure against template attacks. We showed that it was not the case and that parallelism could not be used as protection by itself : it had to be combined with more conventional countermeasures. We also proposed two new GF(2^m) representations we respectively named permuted normal basis (PNB) and Phi-RNS. Those two representations, under some requirements, can offer a great time-area trade-off on FPGAs.
|
124 |
Caractetrização de operadores modulares implementados em FPGAFernandes, Armando Filipe Carvalhido January 2010 (has links)
Tese de mestrado integrado. Engenharia Electrotécnica e de computadores. Faculdade de Engenharia. Universidade do Porto. 2010
|
125 |
Συνδυασμένες μονάδες πολλαπλασιασμού / αθροίσματος τετραγώνων για αριθμητικά συστήματα υπολοίπων / RNS multiplication / sum-of-squares unitsΑδαμίδης, Δημήτριος 16 May 2007 (has links)
Πολλές εφαρμογές ψηφιακής επεξεργασίας σημάτων (DSP) και πολυμέσων μπορούν να ωφεληθούν από τη χρήση ενός αριθμητικού συστήματος υπολοίπων (RNS). Ανάμεσα στους πιο συχνά χρησιμοποιούμενους διαιρέτες σε τέτοια συστήματα είναι αυτοί της μορφής 2^n - 1 και 2^n + 1, ενώ ανάμεσα στις πιο συχνά χρησιμοποιούμενες λειτουργίες βρίσκονται ο πολλαπλασιασμός και το άθροισμα τετραγώνων. Οι λειτουργίες αυτές προς το παρόν υλοποιούνται με τη χρήση ξεχωριστών μονάδων και συνεχόμενων κύκλων μηχανής. Στην παρούσα εργασία προτείνονται δύο αρχιτεκτονικές για μονάδες οι οποίες μπορούν να εκτελέσουν είτε το X * Y είτε το X^2 + Y^2, ανάλογα με την τιμή ενός σήματος ελέγχου. Εξετάζεται τόσο η modulo 2^n - 1, όσο και η ελαττωμένη κατά ένα modulo 2^n + 1 αριθμητική. / Digital signal processing (DSP) and multimedia applications often profit from the use of a Residue Number System (RNS). Among the most commonly used moduli in such systems are those of 2^n - 1 and 2^n + 1 form and among the most commonly used operations are multiplication and sum-of-squares. These operations are currently performed using distinct design units and consecutive machine cycles. In this paper, we propose two architectures for units that perform either the X * Y or the X^2 + Y^2 operation depending on the value of a control signal. Both modulo 2^n - 1 and diminished-1 2^n + 1 arithmetic is considered.
|
126 |
Konformationelle Vielfalt Synthese eines spleißosomalen RNA-Konstruktes, NMR-Strukturen von Minigramicidin und einem molekularen SchalterBockelmann, Dirk Unknown Date (has links)
Univ., Diss., 2006--Frankfurt (Main) / Enth. Sonderabdr. aus versch. Zeitschr. - Zsfassung in dt. und engl. Sprache
|
127 |
Analyses of the archaeal transcription cycle reveal a mosaic of eukaryotic RNA polymerase II and III-like featuresSpitalny, Patrizia January 2008 (has links)
Regensburg, Univ., Diss., 2008
|
128 |
Co-transcriptional recruitment of the U1 snRNPKotovic, Kimberly Marie. Unknown Date (has links) (PDF)
Techn. University, Diss., 2004--Dresden.
|
129 |
Petits ARN non codants dérivant d’ARN de transfert et endoribonucléases impliquées dans leur biogenèse chez Arabidopsis thaliana / tRNA derived small non-coding RNA and endoribonuclease implicated in their biogenesis in Arabidopsis thalianaMegel, Cyrille 29 June 2016 (has links)
Parmi les petits ARN non codants, les fragments dérivant d’ARNt (tRF) ont été identifiés dans tous les embranchements de la vie. Cependant, très peu de donnée existe sur les tRF de plantes. Les populations de tRF issues de plusieurs banques de petits ARN (différents tissus, plantes soumises à des stress abiotiques, ou fractions immunoprécipitées avec la protéine ARGONAUTE1) ont été analysées. Les populations sont essentiellement constituées de tRF-5D ou des tRF-3T (clivage dans la boucle D ou T respectivement) et elles varient d’une banque à l’autre. Par une approche in silico suivie de tests de clivage in vitro, des RNases T2 d’A. thaliana (RNS) ont été identifiées comme étant capables de cliver les ARNt dans la région de l’anticodon, de la boucle D et de la boucle T. Lors de l’étude de l’expression des RNS, nous avons observé que deux d’entre elles sont fortement exprimées à un stade de maturation tardif des siliques. Ainsi, la population en tRF issue de stades de développement avancés des siliques a été analysée. Des expériences de carences en phosphate nous ont permis de démontrer l’implication d’une des RNS dans la genèse de tRF dans A. thaliana. Au final, nos données ouvrent de nouvelles perspectives quant à l’implication des RNS et des tRF comme des acteurs majeurs dans l’expression des gènes chez les plantes. / Among the small ncRNAs, tRNA-derived RNA fragments (tRFs) were identified in all domains of life. However, only few data report on plants tRFs. Short tRF were retrieved from A. thaliana small RNA libraries (various tissues, plants submitted to abiotic stress or argonaute immunoprecipitated fractions). Mainly tRF-5D or tRF-3T (cleavage in the D or T region respectively) were found, and fluctuations in the tRF population were observed.Using in vitro approaches, A. thaliana RNase T2 endoribonucleases (RNS) were shown to cleave tRNAs in the anticodon region but also in the D or T region. Through a whole study of RNS expression, we show that two RNS are also strongly expressed in the siliques at a late stage of development. Thus, we analyzed the tRF population of this particular developmental stage. Upon phosphate starvation, we demonstrate also the implication of one RNS in the production of tRFs in planta. Altogether, our data open new perspectives for RNS and tRFs as major actors of gene expression inplants.
|
130 |
Generation of tools to investigate Chikungunya virus / Entwicklung von Methoden zum Chikungunya-NachweisVu Xuan, Nghia January 2008 (has links) (PDF)
CHIKV is the prototype of Alphaviruses and it causes an acute febrile illness with rash, severely painful arthralgias, and sometimes arthritis. While CHIKV has first been identified in the 1950s in Africa, recent outbreaks of CHIKV in the islands of the Indian Ocean and particular in Italia have re-drawn attention to CHIKV. In the past CHIKV disease was considered self-limiting and non-fatal. However, a number of deaths on Reunion (Anonym, 2006) during the outbreak, which was affected directly or indirectly by CHIKV, have changed this view. To defeat CHIKV outbreaks diagnostic tools and anti CHIKV therapies are urgently needed. In this thesis, we generated tools to investigate CHIKV at the molecular level by serological tests. CHIKV was isolated from a German woman who was infected during her holidays on the Mauritius Island. To characterize this viral isolate the complete viral genome was amplified by PCR and molecular cloned. In order to analyse antibody responses of infected individuals some of the structural and non-structural genes were subcloned in bacterial expression vectors. The NSP2, proteinase, capsid, E1 and E2 were subsequently expressed in E.coli using purified successfully. In this thesis, the structural proteins were used to develop a screening test for anti-CHIKV antibodies in patient derived serum samples. These tests were evaluated with pre-characterized anti-CHIKV sera (30 samples) obtained from the BNI Hamburg and 100 serum samples from German blood donors used as negative controls. Immunoblotting analysis revealed that up to 77% of precharacterised positive sera could recognize the recombinant proteins and there were no detectable reactivity of CHIKV-negative German donor sera. The recombinant proteins were also recognized by 71.4% of positive sera in the newly established ELISA. In order to go further in analyses of the results, an in house IFA was performed. Positive sera (21 samples) were used. The results showed that all of them reacted positive, but this assay was less sensitive than the IFA from BNI. In comparison with the IFA result from BNI Hamburg, the results were not congruent in all test performed. This could be due to various drawbacks of the tests. A cross reaction in Alphaviruses and the different strains are mentioned as well as the denatured forms of the structural proteins. Besides the main structural proteins (E1, E2 and C), other proteins such as non-structural proteins, uncleaved precursor proteins could participate in the different outcomes of serological assays. In order to go further in the CHIKV diagnoses, the CHIKV recombinant proteins were applied to screen the anti-CHIKV antibodies in the Vietnamese population, who are considered to live in the high risk regions. In serological tests, 158 sera of Vietnamese donors were incubated with the recombinant proteins or the fixed CHIKV infected cells. The results showed that 24% of Vietnamese donor sera recognized the recombinant proteins in immunoblot assay, while 36% scored positive in the ELISA assay. In IFA, the sera considered positive were 11.4%. While some discrepancies in serological tests were found, these results showed that the ratio of CHIKV-positive sera seem to be equal to the other regions in the world, which are affected by CHIKV. It is suggested that CHIKV infection in Vietnam has been repeatedly misdiagnosed. This study cohort consisted only of samples originating from Hanoi area of Northern Vietnam, thus, future studies should expand to include samples from other Vietnam areas. To do this the various subtypes of the virus in the different regions should be isolated and the sequences of these viruses should be well characterized.
|
Page generated in 0.18 seconds