Dissertations / Theses on the topic 'Transfection'
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1
Ma, Nan. "Tailoring optical fibers for cell transfection." Thesis, University of St Andrews, 2012. http://hdl.handle.net/10023/3177.
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Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free-space bulk optical setup. Here in this thesis, a few novel fabrication methods are devised to obtain easy and inexpensive fabrication of microlensed optical fibers, which can be used to replace traditional optical setup and perform femtosecond optical transfection. These fabrication methods offer the flexibility to fabricate a microlens which can focus femtosecond laser pulses at 800 nm to a small focal spot whilst keeping a relatively large working distance. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. This thesis reports the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining lensed fiber based optical transfection system with a micro-capillary based microfluidic system. Finally, based on an imaging fiber (coherent optical fiber bundle), the first endoscope-like integrated system for optical transfection with subcellular resolution epifluorescence imaging was built. The transfection efficiency of these fiber based systems is comparable to that of a standard free-space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of genetic material for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, such as the dosing of tissue slices to study neuron activities, targeted drug delivery, and in particular for using endoscope-like integrated systems for targeted in vivo optical microsurgery.
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Finlay, Siân. "Modulation of macrophage phenotype using adenoviral transfection." Thesis, University of Aberdeen, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409252.
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The initial aim of this study was to examine the nature of the interaction between adenovirus and transfected macrophage, and to characterise the molecular mechanisms underlying macrophage response to adenoviral infection. Results showed that adenoviral transfection activated macrophages, promoting production of pro-inflammatory mediators and priming an enhanced response to other inflammatory stimuli. Activation was dependent on the nuclear factor kappa B (NFkB) signalling pathway, which was activated within two hours of transfection, and could be prevented using an inhibitory adenovirus which blocked NFkB signalling. The ultimate phenotype of the transfected macrophage was determined both by non-specific viral activation and by the properties of the transgene expressed. The second aim of this research was to investigate the effect of the local cytokine milieu on transgene expression in vitro. Results showed that transgene expression under the control of two different promoter constructs was subject to regulation by pro-inflammatory mediators, by mechanisms at least partly dependent on the NFkB signalling pathway. the results have important implications for the use of these promoters in gene therapy applications where the gene of interest is delivered into an inflammatory environment. The final stage of the project focused on the use of transfected macrophages in vivo, in a rat model of glomerulonephritis. Results showed that transfected macrophages expressing the anti-inflammatory cytokine IL-4 localised with enhanced efficiency to inflamed glomeruli after intra-renal artery injection of the mechanisms for this were examined. Better understanding of the mechanisms which promote localisation may ultimately permit the design of genetically-modified macrophages which selectively target sites of injury for delivery of anti-inflammatory cytokines.
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Horefti, Ioulia-Christina. "Aeroporation : a new method for cell transfection." Thesis, University of Essex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364549.
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Yingyongnarongkul, Boon-ek. "Transfection agents : from traditional to miniaturised screening." Thesis, University of Southampton, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289566.
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Tsampoula, Xanthi. "Femtosecond cellular transfection using novel laser beam geometries." Thesis, St Andrews, 2009. http://hdl.handle.net/10023/909.
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Mthunzi, Patience. "Optical sorting and photo-transfection of mammalian cells." Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/1254.
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Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.
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Lemoine, Jérôme. "Transfection de l'épithélium respiratoire nasal normal de souris." Reims, 2005. http://www.theses.fr/2005REIMP205.
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La @mucoviscidose est une maladie génétique affectant principalement les poumons, qui est due à la mutation du gène CFTR. La protéine CFTR est un canal chlorique. Le transfert du gène CFTR sauvage dans l'épithélium bronchique peut être obtenu à l'aide de vecteurs de gènes non-viraux. Ces transferts n'ont pas permis, jusqu'à présent, de corriger complètement le transport de chlorure. Dans cette étude, nous avons démontré que de l'ADN nu dissout dans de l'eau déminéralisée transfecte les cellules épithéliales nasales plus efficacement que dans une solution isotonique ou hypertonique. Cette méthode a été baptisée : transfection par choc hypotonique. Le plasmide serait internalisé par les cellules épithéliales au cours de l'étape dite de diminution régulée du volume cellulaire du choc hypotonique. La stratégie la plus simple pour améliorer la transfection a consisté à raccourcir le plasmide, en le coupant dans la portion bactérienne à l'aide d'enzymes de restriction. L'expression du transgène obtenue suite à l'administration de fragments d'ADN nu, dissous dans de l'eau pure, a atteint un très haut niveau. L'amplificateur transcriptionnel IE1 du CMV contient des sites de fixation pour NF-κB et ATF-CREB, qui ont été remplacés par des sites Oct-1. Les sites de fixation non-consensuels NF1 et Sp1, ont été remplacés par leurs homologues consensuels. Ces modifications ont permis de réduire la diminution de l'expression du transgène entre les jours un et dix. La thérapie génique de la mucoviscidose deviendra concevable s'il est possible d'atteindre et de maintenir pendant plusieurs semaines, un niveau élevé d'expression du transgène dans l'épithélium bronchique des patients@Cystic fibrosis is a recessive genetic disease mostly affecting the lung. It is due to the mutation or the loss of the CFTR gene. The CFTR protein is a chloride channel located in the apical membrane of airway epithelial cells. CFTR gene transfer to the bronchial epithelium might be carried out by nonviral vectors, which appear insufficiently efficient to date. In this study, we have demonstrated that naked DNA dissolved in deionized water transfect the nasal airway epithelial cells 100-fold more efficiently than in an isotonic or hypertonic solution. This method has been named transfection by hypotonic shock. The plasmid DNA would be endocytosed by the airway epithelial cells during the so-called phase of regulatory volume decrease of the hypotonic shock. The simplest strategy to further improve gene transfer consisted in shortening the plasmid DNA by deleting a part of its backbone by using some restriction enzymes. The transgene expression achieved by some naked DNA fragments dissolved in pure water was over 21-fold higher than that obtained by the intact plasmid DNA. The CMV IE1 enhancer contains a few NF-κB and ATF-CREB binding sites, which were replaced by Oct-1 binding sites. Also, the non-consensual NF1 and Sp1 binding sites were replaced by their consensual sequences. These modifications reduced the decline of the transgene expression 3. 4-fold between day 1 and day 10. Gene therapy will become a conceivable therapeutic approach, if a high level of transgene expression is sustained for several weeks in the bronchial epithelium of cystic fibrosis patients
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Le, Bolc'h Gwénaelle. "Synthèse de phosphonolipides pour la transfection non-virale." Brest, 1997. http://www.theses.fr/1997BRES2032.
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La therapie genique apparait, de plus en plus, comme une solution envisageable pour vaincre les maladies hereditaires comme la mucoviscidose par exemple. L'objectif de ce travail est la synthese de composes pouvant favoriser la transfection d'un gene correcteur a travers les membranes cellulaires. Le premier chapitre bibliographique fait le point sur la therapie genique et sur les differents vecteurs non-viraux deja connus. Les deux chapitres suivants sont consacres a la synthese de composes susceptibles d'etre utilises comme vecteurs non-viraux de transfection. Les premieres evaluations de la capacite de transfection y sont egalement presentees. Puis nous ferons le bilan de ce travail et evoquerons les perspectives qui en resultent.
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Lemoine, Jérôme Desoize Bernard. "Transfection de l'épithélium respiratoire nasal normal de souris." S.l. : S.n, 2005. http://www.univ-reims.fr/BU//exl-doc/GED00000194.pdf.
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DUGONNET, DURAND FREDERIQUE. "Differentes methodes de transfection appliquees en therapie genique." Strasbourg 1, 1994. http://www.theses.fr/1994STR15097.
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Hogge, Donna Eileen. "Genetic investigations of human hemopoiesis : studies of clonality and gene transfer to hemopoietic progenitors." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/27316.
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In most neoplasms malignant change occurs in a single cell which then proliferates. My purpose was to explore methods to study the cell that gives rise to hemopoietic cancer and to investigate the abnormalities at a molecular level.
Cytogenetic analysis of cells from individual hemopoietic colonies revealed that monosomy 7 syndrome, a hematologic disorder of childhood, arises in a primitive cell capable of differentiating down both myeloid and erythroid pathways.
Long-term bone marrow cultures (LTC) from patients with chronic myelogenous leukemia (CML) favor the growth of Philadelphia chromosome (Ph) negative progenitors which, although cytogenetically normal, could have been part of the malignant clone at a stage prior to the development of the Ph. LTC's were initiated with cells from 2 women with CML who were heterozygous for 2 electrophoretically distinct glucose-6-phosphate dehydrogenase (G6PD) enzyme variants. In one patient, 2/11 progenitors were Ph-negative after 4 to 6 weeks in LTC and 4/30 were nonclonal by G6PD enzyme analysis, i.e. the colonies expressed the enzyme not found in the malignant clone. In this case, karyotypically normal cells were truly normal.
Next, gene transfer to human hemopoietic cells was demonstrated using recombinant retrovirus carrying the selectable marker gene, neor. With the K562 human leukemic cell line as targets up to 60% of infected cells became G418 resistant (G418r). Cloned populations of G418r cells showed unique patterns of retroviral integration in K562 DNA. When the target cells were progenitors from normal marrow, CML blood or fetal liver, the highest frequencies of G418r granulocyte-macrophage or large erythroid colonies was 16% and 5% respectively. Experiments infecting bone marrow cells in LTC with neor virus produced up to 2% G418r colonies after as long as 3 weeks in culture. Using v-src virus to infect LTC failed to perturb hemopoiesis, although infection of bone marrow-derived cells in these cultures was documented.
In summary:
1. Unique populations of hemopoietic progenitors can be identified in culture using several genetic markers including chromosomes, G6PD analysis or gene transfer. 2. The feasibility of retroviral-mediated gene transfer for use on human hemopoietic cells has been demonstrated.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Siu, King-sun, and 蕭景新. "Development of crosslinked poly(ethylenimine) as a potential nucleic acid delivery agent." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43223874.
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Lai, Wing-fu, and 黎永富. "Fabrication and properties of poly(ethylenimine)-based polymers for non-viral plasmid delivery." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46588127.
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Siu, King-sun. "Development of crosslinked poly(ethylenimine) as a potential nucleic acid delivery agent." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43223874.
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Greentree, David. "Gene transfection in satellite cell transplantation for myocardial repair." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0027/MQ50779.pdf.
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Flobak, Åsmund. "Investigation of Transfection Using Silica-coated Cupric Oxide Nanowires." Thesis, Norges Teknisk-Naturvitenskaplige Universitet, 2013. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-20683.
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One-dimensional high aspect ratio nanostructures grown from a copper foil are considered for intracellular delivery of bioactive molecules, such as drugs and genetic material. The cupric oxide (CuO) nanowires are coated with silicon oxides (SiOx), and embedded in a transparent polymer membrane consisting of the photoresist SU-8 and polydimethylsiloxane (PDMS).Nanowires are envisaged as one way two deliver bioactive molecules to many cells in parallel. It has previously been shown that both HeLa and AR42J cells grow and spread on this nanowire surface. The overarching goal of the current project is to test and optimize delivery of genetic material to cells grown on nanowires. It is first shown that nanowires are not able to induce knockdown of a constitutively expressed protein by delivering siRNA, when cells are sedimenting on top of an array of nanowires by gravitation. It is then shown that nanowire-mediated transfection of plasmids perform similar to flat surfaces, which are chemically identical apart from the nanowires, when HeLa cells are sedimenting down on a nanowire surface by gravitational pull. Increasing the sedimenting force by centrifugation has no effect on transfection efficiency, with relative centrifugal forces up to 88,500 × g tested. Tapping the nanowire surface face-down on cells deposited at a high concentration, which is a well documented method in published articles, also performs similar to flat sur faces, with transfection efficiencies of 1% commonly seen, and occasionally higher (5%).Fluorescently labeled plasmid is quite efficiently delivered to cells by both flat surfaces and nanowire surfaces, with a trend towards more efficient delivery by nanowires. Delivered cargo is however confined to small sections of cells. Cells are not transfected in experiments with identical setups, indicating that delivered biomolecules are isolated from the internal machinery of a cell.In conclusion, our transfection efficiency results are on par with most published experiments. In published experiments control experiments with flat surfaces are often lacking, and our results emphasize the importance for proper controls when investigating nanowire-mediated transfection and delivery of biomolecules.
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Chapman, M. R. "The potential of polyethylene imine derivatives as transfection reagents." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597479.
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This work aimed to address these issues by taking a novel approach to both the design and analysis of a library of transfection reagent derivatives. The library was produced by derivatisation of polyethylene imine (PEI), already a relatively efficient transfection reagent. However, rather than viewing derivatisation as an all or nothing process, our libraries apply derivatisation in a stepwise fashion to completely explore the chemical space. By using combinations of three simple derivatisations (methylation, benzylation and dodecylation), a large range of properties are produced from limited chemical diversity, including some polymers which rival the commercial gold standards in activity. The approach taken to analysis of the library properties is also new to the field. The properties most relevant to activity as a transfection reagent (expression of a reporter gene and toxicity) are viewed as the result of a series of steps each of which may have differing and conflicting requirements. The design of high-throughout assays for these properties and for DNA binding is described as are the clear relationships observed between these activities and derivatization levels. Using the DNA binding assay a start is made towards understanding how chemical space maps into the functional space of transfection and toxicity. Measurements of complex size are used to explain some requirements for transfection with polyethylenimine and the effect of derivatisation on DNA binding is investigated further to produce an account of the transfection process from formation of the transfection complex through to cell entry.
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Fischer, Wiebke [Verfasser]. "siRNA transfection with dendritic core-shell nanocarriers / Wiebke Fischer." Berlin : Freie Universität Berlin, 2012. http://d-nb.info/1026695244/34.
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Uduehi, Aimalohi Natasha. "Transfection of mammalian cell lines with polycationic/DNA complexes." Thesis, University of Bath, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338407.
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Lycett, Gareth John. "DNA transfection of an Ades aegypti mosquito cell line." Thesis, University of Liverpool, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314572.
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Rodamporn, Somphop. "Microfluidic systems for cell transfection using sonoporation and electroporation." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/159193/.
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Studies into sonoporation and electroporation have grown rapidly in biotechnology and medicine in recent years. This research presents a microfluidic system for cell transfection using sonoporation and electroporation. This research has studied, designed, developed and tested the sonoporation system and electroporation system with certain biological cells. Ultrasonic standing waves, previously used for ultrasonic particle manipulation, have been used in the development of the sonoporation aspects of the system. MATLAB has also been used to analyse the required acoustic conditions within the chamber. Furthermore, the electroporation system makes use of a relatively simple circuit consisting of a control module, a pulse generation circuit and a high voltage switch using a power MOSFET. The electroporation system has been designed, developed and tested. The system was evaluated with HeLa cells, THP-1 cells and plasmid DNA (pEGFP-N1) to evaluate transfection rates under a variety of sonoporation conditions. This study also determined cell viability under a range of sonoporation and electroporation conditions.
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Brown, Taylor Andrew. "In Vivo Silicon Lance Array Transfection of Plant Cells." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/8398.
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Arrays of silicon lances were made using photolithographic and STS DRIE Bosch techniques. Arrays consist of a 10 mm square grid pattern of lances measuring 100 m tall and having a 3 mm diameter, each lance being spaced 30 mm apart. The tips of lances are pointed, allowing easier penetration through plant cell walls. A nanoinjector device was also made to accept the silicon lance arrays and perform nanoinjections. A nanoinjection consisted of 2 silicon lance arrays, with lances oriented towards each other, being moved into and out of a plant cotyledon placed between them. Prior to the nanoinjection, polar molecules in solution can be attracted to the lances through a process utilizing the nanoinjector device’s ability to control the electrical current between the 2 lance arrays. During the nanoinjection the displacement between the lances, the force exerted on the plant cotyledon and the electrical current between the lance arrays are controlled. Once the lances are inserted into the cells, the electrical current between the lance arrays is reversed, repelling the molecular load from the lance array. Propidium iodide (PI) and Cotton Leaf Crumple Virus (CLCrV) were used as molecular loads in nanoinjections. The nanoinjector also records and outputs data from the nanoinjection for analysis. Nanoinjections were performed on Arabidopsis and Cotton cotyledons. Changes in the force applied during a nanoinjection and varying the number of repeated nanoinjections on the same cotyledon were observed. Too much force or too many repeated injections causes physical damage to the cotyledon. An optimal force and number of repeated injections can be performed without causing physical damage to the cotyledon. Successful transfection of PI and CLCrV was not observed in a relatively small number of performed nanoinjection procedures on either Arabidopsis or Cotton cotyledons. Possible interacting variables and recommendations for further work are discussed.
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Bresler, Brandon G. "Organic Blue TADF Chromophore Tag For Monitoring Transfection Studies." Wright State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=wright161038158466956.
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Churamani-Poulenc, Rebecca. "Assemblages supramoléculaires de cyclodextrines fonctionnalisées comme outil de transfection." Electronic Thesis or Diss., Sorbonne université, 2024. http://www.theses.fr/2024SORUS078.
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La thérapie génique est un domaine scientifique crucial qui doit être davantage développé, et la crise du Covid-19 n'a fait que confirmer cette nécessité. Les virus sont connus pour être des vecteurs de matériel génétique très efficaces. Parmi eux, le virus de la mosaïque du tabac (TMV), dont le manteau protéique peut s'auto-assembler en un co-assemblage coopératif avec de l'ARN, a inspiré notre groupe pour concevoir un virus artificiel à base de cyclodextrines (CDs). Notre groupe a synthétisé une CD cationique portant un pont ammonium et sur le côté duquel est fonctionnalisé un groupement adamantyle (CD 1). Il a été démontré que cette dernière est capable de s'auto-assembler en un polymère supramoléculaire. Puis, le groupe a montré que la CD 1 pouvait induire la transfection de siARN.[1] Pour comprendre son mode d'assemblage, nous avons étudié le mélange CD 1 et ADN double brin 18-mère par Cryo-EM. Etonnamment, nous avons observé que de longues et fines fibres se formaient. Nous avons ensuite montré que ces fibres contenaient de nombreuses molécules d'ADN qui sont entourées de CD 1 auto-assemblées, une organisation qui rappelle celle du TMV. Dans ce travail, nous avons aussi montré que des architectures identiques sont observées avec différentes tailles d'ADN simple brin ou double brin. Cependant, nous avons montré qu'avec de l'ADN simple brin, les fibres prenaient plus de temps à se former et nous avons pu suivre leur mécanisme d'assemblage par cryo-EM. De plus, nous avons étudié différents paramètres en termes de pH, de ratios CD/ADN, de tampons et de concentrations des composés, et avons pu optimiser le système et en apprendre plus sur ce dernier. Incroyablement, un léger changement de structure de la CD 1 pour la CD 2 où l'unité adamantyle est maintenant placée au centre de la cavité de la CD entraîne un changement drastique de l'architecture du co-assemblage : des tubes sont obtenus à la place des fibres. Aussi, nous avons découvert que la construction de ces tubes suit un chemin d'assemblage multi-étapes que nous avons suivi et élucidé par des expériences de cinétique en cryo-EM. Nous avons aussi montré que ces tubes pouvaient se former avec de l'ADN simple ou double brin. Afin d'appliquer notre système à la transfection d'ARNm, nous avons ensuite synthétisé une série d'analogues aux CD 1 et 2, en modifiant leurs propriétés de lipophilie et d'auto-assemblage. Nous avons évalué leur capacité à transfecter de l'ARNm in vitro, mais malheureusement aucune efficacité n'a été observée. Pour résoudre ce problème et appliquer notre système à la transfection d'ARNm, nous avons conçu une nouvelle génération de CD s'auto-assemblant et avons prouvé que certaines d'entre elles pouvaient effectivement transfecter de l'ARNm dans des cellules. Nous avons aussi montré que des CDs analogues mais auxquelles nous avons retiré la propriété de s'auto-assembler en oligomères supramoléculaires ne pouvaient pas transfecter l'ARNm. C'est donc la coopérativité entre l'auto-assemblage des CDs par effet hydrophobe et entre les interactions électrostatiques de ces dernières avec l'ARNm qui permet la transfection. Finalement, nous avons commencé la synthèse d'analogues de ce hit afin d'améliorer l'efficacité de transfection et la viabilité cellulaire et avons obtenu des premiers résultats prometteurs. [1] Evenou P., Rossignol J., Pembouong G., Gothland A., Colesnic D., Barbeyron R., Rudiuk S., Marcelin A-G., Ménand M., Baigl D., Calvez V., Bouteiller L., Sollogoub M., Angew. Chem. Int. Ed. 2018, 57, 7753-7758. PCT/EP2016/070892, 5th september 2016, WO/2018/041377, 8th mars 2018Gene therapy is a crucial scientific field we must further develop, and the Covid-19 crisis only confirmed it. Viruses are known to be highly effective genetic material vectors. Among them, the tobacco mosaic virus (TMV) whose coat proteins can self-assemble in a cooperative hierarchical co-assembly with RNA, inspired our group to design a synthetic artificial virus based on cyclodextrins (CDs). Our group synthesized a cationic adamantyl-functionalized CD (CD 1) where the adamantyl is borne by an ammonium bridge and demonstrated its ability to self-assemble into a supramolecular polymer. They we showed that CD 1 could induce transfection of siRNA.[1] To understand its mode of assembly, we studied the CD 1/DNA mixture by Cryo-EM. To our surprise and delight, we found that very long thin fibers were formed. We further proved that they contain many copies of double stranded DNA 18-mer, which are surrounded by self-assembled CD 1, a structure highly reminiscent of TMV. In this work, we also proved that identical architectures are observed with different sizes of single and double stranded DNA. However, we proved that with single stranded DNA, the fibers take longer to build, and we discovered and followed its mechanism of assembly by cryo-EM. Moreover, we studied different parameters in terms of pH, CD/DNA ratios, buffers, concentrations and we have optimized our system and learnt more about the behavior of our assemblies. Amazingly, a slight change of structure of CD 1 into CD 2, where the adamantyl group is positioned in the center of the cavity induces a drastic modification of the co-assembly architecture: tubes were obtained instead of fibers. Besides, we found out that the construction of those tubes follows a multi-steps pathway that have been elucidated by cryo-EM kinetics experiments. We also showed that same tubes can be formed with ssDNA or dsDNA and with different size of DNAs. We then developed a series of analogues of CD 1 and CD 2, tuning their lipophilicity and self-assembling properties. We evaluated the capacity of CD 1 and CD 2 and their analogues to transfect mRNA in vitro. Unfortunately, no transfection was observed. Because of the lack of mRNA transfection efficiency with these CDs, we designed a new generation of self-assembling CDs and proved that some of them, indeed, transfect mRNA into cells. We also proved that CDs which cannot self-assemble are not able to deliver mRNA and that we need cooperativity between the CDs' hydrophobic interactions, allowing the supramolecular oligomers formation, and the electrostatic interactions between the CDs and the mRNA to transfect. Finally, we started to synthetize analogues of this hit to improve transfection efficiency and cell viability and got promising results. [1] Evenou P., Rossignol J., Pembouong G., Gothland A., Colesnic D., Barbeyron R., Rudiuk S., Marcelin A-G., Ménand M., Baigl D., Calvez V., Bouteiller L., Sollogoub M., Angew. Chem. Int. Ed. 2018, 57, 7753-7758. PCT/EP2016/070892, 5th september 2016, WO/2018/041377, 8th mars 2018
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Fiff, Fabian. "Transfection of baboon dendritic cells with plasmid DNA containing HIV-1C genes : effect of transfection methods on antigen processing and presentation to T lymphocytes." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1022.
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Escoffre, Jean-Michel. "Mécanismes moléculaires et cellulaire de l'électrotransfert de plasmides in vitro." Toulouse 3, 2010. http://thesesups.ups-tlse.fr/1296/.
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Le transfert de plasmides au sein d'une cellule cible représente un outil clé dans l'étude de fonctions biologiques et dans le développement de nouvelles approches thérapeutiques. Cependant, le transfert de plasmides doit être réalisé avec un minimum d'effets secondaires au niveau de la cellule cible. La technique d'électroperméabilisation est une méthode physique fondée sur la modulation du potentiel électrique transmembranaire natif de la cellule par un champ électrique externe. Cependant, l'utilisation rationnelle de l'électroperméabilisation en pharmacologie et en médecine ne pourra se faire que grâce à une parfaite compréhension des mécanismes impliqués lors de l'électroperméabilisation au niveau membranaire et de ses conséquences cellulaires. Le mécanisme de l'électrotransfert de plasmides est un processus multi-étapes avec une étape d'interaction plasmide/membrane perméabilisée pendant l'application des impulsions électriques, suivi après ces dernières, d'une étape de translocation du plasmide à travers la membrane plasmique. Ce travail de recherche pluridisciplinaire vise à une meilleure compréhension du mécanisme de l'électrotransfert de plasmides. Il intègre l'étude des conséquences membranaires de l'électrotransfert de plasmide et l'étude de l'interaction plasmide/membrane perméabilisée. L'application d'impulsions électriques millisecondes et perméabilisantes induit un désordre membranaire et une rapide translocation des phospholipides dans les régions perméabilisées. La translocation électro-induite des phospholipides n'est pas associée à une perte de la viabilité cellulaire. L'existence du processus multi-étapes de l'électrotransfert de plasmide (perméabilisation membranaire, interaction plasmide/membrane et expression génique) a été confirmé dans différentes lignées cellulaires. Pendant l'application de la première impulsion électrique, les molécules de plasmide migrent par électrophorèse et viennent interagir dans des sites distincts de la région membranaire faisant face à la cathode. L'interaction des molécules de plasmide avec la membrane perméabilisée serait donc un processus rapide (de l'ordre d'une centaine de microsecondes). Les complexes plasmide/membrane se stabilisent en un délai de 200 ms. Un rôle distinct de l'intensité du champ électrique et du nombre d'impulsions électriques a été mis en évidence. Si l'intensité du champ électrique définit la surface membranaire où l'interaction des molécules de plasmide a lieu et de fait, le nombre de spots d'interaction, le nombre d'impulsions électriques définit la quantité de molécules de plasmide présente par complexe. Les complexes plasmide/membrane ainsi formés ne diffusent pas latéralement dans la membrane. Le cytosquelette d'actine n'est pas impliqué dans la formation de ces complexes mais pourrait être impliqué dans le transport intracellulaire des molécules de plasmides. L'électroperméabilisation et l'interaction plasmide/membrane perturbent la mobilité latérale des protéines membranaire du feuillet externe de la membrane plasmique. La combinaison de la sonoporation avec l'électroperméabilisation permet d'améliorer l'efficacité de transfection obtenue par l'électroperméabilisation seule. Le transfert de plasmides par électro-sonoporation est une stratégie prometteuse en thérapie géniquePlasmid transfer within a target cell represents a key tool in the study of biological functions and the development of new therapeutic strategies. However, the transfer of plasmids must be carried out with a minimum of side effects on the level of the target cell. The technique of electropermeabilization is a physical method based on the modulation of the native transmembrane electric potential of the cell by an external electric field. However, the rational use of the electropermeabilization in pharmacology and medicine could be done only thanks to one perfect comprehension of the mechanisms involved in the electropermeabilization at the membrane level and its cellular consequences. The mechanism of the plasmid electrotransfer is a multi-steps process with a step of plasmid/membrane interaction during the application of the electric pulses, followed after these last, of a step of plasmid translocation through the plasma membrane. This multi-disciplinary research task aims at a better comprehension of the mechanism of the plasmid electrotransfer. It integrates the study of the membrane consequences of the electropermeabilization and the study of the plasmid/ membrane interaction. The application of milliseconds and permeabilizing electric pulses induces a membrane disorder and a fast phospholipid translocation in the permeabilized regions. The electro-induced translocation of phospholipids is not associated with a loss of cell viability. The existence of the multi-steps process of the plasmid electrotransfer (membrane permeabilization, plasmid/membrane interaction and gene expression) was confirmed in various cell lines. During the application of the first electric pulse, the plasmids migrate by electrophoresis and come to interact in distinct sites from the membrane region facing the cathode. The interaction of plasmids with the permeabilized membrane would be thus a fast process (about a hundred microseconds). The plasmid/membrane complexes are stabilized in a delay of 200 ms. A distinct role from the intensity of the electric field and the number of electric pulses was highlighted. If the intensity of the electric field defines membrane surface where the interaction of the plasmid molecules takes place and in fact, the number of spots of interaction, the number of electric pulses defines the amount of plasmids presents by complex. The plasmid/membrane complexes thus formed do not diffuse laterally in the membrane. The actine cytoskeleton is not involved in the formation of these complexes but could be involved in the intracellular traffic of plasmids. Electropermeabilization and plasmid/membrane interaction disturbed the lateral mobility of membrane proteins of outer leaflet of plasma membrane. The combination of the sonoporation with the electropermeabilization makes it possible to improve the effectiveness of transfection obtained by the electropermeabilization alone. The transfer of plasmids by electro-sonoporation is a promising strategy in gene therapy
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Balcells, García Laura. "Multi-faced study for the development of enhanced transfection systems." Doctoral thesis, Universitat Ramon Llull, 2020. http://hdl.handle.net/10803/669861.
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L'actual transferència de coneixements des de l'escala de laboratori d'investigació als productes farmacèutics i les teràpies humanes és limitada, entre altres raons, a causa de la percepció de les cèl·lules com caixes negres, en què hi ha entrades i sortides però es presta poca atenció als processos cel·lulars interns que regeixen els resultats generals. Aquesta tesi doctoral proposa un mètode per estudiar els mecanismes cel·lulars que intervenen en la internalització NPs i la transfecció de cèl·lules, que sovint passen desapercebuts. És a dir, mitjançant el desenvolupament d'una nova classe de nanopartícules multicomponents derivades d'un sistema polimèric de gene delivery de bon rendiment, es projecta aquí una inspecció des de l'interior de les cèl·lules que permetrà no només comprendre millor els resultats dels experiments cel·lulars, sinó també establir les bases d'una plataforma de teràpia cel·lular innovadora i millorada.
En primer lloc, s'estudiaran els mecanismes intracel·lulars d'autofàgia i producció exosomes utilitzant poliplexes OM-pBAE en combinació amb nanopartícules metàl·liques. S'investigarà el paper del component polimèric i metàl·lic de sistema en aquests dos mecanismes cel·lulars que es indueixen amb la transfecció cel·lular. També es supervisarà l'avaluació de la ubicació intracel·lular dels complexos. A continuació, s'analitzaran les interaccions d'aquests sistemes de gene delivery amb les proteïnes presents en el medi biològic, ja que tenen un gran impacte en la internalització cel·lular. I, finalment, amb el coneixement tant dels mecanismes cel·lulars subjacents a la transfecció cel·lular com de l'efecte de la corona proteica en els perfils d'internalització cel·lular de les diferents nanopartícules, es proposarà l'aplicació dels nostres sistemes de gene delivery a un propòsit més desafiant: la modificació de les cèl·lules mare mesenquimals per al seu posterior ús com a estratègies de teràpia cel·lular.
En conclusió, en aquesta tesi s'han emprat nanopartícules multicomponents per realitzar un estudi exhaustiu dels mecanismes cel·lulars induïts en la transfecció cel·lular. S'han utilitzat sistemes de lliurament de gens compostos de poliplexes fets d'OM-pBAE i pDNA i un component metàl·lic que són nanopartícules d'or o SPIONs per obrir les cèl·lules i analitzar els processos intra i intercel·lulars que dicten el destí dels vectors internalitzats - la autofàgia i la producció de exosomes -, estudiar la seva interacció amb les proteïnes i demostrar que aquest fenomen és clau per a la seva absorció cel·lular i, finalment, aplicar el coneixement de les seves propietats i capacitats per modificar genèticament les cèl·lules reticents a la transfecció que s'utilitzen per a les estratègies de teràpia cel·lular.La actual transferencia de conocimientos desde la escala de laboratorio de investigación a los productos farmacéuticos y las terapias humanas es limitada, entre otras razones, debido a la percepción de las células como cajas negras, en las que hay entradas y salidas pero se presta poca atención a los procesos celulares internos que rigen los resultados generales. Esta tesis doctoral propone un método para estudiar los mecanismos celulares que intervienen en la internalización de NPs y la transfección de células, que a menudo pasan desapercibidos. Es decir, mediante el desarrollo de una nueva clase de nanopartículas multicomponentes derivadas de un sistema polimérico de gene delivery de buen rendimiento, se proyecta aquí una inspección desde el interior de las células que permitirá no sólo comprender mejor los resultados de los experimentos celulares, sino también sentar las bases de una plataforma de terapia celular innovadora y mejorada. En primer lugar, se estudiarán los mecanismos intracelulares de autofagia y producción exosomas utilizando poliplejos OM-pBAE en combinación con nanopartículas metálicas. Se investigará el papel del componente polimérico y metálico del sistema en estos dos mecanismos celulares que se inducen con la transfección celular. También se supervisará la evaluación de la ubicación intracelular de los complejos. A continuación, se analizarán las interacciones de estos sistemas de gene delivery con las proteínas presentes en el medio biológico, ya que tienen un gran impacto en la internalización celular. Y, por último, con el conocimiento tanto de los mecanismos celulares que subyacen a la transfección celular como del efecto de la corona proteínica en los perfiles de internalización celular de las diferentes nanopartículas, se propondrá la aplicación de nuestros sistemas de gene delivery a un propósito más desafiante: la modificación de las células madre mesenquimales para su posterior uso como estrategias de terapia celular. En conclusión, en esta tesis se han empleado nanopartículas multicomponentes para realizar un estudio exhaustivo de los mecanismos celulares inducidos en la transfección celular. Se han utilizado sistemas de entrega de genes compuestos de poliplejos hechos de OM-pBAE y pDNA y un componente metálico que son nanopartículas de oro o SPIONs para abrir las células y analizar los procesos intra e intercelulares que dictan el destino de los vectores internalizados - la autofagia y la producción de exosomas -, estudiar su interacción con las proteínas y demostrar que este fenómeno es clave para su absorción celular y, por último, aplicar el conocimiento de sus propiedades y capacidades para modificar genéticamente las células reacias a la transfección que se utilizan para las estrategias de terapia celular.
The current transfer of knowledge from a research laboratory scale to pharmaceutical products and human therapies is limited, among other reasons, due to the perception of cells as black boxes, where there are inputs and outputs but little attention is devoted to the inner cell processes governing the overall results. This doctoral thesis proposes a method to study the cellular mechanisms involved in cell uptake and transfection that often remain unnoticed. That is, through the development of a new class of multicomponent nanoparticles derived from a well-performing polymeric type gene delivery system, it is here projected an inspection from inside the cells that will allow not only a better understanding of cell experiment results, but also setting the basis for an innovative and improved cell therapy platform. First, the intracellular mechanisms of autophagy and exosomes production will be studied using OM-pBAE polyplexes in combination with metallic nanoparticles. The role of both the polymeric and the metallic component of the system will be investigated in these two cell mechanisms that are induced upon cell transfection. Also, the intracellular location assessment of the complexes will be monitored. Then, the interactions of these gene delivery systems with the proteins present in any given biological medium will be analyzed, since they have a great impact in cellular internalization. And finally, with the knowledge on both the cellular mechanisms behind cell transfection and the effect of protein corona on the cell uptake profiles of different nanoparticles, it will be proposed the application of our gene delivery systems to a more challenging purpose: the modification of mesenchymal stem cells for further use as cell therapy strategies. In conclusion, in this thesis, multicomponent nanoparticles have been employed to perform a thorough study of the cellular mechanisms induced upon cell transfection. Gene delivery systems composed of polyplexes made of OM-pBAEs and pDNA and a metallic component being either gold nanoparticles or SPIONs have been used to unbox cells and analyze the intra and intercellular processes that dictate the fate of internalized vectors – namely autophagy and exosomes production –, study their interaction with proteins and demonstrate that this phenomenon is key for their cellular uptake, and finally apply the knowledge of their properties and abilities to genetically modify reluctant to transfection cells that are used for cell therapy strategies.
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Cartwright, Joseph. "CHO cell genetic instability : from transfection to stable cell line." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/13986/.
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Chinese hamster ovary (CHO) cells are the predominant host cell type used in the production of recombinant therapeutic proteins. They are chosen as hosts, because of their ability to create, fold and modify proteins in a manner that makes them compatible with the human immune system. Moreover, CHO cells are tried and tested model organisms for bioprocess platforms, meaning regulatory body approval for new therapeutics is relatively easy to achieve. CHO cells are inherently genetically unstable, which can lead to a decline in productivity and poses a threat to product quality heterogeneity of stable cell lines. The primary aim of this thesis was to characterise genomic instability of a CHOK1SV cell line and measure directly the impact this genetic instability has on the fidelity of recombinant plasmid copies. The impact of this would be two-fold: Firstly, an accurate quantification of genetic instability type and frequency would be established. Secondly, the techniques used to characterise genetic instability would be evaluated as tools for the detection of instability in cell line development processes. Microsatellite analysis and karyotype analysis were used to assess CHO cell genomic instability at the base pair / gene copy number (GCN) level and the chromosome level respectively. Microsatellites were found to be effective markers for genetic drift and cell line relatedness. However, there was no substantial evidence of microsatellite mutational change, and so it could not be concluded that microsatellites are an effective marker for deficient DNA replication / DNA damage or mismatch repair. Microsatellite change did not correlate with changes in GCN or cell specific productivity (qP). There was substantial evidence of chromosomal aberration from Karyotype analysis, which showed considerable levels of aneuploidy and chromosome breakage/fusion events. It was concluded that CHO cells have an inherent chromosomal instability and that karyotyping is a promising tool for genetic instability cell line development assessments. However, there was no substantial association found between changes in CHO karyotype and changes in qP or GCN. In order to generate a stable GFP cell line for the investigation of recombinant plasmid genetic instability it was necessary to optimise an electroporation protocol. Preliminary experiments indicated that standard industry conditions were suboptimal and so a Design of Experiments (DoE) – based strategy was used to optimise electroporation. Final optimal conditions (termed 320-26) improved transfection efficiency by 17%. The final results chapter outlines a novel single-molecule real time (SMRT) sequencing analysis platform, which maximises the sensitivity of the technology, enabling mutation calling from individual molecules at a 0.01% frequency. One mutation was present at high levels throughout the study, a C T transition in the bacterial origin of replication, which is assumed to have originated from the original plasmid stock. There was no evidence of mutations arising in plasmid cloning or as a result of the pre-integration CHO cell environment. Substantial levels of point mutation were found in recombinant plasmid copies. Mutations were randomly distributed along the length of the plasmid and were apparently not influenced by natural selection. G and C residues were mutated to a greater extent than A and T residues, with G.C A.T transitions predominating. This final assessment of CHO cell genetic instability shows the requirement for product quality checks during cell line development.
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Aravindan, Latha. "Engineering polyethylenimine- Based gene delivery vectors with improved transfection efficiency." Thesis, University of Reading, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533763.
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Hufnagel, Hansjörg. "Overcoming barriers in non-viral gene transfection by PEI-25." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608478.
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Shuang, Liang. "New Approach To DNA Transfection And Genetics In Schistosome Parasites." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1424201510.
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François, Marion. "Mécanismes de transfection par la polyethylenimine : influence de la PEGylation." Université Louis Pasteur (Strasbourg) (1971-2008), 2007. http://www.theses.fr/2007STR13252.
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Le concept de thérapie génique, défini par l'utilisation d'acides nucléiques en tant que médicament, a émergé il y a une trentaine d'années. C'est un concept très attractif, puisque contrairement aux traitements palliatifs, elle permettrait la guérison de pathologies génétiques héréditaires, d'affections acquises, de maladies dégénérescentes ou encore d'infections virales. La mise en oeuvre de la thérapie génique nécessite une vectorisation de l'ADN afin de transporter les acides nucléiques dans le noyau des cellules en utilisant des systèmes de délivrances (vecteurs) d'origine naturel ou synthétique. Si les vecteurs viraux sont efficaces, ils sont délicats à produire et ont montré des propriétés immunogènes et oncogéniques. Les systèmes de délivrance synthétique actuels sont par contre beaucoup plus faciles à produire et offrent des perspectives de modification. Parmi ces vecteurs non-viraux, la polyéthylènimine linéaire de 22 KDa (L-PEI) de masse molaire a été la plus étudiée in vivo en raison de sa grande efficacité (pour un vecteur de synthèse. . . ). Dans ce travail de thèse, nous avons entrepris de recouvrir les polyplexes avec des éléments de protection pour augmenter la résilience des particules in vivo. Pour cela, nous avons d'abord modifié chimiquement de la L-PEI par du N-Succinimidyl-3-(2- pyridyldithio) propionate pour former un polymère fonctionnalisé, le PEI-POP. Nous avons ensuite formé des complexes PEI-POP/ADN de petites tailles et nous les avons recouverts de molécules de PEG par formation de pont disulfure. Nous avons abouti à un protocole fiable et reproductible et obtenu des polyplexes marqués par des fluorophores. Finalement, nous avons étudié, la visualisation de nos polyplexes en cellules vivantes par microscopie confocale, le suivi et le tri par FACS des différentes populations de cellules ainsi que le devenir de l'ADN dans la cellule. Toutes ces études ont été faites en parallèle sur des polyplexes PEGylés et non PEGylés. Cela nous a permis de constater que la transfection est maximale 24 à 48h après application des complexes mais que le phénomène persiste jusqu'à 8 jours. Aussi, des cellules non transfectées peuvent devenir transfectées jusqu'à plus de 4 jours post transfection ou encore que certaines cellules passent du statut de transfectées à celui de non transfectées pour redevenir transfectéesConcept of gene therapy emerged 3 0 years ago and is defi ned as the use of n ucleic acids as drugs and. This concept is very attractive since it should cure hereditary genetic pathologies, acquired or degenerative diseases, viral infections by contrast with palliative cares. Vectorisation allows DNA to be compacted into small particles which can penetrate cells and reach their nucleus. Besides viral vectors, which can cause immunogenic and oncogenic problems, non-viral vectors offers safer use and other advantages. They are easier to produce, they can be chemically modified and they can incorporate larger nucleic acids. Among known non-viral vectors, we have worked with linear polyethyleneimine 22K which seems to be both the most powerful synthetic vector and the one which exhibits the best in vivo results. During this PhD work, we first chemically modified this L-PEI with SPDP (N-succinimidyl-3-(2- pyridyldithio)propionate). Such modification allowed to bind PEG molecules containing a terminal thiol moiety after the polyplex formation. We chose a postPegylation pathway to prevent DNA condensation problems which occur with prePEGylation. We purified our polyplexes at every stage to get rid off excess PEG and released thiopyridone. We set up a reliable and reproducible protocol which also allowed us to label polyplexes with fluorophores. Such fluorophores allowed visualization of the polyplexes inside living cells with confocal microscopy, FACS sorting of different cell strains and observation of DNA fate within cells. That study was carried out both with PEGylated and nonPEGylated polyplexes. We observed that transfection is a long-lived phenomenon (up to 8 days posttransfection), that cells can become transfected up to 4 days after transfection and that some transfected cells became non-transfected and transfected back
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Leonhardt, Carolin Anke [Verfasser], and Joachim [Akademischer Betreuer] Rädler. "On quantitative mRNA transfection / Carolin Anke Leonhardt ; Betreuer: Joachim Rädler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/111514491X/34.
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Venkatesh, Savitha. "Chitosan-mediated transfection and the role of cell surface interactions." Scholarly Commons, 1997. https://scholarlycommons.pacific.edu/uop_etds/2320.
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Transfection is the process of introducing DNA into cells and expression of the gene contained in the DNA. The DNA itself could be a functional gene, part of a gene, or a gene with the regulatory and transcribed sequences intact. The ability to transfect mammalian cells is a powerful tool that can be used to study the function and control of mammalian genes. Transfection of DNA into eukaryotic cells can be done in several ways. One of the methods of introducing foreign DNA into mammalian cells is known as cationic liposome-mediated transfection. Preliminary studies involved the development of a cationic lipid-mediated transfection method using HeLa cells and a plasmid that codes for ~-galactosidase. The cationic lipid used was Transfectam. Transfectam is a commercially available, cationic lipopolyamine. Chitosan, a cationic polymer of glucosamine, was used as an alternative transfecting agent and as a binding agent for polyanions. The transfection efficiency of chitosan was compared to that of Transfectam. Chitosan was found to be comparable to Transfectam in this regard. Polyanions of different chemical structures were used in a chitosan binding study. These include aurin tricarboxylic acid, calf thymus DNA and methyl green DNA. The kinetics of the binding interactions suggest that ionic interactions predominated. Based upon these findings, studies were performed to determine the nature of chitosan interactions with the cell surface. Chitosan beads were prepared to determine the interaction of chitosan with the cell membrane of He La cells and for the isolation of membrane proteins. Membrane proteins which bound to chitosan beads could be effectively eluted with a-0-mannopyranoside but not sodium chloride. Furthermore, HeLa cells bound to the chitosan beads could be eluted with aD- mannopyranoside but not sodium chloride. The results suggested that membrane bound proteins interact with chitosan through carbohydrate moiety interactions which may facilitate the transfection process.
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Leonhardt, Carolin Anke Verfasser], and Joachim [Akademischer Betreuer] [Rädler. "On quantitative mRNA transfection / Carolin Anke Leonhardt ; Betreuer: Joachim Rädler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/111514491X/34.
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Sabraoui, Abbas. "Régulation de la cavitation acoustique appliquée à la transfection cellulaire." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10007.
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Le travail présenté ici porte sur l’étude et le contrôle de la cavitation acoustique dans le but de développer un système de sonoporation efficace pour les cellules en suspension et les cellules adhérentes. Le manuscrit est composé de trois chapitres. Tout d’abord, une revue de la littérature sur les différentes techniques physiques utilisées en transfection cellulaire, et plus particulièrement la sonoporation. Il a été démontré que le principal mécanisme de la sonoporation est étroitement lié au phénomène de cavitation acoustique. Un contrôle de ce phénomène aléatoire apparaît alors intéressant afin d’augmenter le taux de transfection tout en gardant une forte viabilité cellulaire. Dans le second chapitre, un système de régulation de cavitation ultrasonore basé sur un indice acoustique de cavitation a été étudié. Cet indice, est basé sur la mesure de bruit large bande émis lors de l’implosion des bulles de cavitation. Les avantages d’un tel système sont : un suivi en temps réel du niveau de cavitation durant l’irradiation, des informations quasi-instantanées sur les composantes spectrales caractéristiques de la cavitation, une meilleure reproductibilité et stabilité du niveau de cavitation surtout pour les intensités modérés. Dans le troisième chapitre, pour comprendre les mécanismes de la sonoporation, un deuxième système de cavitation contrôlé a été conçu dans le but de permettre une visualisation du milieu en cours d’insonification. Ce nouveau dispositif est adapté à un fonctionnement sous microscope photonique à transmission et à fluorescence. Des essais de transfection de siRNAs, sur les cellules en suspension (RL du lymphome folliculaire) et sur les cellules adhérentes (cancer du sein ; MDA-MB 231) ont permis de valider in vitro l’efficacité de ce système en atteignant un taux de 40 % de transfection pour ces deux types de cellules, avec un très faible taux de mortalité (< 10 %)The aim of the present work, which is based on the study and the control of acoustic cavitation, is to develop an efficient sonoporation system to transfect the cells in suspension and the adherent cells. The manuscript is composed of three chapters. The first one takes a glance on the state of art of different physical techniques used in cells transfection, and more precisely on sonoporation. It has been shown that the principal mechanism of sonoporation is closely linked to acoustic cavitation. Thus, a control of this random phenomenon is important to increase the rate of transfection while keeping strong cell viability. In the second chapter, a regulated cavitation generator based on an acoustic index was studied. This index is based on the measure of broad band noise emitted during the implosion of the cavitation bubbles. The advantage of such a system is: a control in real time of the level cavitation during sonication, leading to a better reproducibility and stability of the cavitation level, especially for the moderate intensities. In the third chapter, in order further study the sonoporation mechanisms, a second regulated cavitation generator was studied; its aim is to be able to visualize the medium during sonication. This new device is adapted to the performance under a fluorescencemicroscope with fluorescence transmission. SiRNAs transfection, was validated in vitro by attending a rate of 40 % of transfection for the two types of cells, with a very low rate of mortality (< 10%), for both suspended cells (RL of follicular lymphoma) and adherent cells (Cancer of breast; MDA-MB 231)
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Sabraoui, Abbas. "Régulation de la cavitation acoustique appliquée à la transfection cellulaire." Electronic Thesis or Diss., Lyon 1, 2012. http://www.theses.fr/2012LYO10007.
Full textAbstract:
Le travail présenté ici porte sur l’étude et le contrôle de la cavitation acoustique dans le but de développer un système de sonoporation efficace pour les cellules en suspension et les cellules adhérentes. Le manuscrit est composé de trois chapitres. Tout d’abord, une revue de la littérature sur les différentes techniques physiques utilisées en transfection cellulaire, et plus particulièrement la sonoporation. Il a été démontré que le principal mécanisme de la sonoporation est étroitement lié au phénomène de cavitation acoustique. Un contrôle de ce phénomène aléatoire apparaît alors intéressant afin d’augmenter le taux de transfection tout en gardant une forte viabilité cellulaire. Dans le second chapitre, un système de régulation de cavitation ultrasonore basé sur un indice acoustique de cavitation a été étudié. Cet indice, est basé sur la mesure de bruit large bande émis lors de l’implosion des bulles de cavitation. Les avantages d’un tel système sont : un suivi en temps réel du niveau de cavitation durant l’irradiation, des informations quasi-instantanées sur les composantes spectrales caractéristiques de la cavitation, une meilleure reproductibilité et stabilité du niveau de cavitation surtout pour les intensités modérés. Dans le troisième chapitre, pour comprendre les mécanismes de la sonoporation, un deuxième système de cavitation contrôlé a été conçu dans le but de permettre une visualisation du milieu en cours d’insonification. Ce nouveau dispositif est adapté à un fonctionnement sous microscope photonique à transmission et à fluorescence. Des essais de transfection de siRNAs, sur les cellules en suspension (RL du lymphome folliculaire) et sur les cellules adhérentes (cancer du sein ; MDA-MB 231) ont permis de valider in vitro l’efficacité de ce système en atteignant un taux de 40 % de transfection pour ces deux types de cellules, avec un très faible taux de mortalité (< 10 %)The aim of the present work, which is based on the study and the control of acoustic cavitation, is to develop an efficient sonoporation system to transfect the cells in suspension and the adherent cells. The manuscript is composed of three chapters. The first one takes a glance on the state of art of different physical techniques used in cells transfection, and more precisely on sonoporation. It has been shown that the principal mechanism of sonoporation is closely linked to acoustic cavitation. Thus, a control of this random phenomenon is important to increase the rate of transfection while keeping strong cell viability. In the second chapter, a regulated cavitation generator based on an acoustic index was studied. This index is based on the measure of broad band noise emitted during the implosion of the cavitation bubbles. The advantage of such a system is: a control in real time of the level cavitation during sonication, leading to a better reproducibility and stability of the cavitation level, especially for the moderate intensities. In the third chapter, in order further study the sonoporation mechanisms, a second regulated cavitation generator was studied; its aim is to be able to visualize the medium during sonication. This new device is adapted to the performance under a fluorescencemicroscope with fluorescence transmission. SiRNAs transfection, was validated in vitro by attending a rate of 40 % of transfection for the two types of cells, with a very low rate of mortality (< 10%), for both suspended cells (RL of follicular lymphoma) and adherent cells (Cancer of breast; MDA-MB 231)
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Dréan, Mathilde. "Controlled synthesis of polyvinylamine-based (co)polymers for gene transfection." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066444.
Full textAbstract:
Le transfert de gènes consiste en l’introduction d’acides nucléiques au sein de cellules afin de modifier leur activité dans un but essentiellement thérapeutique. Pour préserver le matériel génétique de toute dégradation, il faut recourir à des vecteurs. Parmi ceux-ci, les polymères cationiques sont très prometteurs, en particulier, la polyéthylènimine, considérée comme le vecteur non-viral de référence. Néanmoins, il présente une cytotoxicité élevée. Ainsi, de nombreuses recherches ont pour but d’identifier et de développer de nouveaux polymères combinant efficacité de transfection et haute viabilité cellulaire. Cette thèse vise le développement de méthodes d’ingénierie macromoléculaire donnant accès à une large gamme de dérivés à base de polyvinylamine et l’évaluation de leurs performances en tant que vecteurs de transfection. Différentes techniques de polymérisation radicalaire conventionnelle et contrôlée ont été mises au point afin de synthétiser des (co)polymères à base de polyvinylamine constitués d’amines primaires et secondaires. L’efficacité du transfert d’ADN plasmidique et la viabilité cellulaire ont été évaluées sur des cellules HeLa. L’influence de différents paramètres macromoléculaires sur les performances de transfection a été investiguée. Cette étude a permis de démontrer que certains dérivés de polyvinylamine possédaient une efficacité de transfection aussi élevée que la PEI tout en étant moins toxique. De manière générale, ce travail rend compte du haut potentiel des (co)polyvinylamines en tant que vecteurs pour le transfert de gènesGene transfection consists in the introduction of genetic materials (DNA or RNA) in cells in order to modulate the cell activity, with therapeutic purposes in most cases. To deliver the genetic materials into cells without degradation, vectors are necessary. Among them, cationic polymers are promising candidates. For instance, polyethylenimine has emerged as a gold standard due to its high transfection ability. Nevertheless, this polymer exhibits high cytotoxicity, and current research aims at identifying and developing new polymers with improved cell viability and high gene transfer efficiency. In this context, the aim of this thesis was to develop efficient macromolecular engineering tools to prepare a library of polyvinylamine-containing (co)polymers and to evaluate their performances as DNA carriers. Consequently, free radical polymerization (FRP) and controlled radical polymerization (CRP) have been explored and a series of (co)polyvinylamines, containing primary and secondary amines, as well as vinylimidazole and guanidine moieties, have been synthesized. The transfection efficiency of plasmid DNA (pDNA) and cell viability were evaluated on HeLa cells. The influence of different macromolecular parameters such as molar mass, molar mass distribution and composition, was also studied. The most promising polymers for pDNA transfection were also tested for siRNA delivery and on other cell lines. Overall, several polymers were competitive with PEI regarding the transfection efficiency but were much less toxic. (Co)polyvinylamines, which have often been disregarded for transfection purposes, should definitely be considered as valuable gene carriers
39
Dréan, Mathilde. "Controlled synthesis of polyvinylamine-based (co)polymers for gene transfection." Electronic Thesis or Diss., Paris 6, 2016. http://www.theses.fr/2016PA066444.
Full textAbstract:
Le transfert de gènes consiste en l’introduction d’acides nucléiques au sein de cellules afin de modifier leur activité dans un but essentiellement thérapeutique. Pour préserver le matériel génétique de toute dégradation, il faut recourir à des vecteurs. Parmi ceux-ci, les polymères cationiques sont très prometteurs, en particulier, la polyéthylènimine, considérée comme le vecteur non-viral de référence. Néanmoins, il présente une cytotoxicité élevée. Ainsi, de nombreuses recherches ont pour but d’identifier et de développer de nouveaux polymères combinant efficacité de transfection et haute viabilité cellulaire. Cette thèse vise le développement de méthodes d’ingénierie macromoléculaire donnant accès à une large gamme de dérivés à base de polyvinylamine et l’évaluation de leurs performances en tant que vecteurs de transfection. Différentes techniques de polymérisation radicalaire conventionnelle et contrôlée ont été mises au point afin de synthétiser des (co)polymères à base de polyvinylamine constitués d’amines primaires et secondaires. L’efficacité du transfert d’ADN plasmidique et la viabilité cellulaire ont été évaluées sur des cellules HeLa. L’influence de différents paramètres macromoléculaires sur les performances de transfection a été investiguée. Cette étude a permis de démontrer que certains dérivés de polyvinylamine possédaient une efficacité de transfection aussi élevée que la PEI tout en étant moins toxique. De manière générale, ce travail rend compte du haut potentiel des (co)polyvinylamines en tant que vecteurs pour le transfert de gènesGene transfection consists in the introduction of genetic materials (DNA or RNA) in cells in order to modulate the cell activity, with therapeutic purposes in most cases. To deliver the genetic materials into cells without degradation, vectors are necessary. Among them, cationic polymers are promising candidates. For instance, polyethylenimine has emerged as a gold standard due to its high transfection ability. Nevertheless, this polymer exhibits high cytotoxicity, and current research aims at identifying and developing new polymers with improved cell viability and high gene transfer efficiency. In this context, the aim of this thesis was to develop efficient macromolecular engineering tools to prepare a library of polyvinylamine-containing (co)polymers and to evaluate their performances as DNA carriers. Consequently, free radical polymerization (FRP) and controlled radical polymerization (CRP) have been explored and a series of (co)polyvinylamines, containing primary and secondary amines, as well as vinylimidazole and guanidine moieties, have been synthesized. The transfection efficiency of plasmid DNA (pDNA) and cell viability were evaluated on HeLa cells. The influence of different macromolecular parameters such as molar mass, molar mass distribution and composition, was also studied. The most promising polymers for pDNA transfection were also tested for siRNA delivery and on other cell lines. Overall, several polymers were competitive with PEI regarding the transfection efficiency but were much less toxic. (Co)polyvinylamines, which have often been disregarded for transfection purposes, should definitely be considered as valuable gene carriers
40
Wang, Weiguang. "The study of the oncogenic effect of PAC3, PAX3-FKHR and IGF-II genes in rhabdomyosarcoma and medulloblastoma." Thesis, Manchester Metropolitan University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243719.
Full text
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Denoyelle, Séverine. "Synthèses et études physico-chimiques de bolaformes hémifluocarbonés asymétriques : applications à la transfection de gènes." Avignon, 2005. http://www.theses.fr/2005AVIG0224.
Full textAbstract:
Les travaux de recherche portent sur l'élaboration d'une nouvelle famille de composés bolaformes cationiques hémifluorocarbonés asymétriques et l'évaluation de leur aptitude à transfecter l'ADN. Les études physicochimiques réalisées par les techniques de diffusion de la lumière quasi élastique, de zêtamétrie, de microscopie électronique à transmission et d'électrophorèse sur gel d'agarose, ainsi que les études biologiques, ont permis de mieux comprendre la relation entre l'organisation supramoléculaire de ces bolaformes dans l'eau et leur capacité à complexer l'ADN et transfecter les cellules. La modulation de la structure du bolaforme le plus performant a permis de définir la relation structure-activité du vecteur. Enfin, nous avons élaboré un complexe bolaforme/ADN polyfonctionnalisé porteur de différents groupements fonctionnels susceptibles d'améliorer l'efficacité de transfection de ces vecteurs en milieu biologiqueThe subject of the research is the elaboration of a new class of perfluorocarbonated cationic bolaforms and the assessment of their ability to transfect DNA. The physico-chemical analysis, carried out with the techniques such as quasi elastic light scattering, zetametry, transmission electron microscopy and agarose gel electrophoresis, and the biological studies allowed to understand better the relationship between the supramolecular organisation in water of these bolaforms and their ability to complex DNA and transfect cells. The modulation of the structure of the most efficient bolaform allowed us to define the structure-activity relationship of the vector. Last, we elaborated a bolaform/DNA complex polyfunctionalised bearing different functional groups susceptible of improving the transfection efficiency of these vectors in biological environment
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Hlawaty, Hanna. "Effets de siRNA anti MMP-2 et d'inhibiteurs de récepteurs aux leucotriènes sur l'hyperplasie intimale post-angioplastie chez le lapin hypercholestérolémique." Paris 13, 2007. http://www.theses.fr/2007PA132024.
Full textAbstract:
Le premier chapitre porte sur une stratégie de thérapie génique basée sur le siRNA L’application de siRNA en présence des lipides est une solution prometteuse aux problèmes posés par les méthodes virales. Cependant il est nécessaire d’augmenter l’efficacité de transfection dans les cellules musculaires lisses (CML) in vivo. Dans les artères saines les CML sécrètent la métalloprotéase 2 (MMP-2) de façon constitutive. L’angioplastie par ballonnet provoque l’augmentation de la MMP-2, la migration des CML et l’hyperplasie intimale. Le but de cette partie de thèse était d’appliquer le siRNA contre la MMP-2 (MMP-2-siRNA) dans les CML in vitro et in vivo dans les artères carotidiennes chez le lapin hypercholestérolémique avec hyperplasie intimale. Nous avons montré que le transfert de MMP-2-siRNA in vitro dans les CML, provoque l’inhibition d’expression et d’activité de la MMP-2, et l’inhibition de la migration des CML. Nous avons ensuite montré le transfert local de MMP-2-siRNA in vivo dans l’artère carotidienne. Cette étude montre pour la première fois l’application intraluminale de siRNA in vivo et une réduction d’expression et d’activité de MMP-2 de la paroi artérielle en d’hyperplasie intimale. Le deuxième chapitre est consacré à l’étude de la thérapie antileucotriènes B4 (LTB4) dans le même modèle animal avec présence de stent. Les études in vitro montrent que LTB4 induit la migration des CML. L’analyse ex vivo sur les artères mammaires humaines confirme que LTB4 provoque l’augmentation de la sécrétion de MMP-2. L’effet d’application d’un antagoniste du récepteur BLT a montré l’inhibition d’hyperplasie intimale. Nous reportons également les résultats sur la salive humaine. L’analyse de taux de biomarquers comme lysozyme, LTB4, prostaglandine 2, MMP-9, et MMP-2 a montré que la salive est un échantillon biologique où les marqueurs d’inflammation dans les maladies cardiovasculaires sont faciles à détecter et à étudier.
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Goula, Tamafouo Daniel G. "Contribution à l'optimisation du transfert de gènes in vivo : analyse des facteurs pouvant influencer la transfection par des complexes ADN/PEI ainsi que la stabilité de l'expression des transgènes." Paris, Muséum national d'histoire naturelle, 1999. http://www.theses.fr/1999MNHN0021.
Full textAbstract:
Pour pouvoir être applicables à la thérapie, les vecteurs de transfert de gènes doivent d'abord prouver leur efficacité et leur innocuité in vivo. Le polyethylenimine (PEI), polymère cationique ayant une grande capacité de protonation, présente de nombreuses caractéristiques qui en font un bon candidat vecteur pour la transfection somatique in vivo. En effet, son potentiel a été démontré dans de nombreux travaux effectues essentiellement in vitro (voir section ii. 5. 4. C de l'introduction). Nous avons choisi l'injection intracérébrale et l'injection intravasculaire pour analyser la capacité de transfection de complexes formes avec les PEI 25 kDa branche et 22 kDa linéaire. L’analyse biophysique montre que les particules ADN/PEI formulées dans une solution de glucose 5% (par rapport à celles formées en solution saline) sont de petites tailles (60nm). Ces complexes diffusent largement dans les fluides biologiques et ne montrent pas de toxicité lorsqu'ils sont injectes in vivo. L’analyse histologique des organes transfectés révèle que plusieurs types cellulaires dans le cerveau et le poumon contiennent et expriment le transgène. Les complexes ADN/PEI injectes dans la veine caudale de souris traversent l'endothélium vasculaire sans causer ni d'inflammation ni de dommage visible. L’expression des transgènes est détectable des 2h p. I. Dans le poumon. La mort cellulaire et la perte du plasmide contribuent à la perte de l'expression des transgènes. Cependant, la co-expression de protéines anti-apoptotiques ainsi que la diminution de la quantité de plasmide injectée favorisent le maintien de cette expression. L’ensemble de ces résultats suggère que des complexes ADN/PEI, préparés dans une solution de faible force ionique et dans un rapport des amines du PEI aux phosphates de l’ADN de 4 et de 6, sont efficaces in vivo respectivement dans le cerveau et le poumon de souris. De plus, le PEI, par comparaison avec les lipides cationiques, semble favoriser le passage de la paroi capillaire sans causer de lésion apparente. Le PEI constitue donc un vecteur très prometteur pour la thérapie génique.
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PRATAMA, MUHAMMAD YOGI. "TRANSLATIONAL STUDIES ON MICRORNA IN HEPATOCELLULAR CARCINOMA: FROM PREDICTIVE AND PROGNOSTIC BIOMARKERS TO MOLECULAR EFFECTORS." Doctoral thesis, Università degli Studi di Trieste, 2020. http://hdl.handle.net/11368/2973744.
Full textAbstract:
Introduction
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, represents the seventh most frequent cancer and the fourth leading cause of cancer-related death worldwide. Due to unspecific sign and symptoms along with the inefficacy of current non-invasive diagnostic tools for detecting early stages HCC, majority of patients are diagnosed at advanced stages that is no longer eligible for curative treatments. Moreover, the long-term outcome of treatments remain unsatisfactory. To overcome these problems, a better diagnostic and predictive biomarker might be one of the most feasible strategies. The characteristic of MiRNAs, that are proved to have essential roles in various cancer pathways and found to be stable in biological fluids, holds a potential value as non-invasive clinical biomarkers in HCC. The present study include three tasks whose aims are:
• Task 1 - Identify circulating miRNAs as predictor of early HCC occurrence in high-risk population setting, specific in DAA treated chronic HCV patients.
• Task 2 - Identify circulating miRNAs as a prognostic non-invasive biomarker after HCC treatments.
• Task 3 - Identify putative targets of each potential miRNAs and their cellular involvement in HCC pathway by in silico target prediction methods and in vitro approaches using miRNA transfection methods in HCC cell model.
Result and discussion
Task 1: We performed a circulating miRNA profiling analysis of cirrhotic patients treated with DAA before and after therapy initiation. Our group of patients consisted of 15 patients developing HCC after DAA treatment (HCC+) compared to 15 patients nor developing HCC (HCC-) within 12 month after SVR. Through microarray and qRT-PCR analysis, we confirmed differently expressed miR-3197 expression between HCC+ vs HCC− patients at T0 (before the initiation of DAA) (p< 0.05) with diagnostic performance of AUC values of 0.75 with a sensitivity of 80% and specificity of 73% in distinguishing both groups at T0, and AUC values of 0.75 with a sensitivity of 86% and specificity of 73% in distinguishing both groups At T1. Taken together, miR-3197 represent a promising tool for the identification of patients at risk such as HCV patients undergo DAA treatment.
Task 2: We performed a longitudinal study analyzing the expression of serum miRNAs in the cohort of 105 HCC patients before treatments (T0), one (T1) and six months (T6) after treatments. Patients were then separated based on therapy response (TR), disease-free survival (DFS), and overall survival (OS). High expression of miR-4454 (p=0.02) and miR-4530 (p=0.04), and low expression of miR-4443 (P=0.05) at T0 were significantly associated with complete response to curative treatments and able to distinguish complete responder (CR) from partial and non-responder (PR) with an AUC of 0.84, sensitivity and specificity of 72% and 75%. High expression of miR-4454 (p=0.03) and miR-4530 (p=0.015) were also associated with DFS > 6 months in patients receiving curative therapies. For non-curative treatments (TACE), at T0 we observed the potential of miR-4492 distinguishing CR and PR (p=0.01) with AUC=0.84 and sensitivity and specificity of 84.6% and 71%. For OS, we observed significantly different expression of miR-4507 (p=0.00037) and ), and miR-3185 (p=0.014) to distinguish patients with shorter and longer OS. Higher Expression of miR-4507 and miR-3185 was significantly associated with longer OS with HR of 1.98 (p=0.016) and 2.02 (p=0.0086), respectively.
Task 3: We confirmed the presence of some of our miRNAs candidate from task 1 and task 2 in tumoral tissue from HCC patients. We discovered several predicted target genes from in silico prediction approach. This result will be further validated in HCC cellular model using a transfection system with miRNA mimics and inhibitor.
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Torres, Maria Leilani. "Optical transfection and injection techniques applied to mammalian and embryonic cells." Thesis, University of St Andrews, 2011. http://hdl.handle.net/10023/2547.
Full textAbstract:
The delivery of biomolecules into living cells is an important methodology in cell and molecular biology. Optical methods using lasers are attractive tools for such application. However, the interaction of the laser with the cell depends on the laser type and the parameters used. Hence, in this thesis, optical transfection and injection of both mammalian and embryonic cells is demonstrated using a variety of laser sources. Furthermore, some key issues are addressed by demonstrating alternative configurations of optoinjection and transfection systems to develop a robust, user-friendly device with potential for commercialisation. Most optical methods for the delivery of molecules rely on complex and expensive laser systems that occupy a large footprint. In order for the system to be accessible to end-users, transient transfection of plasmid DNA into mammalian cells using an inexpensive continuous wave 405 nm diode laser is demonstrated. In this work, the laser parameters are varied in order to optimise the transfection efficiency. By calculating the temperature change upon irradiation of the focused violet light, the mechanism of violet diode laser transfection is elucidated. Furthermore, the system is used to deliver small interfering RNA molecules to specifically knock down a particular protein within the cell. This work is a major step towards an inexpensive and portable optical transfection system. The critical issue of accurate targeting of the cell membrane is also addressed in conventional near-infrared femtosecond optical transfection systems. A near-infrared femtosecond holographic system is built utilising a spatial light modulator in order to provide fast three dimensional beam translation. Computer control of dosage and targeting allows us to explore the potential of different targeting modalities. An enhanced optoinjection and transfection on mammalian cells is demonstrated. Furthermore, the system is applied to optically manipulate a developing Pomatoceros lamarckii embryo. The holographic system can be employed to optoinject a variety of macromolecules into the embryo, as well as orient and position the embryo by switching to the continuous wave mode of the laser. Such development of optical techniques to deliver biomolecules and orient embryos will benefit the field of developmental biology. Lastly, to achieve controlled cavitation, limiting the mechanical effects of a nanosecond laser source, an optically trapped microsphere undergoes laser induced breakdown in the presence of a cell monolayer. Laser induced breakdown of a trapped microsphere allows control over several parameters, such as the microsphere material, position of the breakdown from the monolayer and the size of the microsphere. Optimising these parameters provide limited mechanical effects, particularly suited for cell transfection. This technique is an excellent tool for plasmid-DNA transfection of multiples of cells with both reduced energy requirements and cell lysis compared to previously reported approaches. Demonstrating optimised and successful delivery of macromolecules with the variety of laser sources used in this thesis will advance the applicability of optical injection and transfection and allow more potential users to access the technique. This thesis advances optical injection and transfection for optimised delivery of macromolecules to both mammalian cells and a developing embryo.
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Wang, T. "Chitosan nanoparticles by ionic coacervation for protein release and gene transfection." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487466.
Full textAbstract:
The Ph.D. dissertation focuses on providing in depth understanding and scientific Iaiowledge usable to realize the potential of chitosan nanoparticles as protein release vehicles and non-viral gene carriers for phannaceutical applications. The preparation ofthe nanoparticles was based on the ability of chitosan to undergo a liquid-gel transition due to ionic interaction with polyanion, such as tripolyphosphate (TPP..). The chitosan-TPP nanoparticles had a particle size range 100-250 nm, positive zeta potential around +28-+52 mV ·and exhibited a high positive surface charge across a wide pH range. The particular imaging analysis of the nanoparticles morphologies revealed that the nanoparticles possessed typical characterization of polyhedrons, indicating an analogy to crystallization mechanism during the nanoparticles formation and growth process. The BSA-Ioaded chitosan nanoparticles formed by both incorporation and incubation methods were in the size range of200-700 nm, and exhibited a positive zeta potential +43-+56 mY. BSA encapsulation efficiency varied from 39 to 88 wt%. Detailed sequential time frame TEM imaging of morphological changes of the nanoparticles showed a swelling and particle degradation process. BSA release showed a typical initial burst within 6 hours followed by an extended slow release. Higher gene transfection efficiency with DNA-loaded chitosan nanoparticles was optimized to reach a peak value of20.2% at chitosan concentration 0.2 mg/ml, which is 5.4% higher than chitosan-DNA complexes. Further analysis of life cycle of gene expression of DNA-loaded chitosan nanoparticles was detectable at 24 days with its compact structure and multiple ionic crosslink, provided better shielding and protection of DNA molecules from enzymatic degradation. .It demonstrates that chitosan-TPP nanoparticles can be a versatile carrier for protein release and gene transfection, and polyanionic crosslink TPP of polycationic chitosan molecules offers simple preparation. conditions and clear processing windows for manipulation of physiochemical properties, especially particle size and surface charge.
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Pan, Fang. "Self-assembly of amphiphilic peptides and their potential in gene transfection." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496700.
Full textAbstract:
The surfactant-like peptides are amphiphillic molecules containing polar or charged amino acids moieties as hydrophilic heads and non-polar ammo acids as hydrophobic tails, their surfactant-like amphiphilicity is attractive to a wide range of biotechnological applications including regenerative medicine and drug delivery. Short and designed peptides are easy to synthesise. They are becoming increasingly popular for self-assembly studies as model systems. It is of bith fundamental and practical significance to explore how these designed peptide molecules aggregate in solution and adsorb at the interface.
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Venter, Chrizelle. "Chitosan and quaternised chitosan polymers as gene transfection agents / Chrizelle Venter." Thesis, North-West University, 2005. http://hdl.handle.net/10394/1015.
Full textAbstract:
Several approaches have been employed for directing the intracellular trafficking
of DNA to the nucleus. Cationic polymers have been used to condense and
deliver DNA and a few specific examples using chitosan as cationic polymer
have been described. The concerted efforts in gene therapy to date have
provided fruitful achievements toward a new era of curing human diseases. A
number of obstacles, however, still must be surmounted for successful clinical
applications.
Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes
(polyplexes) were investigated for their ability to transfect COS-1 cells and the
results were compared with Transfectam/DNA lipoplexes for transfection
efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1
cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which
served as a positive control. Complexes formed with quaternised trimethyl and
triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior
transfecting agents compared to other chitosans. The molecular mass of
chitosan is considered to influence the stability of the chitosan/DNA polyplex, the
efficiency of cell uptake and the dissociation of DNA from the complex after
endocytosis.
In literature it was shown that the toxicity of the chitosan1DNA polyplexes is
relatively low compared to viral gene and lipid non-viral delivery vectors. This
study showed that the percentage viable COS-1 cells when transfected with the
chitosan polymers, oligomers, quaternised chitosan polymers and quaternised
chitosan oligomers (chitoplexes) was higher than the percentage viable cells
when transfected with lipoplexes prepared with Transfectam with the MTT
assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased
viability of COS-1 cells were found. Chitosan/DNA and quaternised
chitosan/DNA complexes did not affect the viability of the cell line. The degree of
quaternisation of the polymers and oligomers and molecular size proved to be
two important factors when considering effective non-viral gene delivery.
It can be concluded that chitosan, especially quaternised oligomeric derivatives
are polysaccharides that demonstrate much potential as a gene delivery system.
The high solubility and low toxicity of chitosan allow its use in a wide variety of
applications in the pharmaceutical industry and, as shown in this study, in gene
delivery.Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.
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Jassal, Ramesh Kumar. "Remodelling recombinant glycoproteins made in CHO cells by transfection of glycosyltranferases." Thesis, De Montfort University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391648.
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Angel, Matthew. "Extended transient transfection by repeated delivery of in vitro-transcribed RNA." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/47895.
Full textAbstract:
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references (p. 53-56).
An experimental study was performed to evaluate a novel method of controlling protein expression by repeatedly transfecting cells with in vitro-transcribed RNA. Transcripts encoding six factors, each known to play an important role in cell-type specification and maintenance, were designed and synthesized. Aspects of the design were optimized, and the intracellular stability and translation efficiency of the ivT RNA were quantified. Transcripts were delivered to cells by electroporation, and a method of increasing the rate at which cells recover from ivT-RNA transfection by combined knockdown of innate-immune-related genes was developed. Using this technique a high, approximately steady-state level of protein expression was transduced in MRC-5 human fetal-lung fibroblasts by repeated transfection with capped, poly(A)+ ivT RNA encoding a protein with an intracellular half-life of approximately three days. Transfection at 24-hour intervals with ivT RNA encoding a less stable protein resulted in protein expression that peaked twelve hours after each transfection, and diminished before the next transfection. In both cases, cells sustained a high rate of proliferation. In this study, extended transient transfection by repeated delivery of ivT RNA was shown to transduce expression of defined factors in cultured cells without genetic modification or the extensive screening required in small-molecule approaches and with significant control over the level of transduced protein. This technique may become a powerful tool in the development of new directed-differentiation and cell-type-conversion protocols.
by Matthew Angel.
S.M.