Le Tumor Necrosis Factor alpha (TNF-[alpha]) est une cytokine pro-inflammatoire qui occupe un rôle central dans la physiopathologie de nombreuses pathologies inflammatoires et particulièrement l'arthrite. La neutralisation de cette cytokine par l'utilisation d'anticorps anti-TNF-[alpha] a montré son efficacité dans la polyarthrite rhumatoïde et est aujourd'hui le traitement de référence pour la prise en charge de cette pathologie. Cependant, un tiers des patients traités par anticorps anti-TNF restent réfractaires ou ne répondent pas à ce traitement. Dans ce contexte, il apparait nécessaire de développer des approches nouvelles ou complémentaires pour renforcer l'arsenal thérapeutique actuellement disponible. L'utilisation d'oligonucléotides triple hélice (TFO) permet de moduler l'expression génique de manière spécifique par interaction avec la double hélice d'ADN. Dans cette étude, nous avons évalué les potentialités anti-inflammatoires d'un TFO anti-TNF-[alpha] in vitro sur les synoviocytes et chondrocytes articulaires et in vivo dans deux modèles d'arthrite expérimentale. Ce TFO interagit avec le promoteur du gène du TNF-[alpha], et son activité inhibitrice a été comparée à celle d'une approche par ARN interférence in vitro. Dans les modèles d'arthrite aigue et chronique, l'injection intra-articulaire préventive de TFO anti-TNF-[alpha] permet une amélioration significative des symptômes arthritiques. Particulièrement, le traitement par le TFO diminue sensiblement l'inflammation synoviale et les lésions ostéocartilagineuses articulaires. Ces résultats sont les premiers à montrer la possibilité d'utiliser un TFO in vivo et offrent d'intéressantes perspectives thérapeutiques / Tumor necrosis factor alpha (TNF-[alpha]), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-[alpha] antibodies has shown its efficacy in rheumatoid arthritis and is now widely used. Nevertheless, some patients currently treated with anti-TNF-[alpha] remain refractory or become non-responder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. Triplex forming oligonucleotides (TFO) can inhibit gene expression with high sequence-specificity by interacting with the DNA double-strand. In this study, we investigated if an anti-TNF-[alpha] TFO had a therapeutic activity on inflammatory processes in vitro and in vivo, as judged from effects on two rat arthritis models. This TFO interacted with the TNF-[alpha] gene promoter, and its inhibitory activity was verified and compared to that of siRNA in vitro. A local intra-articular preventive injection of TFO in both acute and chronic arthritis models significantly reduced the development of the disease. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-[alpha] TFO modulates arthritis in vivo, thus providing proof of concept that it could be used as therapeutic tool for TNF-[alpha]-dependent chronic inflammatory disorders

Oligonucleotides are short fragments of DNA (10-100nt) which are of great interest because their applications in molecular biology, biomedicine and nanotechnology. As a result of their ability to base pairing, oligonucleotides can be used as primers, hybridization probes in biosensors, agents for controlling gene expression, structural material in nanotechnology or as substrates for a variety of biochemical and biophysical studies. Chemical modification of oligonucleotides as well as conjugation to different functional molecules allows for modulation of both therapeutical and biotechnological properties. This thesis is focused in the nucleic acid chemistry field and the main objective is the synthesis of modified oligonucleotides for obtaining structures with therapeutical and/or biotechnological interest. Oligonucleotides capable to form structures other than the canonical DNA double helix have received considerable attention in the last years. The ability of triplex forming oligonucleotides (TFOs) to bind specifically to certain duplex DNA regions provides a strategy for site-directed modification of genomic DNA. Besides, G-quadruplexes are four-stranded DNA structures stabilized by stacking of guanine tetrads which have been found in telomeres and some promoters and play a role in regulation of transcription and translation. In addition, they are also interesting for nanotechnological devices. In this context, the first part of the research work was addressed to synthesize parallel stranded oligonucleotide clamps carrying LNA (locked nucleic acid) residues and study the stability of the triplex formed with DNA and RNA target sequences. Secondly, a novel strategy to obtain parallel clamps using the non-templated chemical ligation of two oligonucleotides by 5’-5’ linkages was developed. For this purpose, several protocols for introduce azido and alkyne moieties in the 5’-end of different sequences were developed so that the modified DNA strands could form a parallel hairpin after their chemical ligation by click chemistry. Thirdly, a system composed of four DNA strands whose 5’ ends are covalently attached was designed to form a monomolecular parallel G-quadruplex, which was used to study the effects of some nucleobase modifications in quadruplex structure. Finally, oligonucleotide conjugates carrying Cu(II) complexes were synthesized to construct arrays of electrochemical oscillators for nanotechnology applications.

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

Mutacijos, atsiradusios atitinkamuose žmogaus genuose, gali lemti pakitusių baltymų atsiradimą, kurie sukelia įvairias ligas (pvz.: vėžį), klaidingą embriono vystymąsi ar priešlaikinę mirtį. Tokios genetinės ligos gali būti gydomos genų terapijos būdu. Labiausiai vystoma genų terapijos strategija yra paremta homologine rekombinacija, kurios metu DNR seka, naudojama geno taisymui, yra patiekiama in trans. Natūraliai žinduolių ląstelėse homologinė rekombinacija (HR) vyksta žemu rekombinacijos dažniu (10-6). Tačiau yra žinoma, kad dvigrandininio trūkio įvedimas žymiai pagreitina HR (10-1). In vivo eksperimentų atveju dvigrandininio trūkio įvedimas turi būti ypač tikslus, todėl šis metodas reikalauja naujų molekulinių įrankių, kurie būtų itin specifiški ir griežtai kontroliuojami. Šiame darbe mes orientavomės į itin specifiškų ir griežtai kontroliuojamų meganukleazių kūrimą naudojant restrikcijos endonukleazių (REazių)-tripleksą formuojančių oligonukleotidų (TFO) konjugatus. REazių-TFO konjugatuose TFO suteikia specifiškumą prailgintam atpažinimo taikiniui per DNR triplekso susidarymą taip nukreipdamas restrikcijos fermentą prie konkretaus taikinio kur norima įvesti dvigrandininį trūkį. Šiuo tyrimu mes parodėme dvi alternatyvias restrikcijos endonukleazių-TFO konjugatų aktyvumo reguliavimo strategijas, kas leistų šias nukleazes panaudoti in vivo tyrimuose. Tuo tikslu buvo pasirinkti ortodoksiniai restrikcijos fermentai MunI ir Bse634I, kurie mūsų laboratorijoje yra gerai... [toliau žr. visą tekstą] / Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease... [to full text]

Simple mutations within the coding region of critical human genes can lead to the formation of abnormal proteins, resulting in various diseases (e.g. cancer), in failure of an embryo to develop, or premature death. Genetic diseases can only be truly cured via restoration of defective gene function and one of the most promising strategies is based on homologous recombination. Naturally homologous recombination occurs with a low frequency (1 in 106 transfected cells), however it is known that DNA double-strand breaks enhance the efficiency of homologous recombination by several orders of magnitude (up to 10,000-fold). Therefore, gene therapy via homologous recombination requires new molecular tools that should be highly specific and rigorously controllable. In this work we have focused on the development of restriction enzyme-triple helix forming oligonucleotide (TFO) conjugates, where TFO provides specificity for the extended recognition site through the triple helix formation and addresses restriction enzyme to a particular target site where it introduces a double stranded break. We provide proof-of-concept demonstrations of two alternative strategies to control the DNA cleavage activity of restriction endonuclease-TFO conjugates, that allows adopt them in in vivo experiments. To this end we used restriction endonucleases MunI and Bse634I, which were structurally and biochemically characterized before in our laboratory. We successfully combined the restriction endonuclease... [to full text] / Mutacijos, atsiradusios atitinkamuose žmogaus genuose, gali lemti pakitusių baltymų atsiradimą, kurie sukelia įvairias ligas (pvz.: vėžį), klaidingą embriono vystymąsi ar priešlaikinę mirtį. Tokios genetinės ligos gali būti gydomos genų terapijos būdu. Labiausiai vystoma genų terapijos strategija yra paremta homologine rekombinacija, kurios metu DNR seka, naudojama geno taisymui, yra patiekiama in trans. Natūraliai žinduolių ląstelėse homologinė rekombinacija (HR) vyksta žemu rekombinacijos dažniu (10-6). Tačiau yra žinoma, kad dvigrandininio trūkio įvedimas žymiai pagreitina HR (10-1). In vivo eksperimentų atveju dvigrandininio trūkio įvedimas turi būti ypač tikslus, todėl šis metodas reikalauja naujų molekulinių įrankių, kurie būtų itin specifiški ir griežtai kontroliuojami. Šiame darbe mes orientavomės į itin specifiškų ir griežtai kontroliuojamų meganukleazių kūrimą naudojant restrikcijos endonukleazių (REazių)-tripleksą formuojančių oligonukleotidų (TFO) konjugatus. REazių-TFO konjugatuose TFO suteikia specifiškumą prailgintam atpažinimo taikiniui per DNR triplekso susidarymą taip nukreipdamas restrikcijos fermentą prie konkretaus taikinio kur norima įvesti dvigrandininį trūkį. Šiuo tyrimu mes parodėme dvi alternatyvias restrikcijos endonukleazių-TFO konjugatų aktyvumo reguliavimo strategijas, kas leistų šias nukleazes panaudoti in vivo tyrimuose. Tuo tikslu buvo pasirinkti ortodoksiniai restrikcijos fermentai MunI ir Bse634I, kurie mūsų laboratorijoje yra gerai... [toliau žr. visą tekstą]

ESPINOZA HERRERA S.J. - DOCTORAL THESIS - FUNCTION OF MAGNESIUM IONS IN THE FORMATION OF BIOLOGICALLY-ACTIVE NUCLEIC ACIDS STRUCTURES ____________________________________________________________________ ABSTRACT Interaction of magnesium ions, the divalent ions with the highest concentration inside living cells, with the key biomolecules of nucleic acids was studied with aim to find out structural details of the interaction mechanism and effect of the magnesium ions presence on the nucleic-acid structure and structural transitions. Raman spectroscopy was chosen as the main experimental method, some complementary measurements of UV absorption spectra were also performed. Basic analyses of the spectral series obtained as a function of temperature and/or for various concentrations of Mg2+ ions were done by means of factor analysis. Where possible the factor analysis outputs were used for fits of appropriate thermodynamic equations. For the studies, various nucleic-acid molecular models were employed, in particular complementary RNA homopolynucleotides polyA and polyU, DNA homopolynucleotides polydA and polydT and RNA 14-mer oligonucleotide representing the apical hairpin of the TAR segment of HIV-1 genomic RNA. We have confirmed via Raman spectroscopy that magnesium ions added to a solution of polyA-polyU...

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The major challenge associated with renewable-energy systems especially solar, is the supply intermittency. One effective solution is to incorporate thermal energy storage components utilizing phase change materials (PCMs). These materials have the potential to store large amounts of energy in relatively small volumes and within nearly an isothermal storage process. The primary drawback of today’s PCMs is that their low thermal conductivity values critically limit their energy storage applications. Also, this grossly reduces the melting/ solidification rates, thus making the system response time to be too long. So, the application of heat transfer enhancement is very important. To improve the PCM storage performance, an efficient performing containment vessel (triplex-tube) along with applications of various heat transfer enhancement techniques was investigated. The techniques were; (i) dispersion of solid nanoparticles, (ii) incorporation of metal foam with nanoparticle dispersion, and (iii) insertion of longitudinal fins with nanoparticle dispersion. Validated simulation models were developed to examine the effects of implementing these techniques on the PCM phase-change rate during the energy storage and recovery modes. The results are presented with detailed model description, analysis, and conclusions. Results show that the use of nanoparticles with metal foam or fins is more efficient than using nanoparticles alone within the same volume usage. Also, employing metal foam or fins alone results in much better improvement for the same system volume.

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