HIV-NC is a multifunctional protein which plays an important role in almost every step of the retroviral life cycle. NC is essential in catalyzing stand transfers of HIV-1 reverse transcription, including the annealing of the transactivation response element (TAR) of the viral genome to the complementary TAR DNA in minus-strong-stop DNA. In this dissertation, the research starts with focus on elucidating the reaction mechanism of NC-facilitated TAR DNA/RNA annealing using single molecule spectroscopy (SMS) approaches. The results indicate that nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the partially open “Y” form associated with multiple NC molecules. This encounter complex leads to annealing through the 3’/5’ termini, namely “zipper” pathway and the annealing through the hairpin loop region, namely “kissing” pathway. By employing target oligonucleotides for specific TAR regions, we directly probed kinetic reversibility and the chaperone role of NC. Concentration-dependence of NC chaperoned melting and annealing of TAR hairpins was investigated and the results further support the proposed reaction mechanism. Additionally, we used a single-stranded DNA (ssDNA) as model to study ssDNA conformational change upon NC binding. Here we present observation of NC binding to d(TG)n and d(T)n, including NC effect on flexibility and conformation of these oligonucleotides chains. Our results reveal that the rigidity of ssDNA chain is dramatically reduced through interaction with NC. Meanwhile the results of NC dissociation experiments indicate the interaction of NC/ssDNA is complex and heterogeneous. Finally, we used SMS in vitro to systematically compare and contrast the RNA/protein interactions for the zinc-finger-binding-motif protein (NC) and the arginine-rich-binding-motif (ARM) protein (Tat) encoded by HIV-1. Tat and NC use different RNA binding motifs to recognize and interact with RNA hairpin, giving rise to very different changes in the RNA secondary structure upon protein binding. Competition experiments show that the presence of Tat can effectively inhibit the NC binding-induced local melting of TAR RNA hairpins. These results indicate that Tat specifically binds and stabilizes the TAR RNA hairpin structure, which likely inhibits the local melting of the hairpin induced by NC. / text

Ternary systems comprising DNA/RNA, proteins and one (or more) metal ion are generating increased interest due to its biological relevance. The knowledge gained from the study of these systems could provide important clues regarding the precise mechanism for transcription factors, repair proteins and metal complexes with anti-tumoral/anti-viral activities.The interactions occurring among the components of these ternary systems can be broadly grouped into covalent and non-covalent. The first kind of interactions can lead to the irreversible transformation of the components in the system, while the second is thought to be reversible leading to transient states and fluxionality. Both kinds of interaction are generally present in living systems, complementing the function of each other.Monofunetional Platinum-nucleobase complexes (MPNs) are synthesized via substitution of a chloride ligand by a nucleobase in platinum complexes with trans geometry. MPNs are particularly interesting for the study of ternary systems since they mimic the first step in the formation of a platinum-DNA adduct and their interaction with aminoacids/proteins provide a good first approach for more complex systems.The presence of the nucleobase as a ligand, significantly modifies the biological activity of these complexes by reducing its cytotoxicity and generating a promising anti-viral activity, especially against HIV-1 virus. The specific role of the nucleobase ligand on these complexes as a non-covalent motif, important for protein recognition, was explored in models involving tryptophan/N-acetyl tryptophan and a small protein domain called zinc finger, containing also a tryptophan residue.The coordination of the nucleobase to a metal ion such as Pt(II) or Pd(II) was found to increase its π-stacking interaction towards aromatic residues in proteins, specifically tryptophan. The enhancing effect was found to depend on the nature of the metal ion, nature of nucleobase and size/complexity of the protein model. Furthermore, DFT studies revealed an important change in the energy for the lowest unoccupied molecular orbital (LUMO) in the coordinated nucleobases, which could place this orbital in an favored position to interact with the highest occupied molecular orbital (HOMO) in the tryptophan residue. Results from calculations showed a good correlation with experimental evidence and could indicate an important role for the frontier molecular orbitals (HOMO/LUMO) of the species involved in the π-stacking interaction.This study was extended to a zinc finger domain from an essential protein in HIV-1 virus, i.e. nucleocapsid protein NCp7. Findings showed that the nucleobase ligand in addition to modulate hydrolysis and reaction rates for MPNs can also be responsible for an initial non-covalent recognition towards a specific protein. This initial recognition has been proposed as the first stage in a two-step mechanism of action for these platinum complexes that ultimately can lead to zinc ejection from the zinc finger domain in the viral NCp7. The significance of the data presented show that is possible to modulate the ligand coordination sphere in metal complexes to can result in great differences in terms of biological effects.The novel chemistry derived from DNA adducts with platinum complexes with a trans geometry was also explored in silico. The molecular dynamics of two free DNA 20-mer is compared with the corresponding metallated-adducts, namely monofunctional, 1,2-bifunctional interstrand and 1,3-bifunctional intrastrand. The differences in terms of structure and energy are compared for these systems, in general the monofunctional adduct exhibited the most interesting feature in terms of structural change in the DNA double strand causing the destacking of the metallated nucleobase. Bifunctional adducts exhibited loss of Watson-crick bonds and localized change in sugar puckering. These results showed that important differences can be found for platinated DNA even at short simulation times < 1 ns.

The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancers (TNBCs) are pathologically defined by the absence of estrogen, progesterone, and HER2 receptors. Compared to other breast cancers, TNBC has a relatively high mortality. In addition, TNBC patients are more likely to relapse in the first few years after treatment, and experiencing a shorter median time from recurrence to death. Detecting the presence of tumor in patients who are technically “disease-free” after neoadjuvant chemotherapy and surgery as early as possible might be able to predict recurrence of patients, and then provide timely intervention for additional therapy. To this end, I applied the analysis of “liquid biopsies” for early detection of minimal residual disease (MRD) on early-stage TNBC patients using next-generation sequencing. For the first part of this study, I focused on detecting circulating tumor DNA (ctDNA) from TNBC patients after neoadjuvant chemotherapy and surgery. First, patient-specific somatic mutations were identified by sequencing primary tumors. From these data, 82% of the patients had at least one TP53 mutation, followed by 16% of the patients having at least one PIK3CA mutation. Next, I sequenced matched plasma samples collected after surgery to identify ctDNA with the same mutations. I observed that by detecting corresponding ctDNA I was able to predict rapid recurrence, but not distant recurrence. To increase the sensitivity of MRD detection, in the second part I developed a strategy to co-detect ctDNA along with circulating tumor RNA (ctRNA). An advantage of ctRNA is its active release into the circulation from living cancer cells. Preliminary data showed that more mutant molecules were identified after incorporating ctRNA with ctDNA detection in a metastatic breast cancer setting. A validation study in early-stage TNBC is in progress. In summary, my study suggests that co-detection of ctDNA and ctRNA could be a potential solution for the early detection of disease recurrence. / 2021-08-05

No description available.

Les rayonnements ionisants (RI) traversent les tissus humains, y déposent de l'énergie et la dissipent en fragmentant des molécules. Les fragments obtenus peuvent être mis en évidence par spectrométrie de masse. Malgré la quantité d'informations obtenue expérimentalement à partir du spectre de masse, l'expérience seule ne peut pas répondre à toutes les questions concernant les mécanismes de fragmentation des bases de l'ADN/ARN, et l'étude issue des méthodes quantiques est un complément précieux à ces informations. L'étude théorique permet de connaître les liaisons moléculaires rendues faibles dans chaque base par ionisation et ainsi de prévoir les mécanismes de fragmentation et les fragments produits. Le but de ce travail est d'étudier l'ionisation et les mécanismes de fragmentation de bases de l'ADN/ARN (Uracile, Cytosine, Adénine et Guanine) et d'identifier les cations correspondants à chaque pic des spectres de masse. Nous avons effectué nos calculs en utilisant la DFT avec la fonctionnelle PBE. Dans cette thèse, pour toutes les bases d'ARN, la réaction de rétro Diels-Alder est une des voies principales de dissociation (élimination de HNCO ou de NCO•) à l'exception de l'adénine qui n'a pas d'atome d'oxygène. La perte d'une molécule NH3 (NH2•) est aussi un chemin commun à toutes les bases contenant un ou plusieurs groupes amine. Egalement, la possibilité de la perte d'un hydrogène à partir des cations est également envisagée, ainsi que la dissociation de ces cations déshydrogénés et des bases protonées, en se limitant à l'uracile. Ce travail montre tout l'intérêt de l'apport de calcul (DFT) à l'interprétation des spectres de masse de bases de l'ADN / Ionizing radiation (IR) cross human tissue, deposit energy and dissipate fragmenting molecules. The resulting fragments may be highlighted by mass spectrometry. Despite the amount of information obtained experimentally by the interpretation of the mass spectrum, experience alone cannot answer all the questions of the mechanism of fragmentation of DNA/RNA bases and a theoretical study is a complement to this information. A theoretical study allows us to know the weakest bonds in the molecule during ionization and thus may help to provide mechanisms of dissociation and produced fragments. The purpose of this work, using the DFT with the PBE functional, is to study the ionization and fragmentation mechanisms of DNA/RNA bases (Uracil, Cytosine, Adenine and Guanine) and to identify the cations corresponding to each peak in mass spectra. For all RNA bases, the retro Diels-Alder reaction (elimination of HNCO or NCO•) is a major route for dissociating, with the exception of adenine for which there is no atom oxygen in its structure. Loss of NH3 (NH2•) molecule is another common way to all bases that contain amine group. The possibility of the loss of hydrogen from the cations is also investigated, as well as the dissociation of dehydrogenated cations and protonated uracil. This work shows the interest of providing DFT calculation in the interpretation of mass spectra of DNA bases

In this doctoral thesis we study novel analogues based on R06 aptamers and targeting TAR hairpins of the HIV virus by means of surface plasmon resonance biosensor, which allows for sensitive and real-time monitoring of molecular interactions. We investigate seven different modifications placed at nine different positions on the R06 aptamer in order to find out their applicability in the construction of efficient and stable anti-TAR oligonucleotides. We also determine which positions are suitable for substitutions with a modification and interpret the results in the context of the local nucleotide geometries and interactions in the TAR/anti-TAR complex. In this doctoral thesis we further develop a new fluidic system. This fluidic system eliminates sample dispersion and intermixing effects and thus enables accurate monitoring of molecular interactions on the surface of an SPR chip. We also characterize experimental conditions on the surface of an oligonucleotide chip and their relations towards bio-molecular assays. Specifically, we study the shielding effect of monovalent and divalent cations, which are crucial for the interaction of negatively charged oligonucleotides.

La chimie des acides boroniques englobe de multiples domaines comme les sciences analytiques, la synthèse organique et inorganique, la chimie médicinale et thérapeutique. L'aptitude des acides boroniques à se lier de façon covalente et réversible avec des fonctions cis-1,2-diol, naturellement présentent en position 2',3' des ribonucléosides, a été mise à profit pour la conception d'un nouveau lien internucléosidique boronate.La synthèse des 4 borononucléotides, analogues des 4 nucléotides 5'-monophosphates, a été effectuée. La formation de dimères entre ces boronoanalogues et les 4 ribonucléotides naturels a été étudiée par RMN 1H et spectroscopie UV, permettant de déterminer leurs constantes d'association. Le borononucléotide analogue de la TMP a ensuite été introduit à l'extrémité 5' d'oligonucléotides. La formation dirigée par une matrice de boronates internucléosidiques a alors été étudiée au niveau oligonucléotidique. Cette ligation réversible apparaît dépendante de stimuli externes tels que la température, le pH, la présence d'ions cyanure ou de fructose. La nature de la matrice, l'influence de mésappariements ainsi que le nombre de jonctions modifiées ont ensuite été évalués. Enfin, à l'instar de la PCR, la polymérisation dirigée de sondes trimériques 3'-ribo 5'-B(OH)2 a été réalisée en présence d'une amorce.Parallèlement à ces travaux, nous avons développé de nouvelles applications du réactif de Bestmann-Ohira en synthèse organique, notamment au travers de réactions monotopes. / The chemistry of boronic acids is involved in many fields such as analytical sciences, organic and inorganic synthesis, medicinal and therapeutic chemistries. The ability of boronic acids to bind covalently and reversibly cis-1,2-diol functions, which naturally occur in 2',3' position of ribonucleosides, has been employed to design a new internucleosidic boronic ester linkages.For this purpose, the synthesis of the borononucleotide analogs of the 4 nucleotide 5'-monophosphates, has been undertaken. Dimeric association of the latters with the 4 natural ribonucleotides has been investigated by 1H NMR as well as UV spectroscopy and the binding constants have been determined. Following this study, the borononucleotide analog of TMP was introduced at the 5'-end of oligonucleotides. Thus, as we did at dimeric state, the formation of the internucleosidic boronic ester as been investigated at the oligonucleotide state in a template-directed fashion. This ligation has been demonstrated to be dependent on various external stimuli, such as, temperature, pH, presence of cyanide ions or fructose. The nature of the template, the influence of mismatches and the number of modified junctions tolerated by the system have also been investigated. Finally, a PCR-like template-directed polymerisation of 3'-ribo 5'-B(OH)2 trimeric probes has been successfully achieved in the presence of a primer.In parallel to this work, we develop new applications of the Bestmann-Ohira reagent in organic synthesis involving especially one-pot procedures.

No description available.

No description available.