Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores

Double stranded DNA multiply charged anions coupled to chromophores were subjected to UV-Vis photoactivation. in a quadrupole ion trap mass spectrometer. The chromophores included noncovalently bound minor groove binders (activated in the near UV), noncovalently bound intercalators (activated with visible light), and covalently linked fluorophores and quenchers (activated at their maximum absorption wavelength). We found that the activation of only chromophores having long fluorescence lifetimes did result in efficient electron photodetachment from the DNA complexes. In the case of ethidium-dsDNA complex excited at 500 nm, photodetachment is a multiphoton process. The MS3 fragmentation of radicals produced by photodetachment at lambda = 260 nm (DNA excitation) and by photodetachment at lambda > 300 nm (chromophore excitation) were compared. The radicals keep no memory of the way they were produced. A weakly bound noncovalent ligand (m-amsacrine) allowed probing experimentally that a fraction of the electronic internal energy was converted into vibrational internal energy. This fragmentation channel was used to demonstrate that excitation of the quencher DABSYL resulted in internal conversion, unlike the fluorophore 6-FAM. Altogether, photodetachment of the DNA complexes upon chromophore excitation can be interpreted by the following mechanism: (1) ligands with sufficiently long excited-state lifetime undergo resonant two-photon excitation to reach the level of the DNA excited states, then (2) the excited-state must be coupled to the DNA excited states for photodetachment to occur. Our experiments also pave the way towards photodissociation probes of biomolecule conformation in the gas-phase by Forster resonance energy transfer (FRET)

Algorithms for Big Data: Graphs and PageRank

This work consists of a study of a set of techniques and strategies related with algorithm's design, whose purpose is the resolution of problems on massive data sets, in an efficient way. This field is known as Algorithms for Big Data. In particular, this work has studied the Streaming Algorithms, which represents the basis of the data structures of sublinear order $o(n)$ in space, known as Sketches. In addition, it has deepened in the study of problems applied to Graphs on the Semi-Streaming model. Next, the PageRank algorithm was analyzed as a concrete case study. Finally, the development of a library for the resolution of graph problems, implemented on the top of the intensive mathematical computation platform known as TensorFlow has been started.Comment: in Spanish, 143 pages, final degree project (bachelor's thesis

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Longitudinal Evaluation of the Hypothalamic-Pituitary-Testicular Function in 8 Boys with Adrenal Hypoplasia Congenita (AHC) Due to NR0B1 Mutations

BACKGROUND:Boys carrying mutations in the NR0B1 gene develop adrenal hypoplasia congenita (AHC) and impaired sexual development due to the combination of hypogonadotropic hypogonadism (HH) and primary defects in spermatogenesis. METHODS:We analysed the evolution of hypothalamic-pituitary-testicular function of 8 boys with AHC due to NR0B1 mutations. Our objective was to characterize and monitor the progressive deterioration of this function. RESULTS:The first symptoms appeared in the neonatal period (n = 5) or between 6 months and 8.7 years (n = 3). Basal plasma adrenocorticotrophic hormone (ACTH) concentrations increased in all boys, whilst cortisol levels decreased in one case. The natremia was equal or below 134 mmol/L and kaliemia was over 5 mmol/L. All had increased plasma renin. In 3 of 4 patients diagnosed in the neonatal period and evaluated during the first year, the basal plasma gonadotropins concentrations, and their response to gonadotropin releasing hormone (GnRH) test (n = 2), and those of testosterone were normal. The plasma inhibin B levels were normal in the first year of life. With the exception of two cases these concentrations decreased to below the normal for age. Anti-Müllerian hormone concentrations were normal for age in all except one case, which had low concentrations before the initiation of testosterone treatment. In 3 of the 8 cases the gene was deleted and the remaining 5 cases carried frameshift mutations that are predicted to introduce a downstream nonsense mutation resulting in a truncated protein. CONCLUSIONS:The decreases in testosterone and inhibin B levels indicated a progressive loss of testicular function in boys carrying NR0B1 mutations. These non-invasive examinations can help to estimate the age of the testicular degradation and cryopreservation of semen may be considered in these cases as investigational procedure with the aim of restoring fertility

Conformation et dynamique de biomolècules en phase gazeuse

We have developed a new experimental set-up which couple a quadrupole ion trap mass spectrometer with a tuneable UV-Visible laser OPO. This thesis's work deals with the optical properties and the photofragmentation of amino acids, peptides, DNA and silver cluster tryptophan complexes in gas phase.In the case of peptides, the UV excitation of chromophores in the systems leads to a specific fragmentation which can be controlled by laser wavelength. The fragmentation schemes obtained after laser excitation depends on the photo-induced charge transfer and can be linked to the system's geometry. The optical spectra of the silver cluster tryptophan complexes supported by TD-DFT calculations allow an elaborate study of the electronics excitation, charge transfer and relaxation in a hybrid metal biomolecule system.The production and use of the radical fragment produce by photofragmentation or by photodetachment are also discussed and exemple on peptides and DNAs are further illustrated.Nous avons développé une nouvelle expérience qui couple un spectromètre de masse de type piège ionique quadripolaire à un laser OPO UV-Visible accordable en longueur d'onde. Le travail qui est présenté dans cette thèse, est une étude des propriétés optiques et de la photofragmentation en phase gazeuse d'acides aminés, de peptides, d'ADN et de systèmes complexes métal-biomolécule. Dans le cas des peptides, l'excitation UV des chromophores contenus dans ces systèmes conduit à une fragmentation spécifique qui peut être contrôlée en fonction de la longueur d'onde. Les schémas de fragmentation obtenus après excitation laser dépendent des transferts de charge photo induits et peuvent être reliés à la géométrie du système. Les spectres optiques des complexes d'agrégat d'argent tryptophane, supportés par des calculs de TD-DFT, ont permis une étude détaillée de l'excitation électronique, des transferts de charge et de la relaxation dans un système hybride métal biomolécule.La production et l'utilisation de fragments radicalaires obtenus par photofragmentation ou par photodétachement sont également discutées et illustrées sur des peptides et de l'ADN

Dynamique et conformation de biomolécules en phase gazeuse

Nous avons développé une nouvelle expérience qui couple un spectromètre de masse de type piège ionique quadripolaire à un laser OPO UV-Visible accordable en longueur d onde. Le travail qui est présenté dans cette thèse, est une étude des propriétés optiques et de la photofragmentation en phase gazeuse d acides aminés, de peptides, d ADN et de systèmes complexes métal-biomolécule. Dans le cas des peptides, l excitation UV des chromophores contenus dans ces systèmes conduit à une fragmentation spécifique qui peut être contrôlée en fonction de la longueur d onde. Les schémas de fragmentation obtenus après excitation laser dépendent des transferts de charge photo induits et peuvent être reliés à la géométrie du système. Les spectres optiques des complexes d agrégat d argent tryptophane, supportés par des calculs de TD-DFT, ont permis une étude détaillée de l excitation électronique, des transferts de charge et de la relaxation dans un système hybride métal biomolécule.La production et l utilisation de fragments radicalaires obtenus par photofragmentation ou par photodétachement sont également discutées et illustrées sur des peptides et de l ADNWe have developed a new experimental set-up which couple a quadrupole ion trap mass spectrometer with a tuneable UV-Visible laser OPO. This thesis s work deals with the optical properties and the photofragmentation of amino acids, peptides, DNA and silver cluster tryptophan complexes in gas phase.In the case of peptides, the UV excitation of chromophores in the systems leads to a specific fragmentation which can be controlled by laser wavelength. The fragmentation schemes obtained after laser excitation depends on the photo-induced charge transfer and can be linked to the system s geometry. The optical spectra of the silver cluster tryptophan complexes supported by TD-DFT calculations allow an elaborate study of the electronics excitation, charge transfer and relaxation in a hybrid metal biomolecule system.The production and use of the radical fragment produce by photofragmentation or by photodetachment are also discussed and exemple on peptides and DNAs are further illustratedLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins

Base-dependent electron photodetachment from negatively charged DNA strands upon 260-nm laser irradiation

International audienceDNA multiply charged anions stored in a quadrupole ion trap undergo one-photon electron ejection (oxidation) when subjected to laser irradiation at 260 nm (4.77 eV). Electron photodetachment is likely a fast process, given that photodetachment is able to compete with internal conversion or radiative relaxation to the ground state. The DNA [6-mer](3-) ions studied here show a marked sequence dependence of electron photodetachment yield. Remarkably, the photodetachment yield (dG(6) > dA(6) > dC(6) > dT(6)) is inversely correlated with the base ionization potentials (

Electron photodetachment dissociation of DNA polyanions in a quadrupole ion trap mass spectrometer

We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer single strands and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photodetachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Subsequent collision-induced dissociation (CID) of the radical anions produced by electron photodetachment results in extensive fragmentation. In addition to neutral losses, a large number of fragments from the w, d, a*, and z* ion series are obtained, contrasting with the w and (a-base) ion series observed in regular CID. The major advantage of this technique, coined electron photodetachment dissociation (EPD) is the absence of internal fragments, combined with good sequence coverage. EPD is therefore a highly promising approach for de novo sequencing of oligonucleotides. EPD of nucleic acids is also expected to give specific radical-induced strand cleavages, with conservation of other fragile bonds, including noncovalent bonds. In effect, preliminary results on a DNA hairpin and on double strands suggest that EPD could also be used to probe intra- and intermolecular interactions in nucleic acids