Pulsed Laser Manipulation of Cells Decorated by Plasmonic Nanoparticles

Au cours des dernières décennies, la thérapie cellulaire a suscité un intérêt considérable dans les domaines de la recherche biomédicale et théranostique. Une large gamme d’applications biomédicales a obtenu des avantages fructueux par une manipulation directe de cellules (cellules seuls ou groupes de cellules individuelles) en contrôlant la concentration de nanoparticules plasmoniques fonctionnelles et de lasers à impulsions. Cependant, tous ces protocoles de transfection, accompagnés d’une efficacité et d’une toxicité de transfection cellulaire variées, sont applicables pour une condition spécifique. Ainsi, l’optoporation doit être explorée, optimisée et généralisée pour un large éventail de thérapies cellulaires délicates et complexes par le laser. Un suivi en temps réel des cellules optoporées révèle les mécanismes impliqués dans une transfection réussie sans induire de cytotoxicité. Dans cette thèse, le laser pulsé nanoseconde (532 nm) utilisé pour optoporer une seule cellule cancéreuse humaine du sein (MDA-MB-231) montre clairement les effets des fluences du laser pulsé. L’optoporation intériorise les molécules exogènes (Iodure de Propidium) dans l’intervalle fonctionnel compris entre 0.3 et 0.7 J/cm2 sans provoquer d’effet secondaire (confirmé par Calcein AcetoxyMethyl). La position du faisceau détermine clairement dans quels compartiments subcellulaires les molécules exogènes à intérioriser avec précision. Le faisceau laser focalisé près du noyau dirige intensément l’Iodure de Propidium (PI) pour qu’il réagisse avec les nucléotides cellulaires, alors que pour le faisceau focalisé loin du noyau, le PI se déverse à peine dans le site d’action. Ce protocole d’optoporation, indiquant le rôle critique des nanoparticules plasmoniques liées, nécessite un faisceau laser dirigé vers la position de la nanoparticule plasmonique sur la membrane cellulaire. L’éclairage latéral par LED développé ici visualise simplement la nanoparticule plasmonique liée sur la membrane cellulaire dans une grande zone. Par la suite, un laser femtoseconde dans le proche infrarouge (800 nm) est également utilisé pour optoporer des cellules de lymphocyte T humaines supersensibles (Jurkat) avec un taux de survie négligeable après une exposition de courte durée à une fluence relativement élevée (252 mJ/cm2). En explorant plusieurs fluences laser et durées d’irradiation, nous obtenons donc une gamme applicable de fluences laser (63 à 71 mJ/cm2) et de temps d’irradiation (10 ms) pour l’optoporation de cellules de Jurkat liées à des nanoparticules plasmoniques sans réduire leur viabilité cellulaire. Ces résultats fondamentaux indiquent comment effectuer une optoporation réussie en ajustant les paramètres du laser (fluence, durée d’irradiation, position, etc.) sur différentes lignées cellulaires afin d’atteindre une haute transfection pour une large gamme d’applications biomédicales.----------ABSTRACT In the last decades, cell therapy has attracted tremendous interests in biomedical and theranostic research fields. A wide range of biomedical applications has gained fruitful benefits by a direct manipulation of cells (whether single cell or bunch of individual cells) by controlling the concentration of functional plasmonic nanoparticles and pulsed lasers. However, all these transfection protocols, accompanied by a varied cellular transfection efficiency and toxicity, are applicable for a specific condition. Thus, the optoporation needs to be explored, optimized, and generalized for a broad range of delicate and complex laser mediated cell therapies. A real-time monitoring of the optoporated cells reveals mechanisms involved in a successful transfection without inducing cytotoxicity. In this thesis, the nanosecond pulsed laser (532 nm) used to optoporate a single adherent human breast cancer cell (MDA-MB-231) clearly shows effects of the pulsed laser fluences. The optoporation internalizes the exogenous molecules (Propidium Iodide) at the functional range between 0.3 to 0.7 J/cm2 without causing a side effect (confirmed by Calcein AcetoxyMethyl). The beam pointing location clearly determines in which subcellular compartments the exogenous molecules to be internalized precisely. The laser beam localized close to the nucleus intensively directs the Propidium Iodide there to react with cellular nucleotides, whereas the beam far away from the nucleus barely fluxes into the site of action. This optoporation protocol, indicates the critical role of the bound plasmonic nanoparticles, is required a laser beam to be directed to the position of the plasmonic nanoparticle on the cellular membrane. The lateral LED illumination developed here simply visualizes the bound plasmonic nanoparticle on the cell membrane in a large area. Afterward, near-infrared femtosecond laser (800 nm) is also employed to optoporate supersensitive human T lymphocyte cells (Jurkat) with a negligible survival rate after a short time exposure to a relatively high fluence (252 mJ/cm2). Exploring several laser fluences and irradiation durations, we therefore obtain an applicable range of laser fluences (63 to 71 mJ/cm2) and irradiation time (10 ms) for the optoporation of plasmonic nanoparticles bound Jurkat cells without reducing their cell viability. These fundamental results indicate how to perform a successful optoporation by adjusting laser parameters (i.e., fluence, irradiation duration, position, etc.) on different cell lines in order to achieve high transfection for a wide range of biomedical applications

Genome-wide identification and analysis of heterotic loci in three maize hybrids

Heterosis, or hybrid vigour, is a predominant phenomenon in plant genetics, serving as the basis of crop hybrid breeding, but the causative loci and genes underlying heterosis remain unclear in many crops. Here, we present a large-scale genetic analysis using 5360 offsprings from three elite maize hybrids, which identifies 628 loci underlying 19 yield-related traits with relatively high mapping resolutions. Heterotic pattern investigations of the 628 loci show that numerous loci, mostly with complete–incomplete dominance (the major one) or overdominance effects (the secondary one) for heterozygous genotypes and nearly equal proportion of advantageous alleles from both parental lines, are the major causes of strong heterosis in these hybrids. Follow-up studies for 17 heterotic loci in an independent experiment using 2225 F2 individuals suggest most heterotic effects are roughly stable between environments with a small variation. Candidate gene analysis for one major heterotic locus (ub3) in maize implies that there may exist some common genes contributing to crop heterosis. These results provide a community resource for genetics studies in maize and new implications for heterosis in plants

Search for light dark matter from atmosphere in PandaX-4T

We report a search for light dark matter produced through the cascading decay of $\eta$ mesons, which are created as a result of inelastic collisions between cosmic rays and Earth's atmosphere. We introduce a new and general framework, publicly accessible, designed to address boosted dark matter specifically, with which a full and dedicated simulation including both elastic and quasi-elastic processes of Earth attenuation effect on the dark matter particles arriving at the detector is performed. In the PandaX-4T commissioning data of 0.63 tonne$\cdot$year exposure, no significant excess over background is observed. The first constraints on the interaction between light dark matter generated in the atmosphere and nucleus through a light scalar mediator are obtained. The lowest excluded cross-section is set at $5.9 \times 10^{-37}{\rm cm^2}$ for dark matter mass of $0.1$ MeV$/c^2$ and mediator mass of 300 MeV$/c^2$. The lowest upper limit of $\eta$ to dark matter decay branching ratio is $1.6 \times 10^{-7}$

A Search for Light Fermionic Dark Matter Absorption on Electrons in PandaX-4T

We report a search on a sub-MeV fermionic dark matter absorbed by electrons with an outgoing active neutrino using the 0.63 tonne-year exposure collected by PandaX-4T liquid xenon experiment. No significant signals are observed over the expected background. The data are interpreted into limits to the effective couplings between such dark matter and electrons. For axial-vector or vector interactions, our sensitivity is competitive in comparison to existing astrophysical bounds on the decay of such dark matter into photon final states. In particular, we present the first direct detection limits for an axial-vector (vector) interaction which are the strongest in the mass range from 25 to 45 (35 to 50) keV/c$^2$

Readout electronics of a high-resolution gamma camera

To develop high resolution gamma camera, a novel readout method is adopted. The detector of the gamma camera is based on CsI(Na) scintillation crystal array coupling with position-sensitive photomultiplier (PSPMT). The novel readout scheme utilizes the truncated center-of-gravity (TCOG) method, which hybridizes the conventional resistive readout and the individual analogue readout. The TCOG design was tested on a readout circuit board made by ourselves. As a result, the gamma camera could achieve an intrinsic spatial-resolution of 1 mm. The field-of-view (FOV) was also improved to cover the active area of the detector. In conclusion, the TCOG method is an effective approach to developing high-resolution compact gamma camera. This method can also be used in the newly high-integrated multi-anode PSPMT

A novel trigger circuit of signal sampling for modularized high-resolution compact gamma camera

A novel trigger circuit of signal sampling was designed for developing modularized high-resolution compact gamma camera. The design idea is to transfer the peak position detection of the signal of gamma camera into high-precision measurement of pole-zero timing. The unipolar input signal is shaped by a full-pass filter into a bipolar signal. The peak position of signal is determined by measuring the polar-zero position while a trigger pulse is generated corresponding to the peak position. The width and delay of the trigger pulse are adjustable by mono-stable trigger circuits. The pulse is used to trigger the data acquisition system. The experimental results show that the performance of the trigger circuit is stable and reliable. It can provide a high-precision determination of the peak position and well meet the requirements of the high-resolution compact gamma camera

Daily RF AOD dataset in the Sichuan Basin, China (2015-2020)

The random forest (RF) machine learning method and multiple datasets are used to establish aerosol optical depth (AOD) dataset in the cloudy Sichuan Basin. Multiple datasets include ground-based PM10 and PM2.5, the AOD from the Sun-sky radiometer Observation Network (SONET) and the Second Modern-Era Retrospective analysis for Research and Applications (MERRA-2) aerosol reanalysis, and several meteorological variables

Transcriptomic and Targeted Metabolomics Analysis of Detached <i>Lycium ruthenicum</i> Leaves Reveals Mechanisms of Anthocyanin Biosynthesis Induction through Light Quality and Sucrose Treatments

Light quality and sucrose-induced osmotic stress are known to cause anthocyanin synthesis in detached Lycium ruthenicum leaves. To identify the mechanisms by which the kind of light quality and sucrose concentration are induced, here, we conducted transcriptome sequencing in detached L. ruthenicum leaves treated with different qualities of light and sucrose concentrations. Leaves treated with blue light or sucrose showed a significantly increased total anthocyanins content compared to those treated with white light. Delphinidin-3-O-rutinoside and delphinidin-3-O-glucoside production were differentially regulated by the BL(−S), BL(+S), and WL(+S) treatments. The structural genes CHS, CHI, F3′H, F3′5′H, ANS, and UFGT were significantly up-regulated in leaves treated with blue light or sucrose. Leaves treated with blue light additionally showed up-regulation of the light photoreceptors CRY1, PIF3, COP1, and HY5. The anthocyanin-related genes NCED1, PYR/PYL, PP2C, SnRK2, and ABI5 were significantly up-regulated in leaves treated with sucrose, promoting adaptability to sucrose osmotic stress. Co-expression and cis-regulatory analyses suggested that HY5 and ABI5 could regulate LrMYB44 and LrMYB48 through binding to the G-box element and ABRE element, respectively, inducing anthocyanin synthesis in response to blue light or sucrose treatment. Candidate genes responsive to blue light or sucrose osmotic stress in the anthocyanin biosynthesis pathway were validated through quantitative reverse transcription PCR. These findings deepen our understanding of the mechanisms by which blue light and sucrose-induced osmotic stress regulate anthocyanin synthesis, providing valuable target genes for the future improvement in anthocyanin production in L. ruthenicum