Gotowa bibliografia na temat „Fluorescent polymeric microbeads”

I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.

The appearance of microbes resistant to antibiotics requires the development of alternative therapies for the treatment of infectious diseases. In this work two polymers, PTPPF16-EDA and PZnTPPF16-EDA, were synthesized by the nucleophilic aromatic substitution of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin and its Zn(II) complex with ethylenediamine, respectively. In these structures, the tetrapyrrolic macrocycles were N,N′-ethylene crosslinked, which gives them greater mobility. The absorption spectra of the polymers showed a bathochromic shift of the Soret band of ~10 nm with respect to the monomers. This effect was also found in the red fluorescence emission peaks. Furthermore, both polymeric materials produced singlet molecular oxygen with high quantum yields. In addition, they were capable of generating superoxide anion radicals. Photodynamic inactivation sensitized by these polymers was tested in Staphylococcus aureus and Escherichia coli bacteria. A decrease in cell viability greater than 7 log (99.9999%) was observed in S. aureus incubated with 0.5 μM photosensitizer upon 30 min of irradiation. Under these conditions, a low inactivation of E. coli (0.5 log) was found. However, when the cells were treated with KI, the elimination of the Gram-negative bacteria was achieved. Therefore, these polymeric structures are interesting antimicrobial photosensitizing materials for the inactivation of pathogens.

Abstract. Microbes play an essential role in soil functioning including biogeochemical cycling and soil aggregate formation. Yet, a major challenge is to link microbes to higher trophic levels and assess consequences for soil functioning. Here, we aimed to assess how microbial consumers modify microbial community composition (PLFA markers), as well as C dynamics (microbial C use, SOC concentration and CO2 emission) and soil aggregation. We rebuilt two simplified soil consumer–prey systems: a bacterial-based system comprising amoebae (Acanthamoeba castellanii) feeding on a microbial community dominated by the free-living bacterium Pseudomonas fluorescens and a fungal-based system comprising collembolans (Heteromurus nitidus) grazing on a microbial community dominated by the saprotrophic fungus Chaetomium globosum. The amoeba A. castellanii did not affect microbial biomass and composition, but it enhanced the formation of soil aggregates and tended to reduce their stability. Presumably, the dominance of P. fluorescens, able to produce antibiotic toxins in response to the attack by A. castellanii, was the main cause of the unchanged microbial community composition, and the release of bacterial extracellular compounds, such as long-chained polymeric substances or proteases, in reaction to predation was responsible for the changes in soil aggregation as a side effect. In the fungal system, collembolans significantly modified microbial community composition via consumptive and non-consumptive effects including the transport of microbes on the body surface. As expected, fungal biomass promoted soil aggregation and was reduced in the presence of H. nitidus. Remarkably, we also found an unexpected contribution of changes in bacterial community composition to soil aggregation. In both the bacterial and fungal systems, bacterial and fungal communities mainly consumed C from soil organic matter (rather than the litter added). Increased fungal biomass was associated with an increased capture of C from added litter, and the presence of collembolans levelled off this effect. Neither amoebae nor collembolans altered SOC concentrations and CO2 production. Overall, the results demonstrated that trophic interactions are important for achieving a mechanistic understanding of biological contributions to soil aggregation and may occur without major changes in C dynamics and with or without changes in the composition of the microbial community.

Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

Biofilm is a structured community of microorganisms adhering to surfaces of various polymeric materials used in food packaging. Microbes in the biofilm may affect food quality. However, the presence of biofilm can ensure biodegradation of discarded packaging. This work aims to evaluate a biofilm formation on the selected biodegradable polymer films: poly (lactic acid) (PLA), poly (butylene adipate-co-terephthalate) (PBAT), and poly (butylene succinate) (PBS) by selected bacterial strains; collection strains of Escherichiacoli, Staphylococcusaureus; and Bacillus pumilus, Bacillussubtilis, Bacillustequilensis, and Stenotrophomonasmaltophilia isolated from dairy products. Three different methods for biofilm evaluation were performed: the Christensen method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and fluorescence microscopy. High biofilm formation was confirmed on the control PBS film, whereas low biofilm formation ability was observed on the PLA polymer sample. Furthermore, the films with incorporated antimicrobial compounds (thymol or eugenol) were also prepared. Antimicrobial activity and also reduction in biofilm formation on enriched polymer films were determined. Therefore, they were all proved to be antimicrobial and effective in reducing biofilm formation. These films can be used to prepare novel active food packaging for the dairy industry to prevent biofilm formation and enhance food quality and safety in the future.

Many efforts have focused on the adsorption of metals from contaminated water by microbes. Synechococcus PCC7002, a major marine cyanobacteria, is widely applied to remove metals from the ocean’s photic zone. However, its ability to adsorb cesium (Cs) nuclides has received little attention. In this study, the biosorption behavior of Cs(I) from ultrapure distilled water by living Synechococcus PCC7002 was investigated based on kinetic and isotherm studies, and the biosorption mechanism was characterized by Fourier-transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectrometry, and three-dimensional excitation emission matrix fluorescence spectroscopy. Synechococcus PCC7002 showed extremely high tolerance to Cs ions and its minimal inhibitory concentration was 8.6 g/L. Extracellular polymeric substances (EPS) in Synechococcus PCC7002 played a vital role in this tolerance. The biosorption of Cs by Synechococcus PCC7002 conformed to a Freundlich-type isotherm model and pseudo-second-order kinetics. The binding of Cs(I) was primarily attributed to the extracellular proteins in EPS, with the amino, hydroxyl, and phosphate groups on the cell walls contributing to Cs adsorption. The biosorption of Cs involved two mechanisms: Passive adsorption on the cell surface at low Cs concentrations and active intracellular adsorption at high Cs concentrations. The results demonstrate that the behavior and mechanism of Cs adsorption by Synechococcus PCC7002 differ based on the Cs ions concentration.

A gelator 1 containing functional quinoline and Schiff base groups that could form organogels in DMF, DMSO, acetone, ethanol and 1,4-dioxane was designed and synthesized. The self-assembly process of geator 1 was characterized by field emission scanning electron microscopy (FESEM), UV-vis absorption spectroscopy, fluorescence emission spectroscopy, Fourier transform infrared spectroscopy(FTIR), X-ray powder diffraction (XRD) and water contact angle. Under non-covalent interactions, gelator 1 self-assembled into microbelts and nanofiber structures with different surface wettability. Weak fluorescence was emitted from the solution and gel state of 1. Interestingly, gelator 1 exhibited good selectivity and sensitivity towards Zn2+ in solution and gel states along with its emission enhancement and change. The emission intensity at 423 nm of solution 1 in 1,4-dioxane was slightly enhanced, and a new emission peak appeared at 545 nm along with its intensity sequentially strengthened in the titration process. The obvious ratiometric detection process was presented with a limit of detection (LOD) of 5.51 μM. The detection mechanism was revealed by a theoretical calculation and NMR titration experiment, which was that Zn2+ induced the transition from trans- to cis- of molecule 1 and further coordinated with 1. This study will introduce a new method for the construction of functional self-assembly gel sensors for the detection of Zn2+.

ABSTRACTMicroorganisms have been observed to oxidize Fe(II) at neutral pH under anoxic and microoxic conditions. While most of the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrusted with Fe(III)-rich minerals, photoautotrophic and microaerophilic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidation mechanisms and the reasons for encrustation remain largely unresolved. Here we used cultivation-based methods and electron microscopy to compare two previously described nitrate-reducing Fe(II) oxidizers (Acidovoraxsp. strain BoFeN1 andPseudogulbenkianiasp. strain 2002) and two heterotrophic nitrate reducers (Paracoccus denitrificansATCC 19367 andP. denitrificansPd 1222). All four strains oxidized ∼8 mM Fe(II) within 5 days in the presence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) minerals, mainly goethite, formed and precipitated extracellularly in close proximity to the cell surface. Interestingly, mineral formation was also observed within the periplasm and cytoplasm; intracellular mineralization is expected to be physiologically disadvantageous, yet acetate consumption continued to be observed even at an advanced stage of Fe(II) oxidation. Extracellular polymeric substances (EPS) were detected by lectin staining with fluorescence microscopy, particularly in the presence of Fe(II), suggesting that EPS production is a response to Fe(II) toxicity or a strategy to decrease encrustation. Based on the data presented here, we propose a nitrite-driven, indirect mechanism of cell encrustation whereby nitrite forms during heterotrophic denitrification and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and complicates our ability to delineate microbial Fe(II) oxidation in ancient microbes preserved as fossils in the geological record.

Die Dissertation umfasst zwei Themenblöcke: Zum einen wurden die optisch-spektroskopischen Eigenschaften von fluoreszenten, farbstoffbeladenen Polymer-Mikropartikeln untersucht. Zum anderen wurde die Anwendbarkeit solcher Partikel für die Lumineszenzlebensdauer-Kodierung in der Durchflusszytometrie evaluiert. Die Charakterisierung der farbstoffbeladenen Mikropartikel erfolgte mittels optischer Spektroskopie. Am Beispiel mit Rhodamin 6G beladener Polymethylmethacrylat-Partikel konnte ein besseres Verständnis des Einbaus der Farbstoffmoleküle und der resultierenden Fluoreszenz-Charakteristika gewonnen werden. Es stellte sich heraus, dass die Beladungseffizienz stark vom mittleren Partikeldurchmesser und den Synthesebedingungen abhängt. In Verbindung mit den beobachteten optisch-spektroskopischen Eigenschaften wurde geschlussfolgert, dass sich eine farbstoffreiche Schicht an der Oberfläche der Partikel bildet, die sich wesentlich von den sterisch eingebauten Farbstoffmoleküle im Partikelvolumen unterscheidet. Hohe Farbstoffkonzentrationen in dieser Oberflächenschicht führen vermutlich zu Aggregation. Des Weiteren deuten Veränderungen der Fluoreszenzeigenschaften auf intrapartikuläre Energiewanderung bei zunehmender Farbstoffkonzentration hin. Diese Interpretation der experimentellen Ergebnisse konnte qualitativ durch einen Algorithmus zur Simulation der Energiewanderung bestätigt werden. Die Anwendbarkeit der Lumineszenzlebensdauer als Kodierungsparameter in der Zeitdomäne konnte unter Verwendung eines Durchflusszytometer-Prototypen analysiert werden. Die wohl größte Herausforderung bei der Lebensdauermessung in der Durchflusszytometrie ist die kurze Interaktionszeit zwischen Objekt und Anregungslicht. Synthetische Daten wurden herangezogen, um den Einfluss einzelner Messparameter und -bedingungen unabhängig voneinander abzuschätzen. Es konnte festgestellt werden, dass die Lumineszenzlebensdauer als Kodierungsparameter in der Zeitdomäne prinzipiell zugänglich ist.
This thesis comprises two main topics. First, the optical-spectroscopic properties of fluorescent microbeads loaded with organic dyes were studied. In the second part, the feasibility of time-domain luminescence lifetime encoding in flow cytometry based on such microbeads was assessed. The study of the dye-loaded polymer microbeads was based on optical spectroscopy. Poly(methyl methacrylate) beads loaded with rhodamine 6G were used as an example system to achieve a better understanding of the dye incorporation procedure. The dye loading efficiency turned out to be strongly dependent on the mean diameter of the beads and on the amounts of certain compounds used for the bead synthesis. In correlation with the observed fluorescence characteristics, it was deduced that a layer with high local dye concentration forms around each bead. The properties of this layer substantially differ from those of the sterically incorporated dye molecules in the bead core. The high dye concentration in this layer results in aggregation accompanied by the respective changes of the fluorescence characteristics of the beads. Moreover, the observed changes in fluorescence properties indicated the existence of an intra-particulate energy migration process at increased dye loading concentrations. A simulation of the energy migration process based on a random walk algorithm confirmed the interpretation of the experimental results. For the assessment of luminescence lifetime encoding in time-domain flow cytometry, a prototype setup was used. The main issue of lifetime determination in flow cytometry is represented by the short interaction time of only tens of microseconds of the objects with the excitation light spot. Synthetic data were used to study certain measurement parameters and conditions as well as the data analysis procedure independently of other influences. As a result, luminescence lifetime is generally applicable as an encoding parameter in time-domain flow cytometry.

Abstract Polyacrylamide-based friction reducer is commonly used in well completion for unconventional reservoirs. However, residual polymer trapped in the near well-bore region could create unintended flow restrictions and could negatively impact oil production. An eco-friendly approach to regain conductivity was developed by stimulating indigenous bacteria for residual polymer biodegradation. In this work, a series of laboratory experiments were conducted using produced water and oil from Permian Basin, polyacrylamide-based polymer, and a modified nutrient recipe that contained 100 to 300 ppm of inorganic salts. The sealed sample vials containing water, oil, and polymer were prepared in a sterilized anaerobic chamber and then kept in a 160° F incubator to simulate the reservoir condition. Feasibility tests of bacteria growth and biodegradation evaluation of polymer were conducted using an optical laser microscopic system with bacteria tagged with fluorescent dye. Size regression was calculated and applied to a mathematical model based on actual fracture aperture distribution data from shale formation. The indigenous bacteria were successfully stimulated with and without the existence of the friction reducer. It was observed that the size of polymer particles decreased from over 300 µm to less than 20 µm after 15 days. Under the condition of produced water injection, 140° F reservoir temperature, and anaerobic environment, about 30% of the natural fractures in shale were calculated to be damaged and remediated within 15 days. This work is a pioneer research on microbial EOR application in unconventional reservoirs with only indigenous bacteria involved. In field applications, only an extremely low amount of nutrient is required in this process which provides great economic potential. Additionally, the nutrients introduced into the reservoirs will be fully consumed by bacteria during treatment, and the bacteria will be decomposed into organic molecules soon after the treatment. Thus, this technique is environmental- and economical- friendly for the purpose of polymer damage remediation to maximize the recoverable.