Elucidating Zinc Distribution In Cancerous Prostate Cells Using Novel FRET Sensors

Zinc (Zn2+) is an important trace element that is found throughout the human body and plays essential roles in proteins to ensure structural integrity and catalytic activity. It is the second most abundant transition metal in the body. Transition metals are required for numerous enzymes, proteins, and cellular processes. It is important for cells to regulate distribution of metals because numerous enzymes and cellular processes depend on transition metals, yet metal imbalance leads to a wide range of diseases. Prostate tissue has the highest Zn2+ concentration in the body compared to other tissues but Zn2+ is significantly depleted upon onset of malignancy. Numerous studies have revealed that cancerous prostate tissue exhibits dramatically reduced levels of Zn2+ and that the levels of Zn2+ appear to correlate with progression from benign to invasive and metastatic cancer. The mechanistic correlation between malignancy and Zn2+ levels is not well understood and in particular, it is not clear whether Zn2+ depletion contributes to or is a consequence of disease progression. Although the majority of Zn2+ found in cells in bound by proteins, enzymes, and cellular ligands, pools of free Zn2+ have been identified in pancreatic islet cells, brain, and prostate. In this current study we used normal and cancerous prostate cells as a model system to map free Zn2+ levels in the nucleus, cytosol, endoplasmic reticulum, and mitochondria to identify the differences in Zn2+ at the subcellular level. This was accomplished by using novel Zn2+ sensors based on fluorescence resonance energy transfer (FRET) targeted to each organelle compartment and carrying out live cell imaging experiments. FRET sensors are powerful tools to monitor Zn2+ dynamics in cells. This type of probes gives us the ability to monitor Zn2+ in multiple cellular comparments or simultaneously track two cellular signals using orthogonal FRET sensors. Such studies allow us to precisely correlate the timing of two interdependent cellular events or to track the movement of ions or molecules from one compartment to another. We discovered that the free Zn2+ pool in the nucleus and cytosol is higher in our prostate cancer cell line models compared to normal cell line. In the ER we observed that the free Zn2+ level in normal prostate cells is about 4 times higher than all three prostate cancer cell lines models used. Similar results were observed in mitochondria, revealing 3-4 times higher Zn2+ in the normal cells compared to all three prostate cancer cell lines. Although all three prostate cancer cell lines used in this study were characterized by over 50% reduction in total cellular Zn2+, surprisingly we discovered this reduction did not translate into across-the-board deficiency in intracellular Zn2+ stores, but rather there was substantial redistribution of Zn2+ between subcellular locations. In an effort to define how and why Zn2+ pools are redistributed in prostate cancer, we measured the changes in expression levels of key Zn2+ regulatory proteins such as transporters (hZIP1, hZIP2, hZIP3, ZnT1, ZnT2, ZnT4, and ZnT7). Our results suggest that there is a dysregulation of the Zn2+ transporters in cancerous prostate cells and there is some heterogeneity between the prostate carcinoma cell lines. To complement existing tools, we developed a suite of sensors using alternately colored FRET pairs using tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE that were used simultaneously with CFP-YFP sensors. Using these combinations of FRET sensors we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600-700 sec. These new green-red sensors provided the starting point for developing vesicle-targeted sensors. In summary, this thesis details efforts to develop new fluorescent sensors for defining free Zn2+ in cells and applies these sensors to quantify free Zn2+ in normal prostate and cancerous cells at the subcellular level. We discovered unprecedented redistribution of Zn2+ stores in all three cancer cell lines and discovered this correlates, at least in part, to alteration of Zn2+ transporters. This work not only provides the first quantitative maps of Zn2+ distribution in cells, it also provides a critical glimpse of how Zn2+ is altered with disease progression

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ESTRUCTURAS CRISTALINAS

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Electrochemical Properties of Al2O3-Fe/Si Composites Prepared by High-Energy Mechanical Milling

open accessThe growing demand in the manufacture of advanced materials with desired and unique properties (e.g. high mechanical strength, durability, good corrosion resistance and low cost of maintenance/replacing) is one of reasons to motivate the researchers to pay special attention in Ceramics as high performance materials for industrial applications. This is because conventional materials cannot meet the engineering requirements during their service that is why the need for advanced ceramic materials to achieve these industrial requirements. In the present work a study was made on Al2O3-matrix ceramic composites reinforced with 2%wt. or 5% wt. of Fe/Si particulates that were produced using a mechanical ball milling at high-energy condition. The electrochemical behavior of these ceramics was investigated by anodic polarization and Electrochemical Impedance Spectroscopy (EIS) measurements in a solution containing 0.5N NaCl, whereas the morphology and microstructural features were examined by optical or Scanning Electron Microscopy (SEM)

Alumina-Based Composites Reinforced With Silver Particles

Al2O3/Ag composite ceramics were fabricated by the use of mechanical milling and pressureless sintering. Al2O3 + 10 wt.% Ag were mixed and milled during 12 h at 300 rpm in a horizontal mill, then with the powder mixture it was conformed cylindrical samples by uniaxial pressing using 300 MPa. The pressed samples were sintered during 1 h in an electrical furnace at 1300, 1400 and 1500°C respectively. Sinter was performed using an argon atmosphere inside the furnace in order to inhibit silver oxidation. XRD results established that silver retains its crystalline structure. On the other hand, density of samples is better with increments in temperature. However, the final relative density is small and about of 91%. Scanning electron microscopy observations show alumina’s microstructure with very fine and homogeneous distributions of silver particles. Increments in sintering temperature are reflected as enhancements of the density and consequently of the fracture toughness of the Al2O3/Ag composite ceramics

Determination of Allura Red and Tartrazine in Food Samples by Sequential Injection Analysis Combined with Voltammetric Detection at Antimony Film Electrode

An antimony film electrode prepared on-line and installed as part of a sequential injection system, was used as an electrochemical detector to determine azo dyes in food samples.The influence of several flow varia-bles were evaluated using a centra lcomposite design.In optimal conditions, the linear range of the calibration curve varied from 1–5 mM, with a limit of detection limit of 0.3 mM. The relative standard deviation of analytical repeatability was < 5.0 %.The method was validated by comparing the results obtained with those provided by HPLC; no significant difference were seen.Junta de Castilla y León Proyecto VA171U1

Synthesis and Electrical Characterization of PLZT Piezoelectric-Ceramic

This research describes the synthesis of an outstanding ceramic-composite piezoelectric CCP (lead-lanthanum-zirconate-titanate, PLZT) by means of powders technique procedures. Full dense CCP compacts were obtained with a platinum wire implanted in the center of the piezoelectric, which were used to investigate the microstructural and opto-thermal properties. The microstructural details of this ceramic were investigated by optical microscopy; whereas the opto-thermal characterization was performed by measuring the electrical signal in a bidimensional setup under four different temperatures: 20°C, 35°C, 50°C and 75°C. A 160mW/cm2 LASER beam was used in order to produce the optical energy which is detected by the CCP. A total of one hundred of measurements were registered. Measurements showed that, in the explored thermal range, the CCP signal magnitude increased from 87.2 to 147.2 pA. About the microstructural analysis, the microstructure obtained show different phases as characteristic of the processing method, including porosity. The peculiar optical and thermal properties observed in the piezoelectric ceramic are promising for possible applications in temperature-controlled optical devices that require electrical outputs

Microstructural Study of a Zn-Ni Alloy Prepared by Ball Milling Using Two Different Devices

Metallic zinc (Zn) has ben extensively used as protective coating of iron and steel for decades, but problems related with its high permeability has reduced its application. It was found that the corrosion resistance of zinc in form of alloys is increased by adding some transition metals. Recently, the Zn-Ni system is under research as an efficient alternative as anticorrosion coating for metals. Zn-Ni is mostly prepared by electroplating or thermal spray technologies, but there are some problems like: (i) fluctuation of Zn-Ni contents, (ii) the pollution caused by plating solutions and (iii) irregular thickness of the coating. The mechanical alloying (MA) technique can be used to fulfill the above issues because this route facilitates the synthesis of homogeneous materials from powder mixtures . Also, MA is frequently employed for the preparation of new materials based on mechanochemical reactions performed at room temperature, while avoiding the conventional ingot metallurgy [3]. This work deals with the preparation and study of a Zn-Ni alloy prepared by MA using two types of milling devices: a planetary which works manly through abrasion and impact of grinding balls and the Spex which works through the high-energy impact of balls. Zn-Ni was prepared from pure Ni and Zn powders, the equiatomic compositions were weighed and milled for 4 hours followed by sintering at 357°C during 1h