Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc

Zinc (Zn2+) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn2+ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn2+, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn2+ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn2+ sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn2+ sensors by introducing the first generation of red-shifted cpFP Zn2+ sensors. We demonstrate that not only can these Zn2+ sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn2+ concentration. We also show that red-shifted cpFP Zn2+ sensors display high sensitivity for detecting labile Zn2+, similar to the green-shifted cpFP Zn2+ sensors. These discoveries add to the current knowledge of labile Zn2+ within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn2+ in cells that was previously unavailable

Raman Spectroscopy of n-Type and p-Type GaSb with Multiple Excitation Wavelengths

The interpretation of Raman spectra of GaSb can be complicated by the presence of a so-called surface space charge region (SSCR), resulting in an inhomogeneous near-surface Raman scattering environment. To fully interpret Raman spectra, it is important to have an understanding of the SSCR profile relative to the Raman probe depth. However, a priori determination of even the actual SSCR width is not always possible for GaSb under a wide range of doping levels. The primary objective of this report is to provide a convenient reference to aid in the determination of relative contributions to an observed GaSb Raman spectrum of SSCR scattering and bulk scattering for a range of excitation wavelengths, doping levels, and SSCR widths and types. Hence, Raman spectra of both n-type and p-type doped GaSb epilayers were obtained using 488 nm, 514.5 nm, 647.1 nm, and 752.55 nm excitation radiation. Both n-type and p-type doped GaSb epilayers were selected for investigation because these layers exhibit the two different SSCR types that are typically encountered with as-grown GaSb and related materials. A range of doping levels were examined for each doping type so as to examine the effects of a varying SSCR width on the observed spectra. A secondary objective of this report is to demonstrate the performance of a spectroscopic system based on 752.55 nm excitation that is sensitive to bulk carrier properties in n-type and p-type doped GaSb epilayers over a wide doping range, unlike visible wavelength-based optical systems

Raman Spectroscopy Determination of Hole Concentration in p-Type GaSb

Room temperature p-type GaSb bulk coupled mode spectra were measured as a function of hole concentration. These spectra were obtained using an optical system based on 752.55 nm excitation in order to obtain more sensitivity to bulk GaSb coupled mode scattering than possible with visible wavelength excitation-based systems. A relatively simple spectral model for the electronic contribution to the dielectric function was evaluated for determination of hole concentration from the bulk coupled mode spectra. Optically-derived values for hole concentration were determined by minimizing the sum of the residuals squared between an experimental and simulated spectrum as a function of total hole concentration and a plasmon damping parameter. Hole concentrations obtained from the Raman spectroscopic measurements deviated from the values determined from single field Hall effect measurements that were corrected to account for two band conduction by {approx}20% to {approx}65%. These deviations were attributed to the limitations of the spectral model employed and uncertainties in GaSb materials properties

Optical Cell for Combinatorial \u3ci\u3eIn Situ\u3c/i\u3e Raman Spectroscopic Measurements of Hydrogen Storage Materials at High Pressures and Temperatures

An optical cell is described for high-throughput backscattering Raman spectroscopic measurements of hydrogen storagematerials at pressures up to 10 MPa and temperatures up to 823 K. High throughput is obtained by employing a 60 mm diameter × 9 mm thick sapphire window, with a corresponding 50 mm diameter unobstructed optical aperture. To reproducibly seal this relatively large window to the cell body at elevated temperatures and pressures, a gold o-ring is employed. The sample holder-to-window distance is adjustable, making this cell design compatible with optical measurement systems incorporating lenses of significantly different focal lengths, e.g., microscope objectives and single element lenses. For combinatorial investigations, up to 19 individual powder samples can be loaded into the optical cell at one time. This cell design is also compatible with thin-film samples. To demonstrate the capabilities of the cell,in situ measurements of the Ca(BH4)2 and nano-LiBH4–LiNH2–MgH2hydrogen storage systems at elevated temperatures and pressures are reported

First-order Raman spectra of double perovskites AB′1/2B'{1/2}B''{1/2}O3

First principles computations of Raman intensities were performed for perovskite-family compound CaAl$_{1/2}$Nb$_{1/2}$O$_3$ (CAN). This compound features 1:1 (NaCl-type) ordering of Al and Nb superimposed onto the $b^-b^-c+$ octahedral tilting. Raman tensor for CAN was computed using the package for first-principles computations ABINIT (URL \underline {http://www.abinit.org}). Computations performed for both untilted cubic ($Fm\bar{3}m$) and tilted monoclinic ($P2_1/n$) CAN structures showed that the strongest Raman lines are associated with the ordering of Al and Nb. The computed spectrum agreed qualitatively with the experimental data measured on powder (CAN is available in polycrystalline form only). The effect of cation disorder on the Raman intensities was considered using phenomenological theory of light scattering in the vicinity of a phase transition. We suggest that, for certain modes, the corresponding Raman intensities depend primarily on the average long range order while, for other modes, the intensities are determined by fluctuations of the order parameter.Comment: 4 figures, submitte

A thermosyphon-driven hydrothermal flow-through cell for in situ and time-resolved neutron diffraction studies

A flow-through cell for hydrothermal phase transformation studies by in situ and time-resolved neutron diffraction has been designed and constructed. The cell has a large internal volume of 320 ml and can operate at temperatures up to 573 K under autogenous vapor pressures (ca 8.5 106 Pa). The fluid flow is driven by a thermosyphon, which is achieved by the proper design of temperature difference around the closed loop. The main body of the cell is made of stainless steel (316 type), but the sample compartment is constructed from non-scattering Ti–Zr alloy. The cell has been successfully commissioned on Australia’s new high-intensity powder diffractometer WOMBAT at the Australian Nuclear Science and Technology Organization, using two simple phase transformation reactions from KAlSi2O6 (leucite) to NaAlSi2O6H2O (analcime) and then back from NaAlSi2O6H2O to KAlSi2O6 as examples. The demonstration proved that the cell is an excellent tool for probing hydrothermal crystallization. By collecting diffraction data every 5 min, it was clearly seen that KAlSi2O6 was progressively transformed to NaAlSi2O6H2O in a sodium chloride solution, and the produced NaAlSi2O6H2O was progressively transformed back to KAlSi2O6 in a potassium carbonate solution

Non-Contact Determination of Free Carrier Concentration in n-GaInAsSb

GaSb-based semiconductors are of interest for mid-infrared optoelectronic and high-speed electronic devices. Accurate determination of electrical properties is essential for optimizing the performance of these devices. However, electrical characterization of these semiconductors is not straightforward since semi-insulating (SI) GaSb substrates for Hall measurements are not available. In this work, the capability of Raman spectroscopy for determination of the majority carrier concentration in n-GaInAsSb epilayers was investigated. Raman spectroscopy offers the advantage of being non-contact and spatially resolved. Furthermore, the type of substrate used for the epilayer does not affect the measurement. However, for antimonide-based materials, traditionally employed Raman laser sources and detectors are not optimized for the analysis wavelength range dictated by the narrow band gap of these materials. Therefore, a near-infrared Raman spectroscopic system, optimized for antimonide-based materials, was developed. Ga{sub 0.85}In{sub 0.15}As{sub 0.13}Sb{sub 0.87} epilayers were grown by organometallic vapor phase epitaxy with doping levels in the range 2 to 80 x 10{sup 17} cm{sup -3}, as measured by secondary ion mass spectrometry. For a particular nominal doping level, epilayers were grown both lattice matched to n-GaSb substrates and lattice-mismatched to SI GaAs substrates under nominally identical conditions. Single magnetic field Hall measurements were performed on the epilayers grown on SI GaAs substrates, while Raman spectroscopy was used to measure the carrier concentration of epilayers grown on GaSb and the corresponding SI GaAs substrates. Compared to Hall measurements, Raman spectra indicated that the GaInAs/Sb epilayers grown on GaSb substrates have higher free carrier concentrations than the corresponding epilayers grown on SI GaAs substrates under nominally identical conditions. This is contrary to the assumption that for nominally identical growth conditions, the resulting carrier concentration is independent of substrate, and possible mechanisms will be discussed

HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes

During the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization

Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states