In this thesis I present a microfluidic platform developed to control dissolved gases and monitor dissolved oxygen concentrations within the microenvironment of isolated small blood vessels. Dissolved gas concentrations are controlled via permeation through the device substrate material using a 3D network of gas and liquid channels. Dissolved oxygen concentrations are measured on-chip via fluorescence quenching of an oxygen sensitive probe embedded in the device. Dissolved oxygen control was validated using the on-chip sensors as well as a 3D computational model. The platform was used in a series of preliminary experiments using olfactory resistance arteries from the mouse cerebral vascular bed. The presented platform provides the unique opportunity to control dissolved oxygen concentrations at high temporal resolutions (<1 min) and monitor dissolved oxygen concentrations in the microenvironment surrounding isolated blood vessels.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

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No description available.

This thesis investigates the impact of doping on the optical and structural characteristics of cerium oxide (ceria) nanoparticles synthesized using chemical precipitation. The dopants selected are samarium and neodymium, which have positive association energy with oxygen vacancies in the ceria host, and negative association lanthanides, holmium and erbium, as well as two metal dopants, aluminum and iron. Characteristics measured are absorption and fluorescence spectra and the diameter and lattice parameter of ceria. Analysis of the characteristics indicates qualitatively that the dopant controls the O-vacancy concentration and the ratio of the two cerium ionization states: Ce+3 and Ce+4. A novel conclusion is proposed that the negative association lanthanide dopants can act as O-vacancies scavengers in ceria while the O-vacancy concentration increases in ceria doped with positive association lanthanide elements. Doped ceria nanoparticles are evaluated in two applications: dissolved oxygen (DO) sensing and up-conversion. In the first application, ceria doped with either Sm or Nd and ceria doped with aluminum have a strong correlation between the fluorescence quenching with the DO concentration in the aqueous solution in which the ceria nanoparticles are suspended. Stern-Volmer constants (KSV) of doped ceria are found to strongly depend upon the O-vacancy concentration and are larger than some of the fluorescent molecular probes currently used to measure DO. The KSV measured between 25-50oC is found to be significantly less temperature dependent as compared to the constants of commercially-available DO molecular probes. In the second application, up-conversion, ceria nanoparticles doped with erbium and an additional lanthanide, either Sm or Nd, are exposed to IR radiation at 780 nm. Visible emission is only observed after the nanoparticles are calcinated at high temperature, greatly diminishing the concentration of O-vacancies. It is concluded that O-vacancies do not play a dominant role in up-conversion, unlike that drawn for down-conversion, where the fluorescence intensity is strongly correlated with the O-vacancy concentration. Correlations between annealing temperatures, dopant, and dopant concentrations with the power dependence of up-conversion on the pump and the origin of the intensities of the visible emission are presented. These studies show the promise of doped ceria nanoparticles. / Ph. D.

This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The &alpha;/&beta;-heterodimer HIF (<u>H</u>ypoxia <u>I</u>nducible <u>F</u>actor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF &alpha;-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 Å), one containing a Trichoplax adhaerens HIF&alpha; subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C⁴-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIF&alpha; hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.

No description available.

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

Perovskite oxides show wide range of applications in the area of magnetism, ferroelectricity, piezoelectricity, thermoelectricity, gas sensing, catalyst development, solid oxide fuel cell, etc. This is due to flexibility in the structure and compositions that can be tuned by specific element doping. In the perovskite oxide (ABO3), large cation (A) is 12 -coordinated and smaller B-cation is 6 coordinated with oxide ions. Oxide materials are considered as better candidates for thermoelectric applications (interconversion of thermal into electrical energy) due to its non-toxicity and thermal stability at elevated temperature. These are insulating in nature and the conductivity can be increased by doping A and / or B –sites. Perovskite oxides are also used for oxygen monitoring in different applications including control and optimization of combustion of fossil fuels in industries and automobiles, biological and defines places, etc. In the present study, we focused on thermoelectric properties in single perovskite oxides of lanthanum cobaltite and calcium manganite and a double perovskite oxide of dysprosium barium cobaltite. Also, the oxygen sensing behaviour of dysprosium barium cobaltite at elevated temperatures is studied. The thesis contains seven chapters and a summary of respective chapters are given below. The first chapter outlines the basics of thermoelectric and gas sensing applications of both perovskite and double perovskite oxides. In the initial part, thermoelectric phenomena are explained. Thermoelectric effect is the conversion of thermal energy to electrical energy and vice-versa. Higher thermoelectric efficiency (η) can be achieved by maintaining a large temperature difference across the material. The efficiency depends on the thermoelectric figure of merit (zT) of material, which depends on thermopower (S), electrical resistivity (ρ) and thermal conductivity (κ) of the material and hence needs to be optimized. The latter part discusses the oxygen sensing property of distorted double perovskite 112 structure type as it shows advantages over other materials due to oxygen nonstoichiometric. Further, an overview of the relevant literature, objective and scope of the thesis are mentioned. The second chapter elucidates the materials and methods used for the present work. The materials viz. LaCoO3, CaMnO3-δ and DyBaCo2O5+δ, were selected for thermoelectric and oxygen sensing studies. Both the conventional solid state and soft chemistry methods were adopted for the synthesis of these materials. Powders were densified into pellets by hot uniaxial pressing / cold isostatic pressing and various heat treatments were carried out. Samples thus prepared were phase pure as confirmed using powder x-ray diffraction and Rietveld refinement performed for structural analysis. Morphological studies were carried out using scanning electron microscopy and transmission electron microscopy. Further Raman and x-ray photoelectron spectroscopic characterization of these materials were discussed. The transport properties viz. electrical resistivity, thermopower and thermal conductivity of compact pellets were measured at elevated temperatures. Further, the home-built apparatus for room temperature See beck measurements and chemo resistive oxygen sensing were explained in detail as a part of this work. The third chapter describes the effect of monovalent ion doping (Na+ and K+) at A-site of lanthanum cobaltite on thermoelectric properties. Lanthanum cobaltite system exhibit exotic behaviour due to commensuration phenomena of spin, lattice, charge and metal insulator transition. The synthesis, followed by structural refinements by Rietveld method using Fullprof suit program are explained. The results of the transport properties indicate that there is no appreciable change in the See beck Coefficient of K-doped samples throughout the studied temperature range. The Na-doped samples exhibit a decrease in the Seebeck value with increasing Na content at room temperature. At higher temperatures Seebeck value matches with that of the parent sample. This may be due to a change in the ratio of the concentration of Co4+/Co3+ ions which increases the configurational entropy of the system. In conclusion, the highest figure of merit (0.01) found for the Na / K- doped lanthanum cobaltite is for 15 atomic wt. % of doping amongst the studied samples. The fourth chapter explains about Tb/Nb co-doped calcium manganite for thermoelectric applications. The CaMnO3-δ shows enhanced thermoelectric properties, exhibits n-type behavior and the absolute thermopower is found to be 129 µV/K. Here, we investigated the Terbium and Niobium codoped at Ca and Mn-sites respectively. The presence of oxygen non-stoichiometry was confirmed using Raman spectroscopy (Mn3+ peak at 614 cm-1) and δ value was evaluated by iodometric titration. The thermoelectric properties of cold isostatic pressed (CIP) pellets prepared by the solid state and soft chemistry routes are compared. The non-monotonous behavior of absolute thermopower may be due to the increase of Mn3+ in the Mn4+ matrix and also the presence of oxygen defects in compounds. The thermoelectric figure of merit of solid state sample CaMnO3-δ estimated of 0.036 at 825K. The fifth chapter describes the thermoelectric properties of double Perovskite AA’B2O6 (112 type): (RE)BaCo2O5+δ. It is a disordered double perovskite with non-stoichiometry in oxygen and exhibits mixed valences of Cobalt. Resistivity of DyBaCo2O5+δ was found to be 0.09 Ω cm and Seebeck coefficient is found to be 42 µV/K. In order to improve the thermopower value, the Fe is substituted at Co-site. This varies the valences of Cobalt that in turn leads to a higher thermopower. Also, the morphology of thermally etched CIP pellets recorded and correlated with the transport properties. It shows the highest thermoelectric figure of merit of 0.25 at 773 K for 20 at wt % of Fe substituted sample. The sixth chapter explains about oxygen sensing studies of DyBaCo2O5+δ (112 type). The detailed structural and morphological characterization studies were carried out. Thermogravimetric analysis at isothermal temperature 873 K shows fast intake/release of oxygen of this disordered double perovskite structure. The higher chemo resistive oxygen sensitivity at the elevated temperature was measured. Further, the systematic study on the effect of oxygen sensing on the substitution of Fe and Cu at Co-site in DyBaCo2-xM xO5+δ was investigated. The possible bulk diffusion mechanism at higher temperature due to movement of oxygen defects were explained. The highest sensitivity was obtained for x = 0.4 at % of Fe and 0.2 at % of Cu at 973 K and 823 K respectively. The key findings and future aspects are summarized in the chapter-7.