The Gram-positive pathogens Corynebacterium diphtheriae and Streptococcus pyogenes both require iron for survival. These bacteria have developed sophisticated heme uptake and transport protein machinery responsible for the import of iron into the cell, in the form of heme from the human host. The heme utilization pathway (hmu) of C. diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. The axial ligation of the heme in HmuT was probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, R237A, Y272A, M292A, Y349A, and Y349F. Characterization by UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopies indicated that H136 and Y235 are the axial ligands in HmuT. Electrospray ionization mass spectrometry was also utilized to assess the roles of conserved residues in contribution to heme binding. The S. pyogenes streptococcal iron acquisition (sia)/heme transport system (hts) utilizes multiple proteins to bring host heme to the intracellular space. Both the substrate binding protein SiaA and the hemoprotein surface receptor Shr were investigated. The kinetic effects on SiaA heme release were probed through chemical unfolding of axial ligand mutants M79A and H229A, as well mutants thought to contribute to heme binding, K61A and C58A, and a control mutant, C47A. The unfolding pathways showed two processes for protein denaturation. This is consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket. The ease of protein unfolding is related to the strength of interaction of the residues with the heme. Shr contains two NEAT (near-iron transporter) domains (Shr-N1 and Shr-N2) which can both bind heme. Biophysical studies of both Shr-N1 and Shr-N2 indicated a new class of NEAT domains which utilize methionine as an axial ligand, rather than a tyrosine. Thermal and chemical unfolding showed ferrous Shr-N1 and Shr-N2 to be most resistant to denaturation. Shr-N2 was prone to autoreduction. Together, sequence alignment, homology modeling, and spectral signatures are all consistent with two methionines as the heme ligands of this novel type of NEAT heme-binding domain.

The main motivation of this thesis is to study the long standing problem of mixed alkali effect (MAE) in oxyfluro vanadate glass systems from the point of view of structural arrangement and to investigate the effect of two rare earth ions, namely, erbium and europium on the structure of tellurium dioxide based glass. In glass science, it is well known that when one alkali in a glass matrix is gradually replaced by another alkali, leads to a non-linear variation in some of the physical properties. There are many a theories trying to explain this effect. Recently the renewed interest lies in explaining MAE in-terms of structural consideration. Rare earth (RE) ion doped glasses are of interest in a variety of applications in photonics because of the special optical properties exhibited by these materials. Atomic like f-f transitions of RE ions depend on the local environment of the these ions. A particular glass matrix may be able to bring out the optical properties of a RE ion better than the other matrix. In this regard structural characterization of a glass matrix with different RE ions for optical properties is of importance. Spectroscopic tech-niques are best suited to investigate structural arrangement in glasses and in this thesis we have used techniques like Raman, electron paramagnetic resonance (EPR), impedance, photoluminescence and UV- visible absorption spectroscopies. The thesis comprises of five chapters and an appendix. Chapter 1 consists of brief introduction of general properties of glasses and their behavior under different spectroscopic techniques. Chapter 2 deals with the sample preparation and the experimental techniques used in this work. Chapter 3 elaborates on the Raman and EPR spectroscopic studies on the structural arrangement of the mixed alkali oxyfluro vanadate glass systems. Chapter 4 focuses on the ac and electrical modulus analysis to study the MAE in the above samples. Chapter 5 deals with the optical spectroscopic techniques used to study the compatibility of RE ions (erbium and europium) with the tellurium dioxide based glass matrix. Chapter 1: Disordered materials pose a challenge to understand their structure mainly because of their random arrangement of the constituent units. In this chapter the glassy systems and the different experimental techniques used to study them are discussed in general. The behavior of glassy systems to the external stimuli in various frequency ranges is highlighted. A short review of mixed alkali effect in glasses mainly covering the advances in the last two decades is given. Brief outline of the theory of Raman, EPR and ac impedance spectroscopy are given. Chapter 2: This chapter discusses the major experimental techniques used in the thesis to study the glass systems at block diagram level. The Raman and EPR spectrometers are discussed. Experimental technique used in ac impedance measurement is outlined. Different methods of preparing glass are listed and melt quenching technique is discussed in detail. Chapter 3: This chapter discusses the results and analysis of Raman and EPR study in oxyfluoro vanadate glasses emphasizing MAE. The glass having batch formula 40V2O5 - 30BaF2 - (30 - x) LiF - xRbF (x = 0 – 30) is prepared by melt quenching technique. Raman spectroscopic study in back scattering geometry is performed to see the effect of alkali ions on the V – O bond length of VO6 polyhedra in the glass. The de-convoluted Raman peaks corresponding to V = O and VO2 are considered and the effect of alkali mixture on these bonds are studied. • The peak shift of V = O and VO2 bonds shows that V = O is affected only a little by the replacement of lithium (Li) by rubidium (Rb), while VO2 bond gets affected to a larger extent. • From the peak shift the most probable value of the bond length and the spread in it are estimated. The bond length corresponding to V = O is found to increase and that of VO2 decrease as a consequence of alkali replacement. • From the FWHM of the corresponding Raman peaks, it is concluded that O - Rb coordination sphere around VO6 polyhedra is more homogeneous than either O – Li or O - Li /Rb coordination. These results are published in J. Non-Cryst. Solids 370 (2013) 6. EPR studies on the samples are carried out in X band frequency and spin – Hamilto-nian parameters were extracted by simulating and fitting the EPR spectra to experimental data using EasySpin which is a Matlab toolbox. • it is observed that the ratio 4gjj=4g?, which is a measure of tetragonality of octa-hedral crystal symmetry of V2O5, varies non-monotonically with Rb content. • A model based on this observation is proposed. The essential idea of this model is that Rb atoms that are substituted for Li atoms initially prefer terminal positions over planar positions. Continued substitution then replaces planar Li atoms. It is seen that this model of “preferential substitution” explains the observation very well. • Another observation is that the EPR signal intensity, which is due to concentration of V4+ ions, also shows non-monotonous behavior with Rb content. This is also explained using preferential substitution, taking into consideration the oxidation states of the vanadium ions. • The value of 4gjj=4g?, is a minimum for all rubidium environments around V2O5, which infers that Rb coordination is more symmetric than all Li or Li−Rb. • A good correlation is found between Raman and EPR study of the above system. These results are published in J. Phys. Chem. A 118 (2014) 573. Chapter 4: The chapter brings out the results of ac conductivity and electrical mod-ulus study of MAE in the glass system mentioned above. The Agilent 4294A precision impedance analyzer operating in frequency range 40 Hz to 110 MHz, is used for per-forming impedance and capacitance experiments carried out in this thesis. Impedance measurements in our studies are performed in sandwich geometry. • Room temperature dc conductivity shows a decrease as Li is replaced by Rb and reaches its minimum - five orders less than its all Li value at 0.33 molar fraction of Rb, which is attributed to MAE. This observation is explained using the structural aspect. • Using the linear response theory the number of mobile ions participating in the conduction is estimated. • Imaginary part of the electrical modulus is fitted to Kohlrausch – Williams – Watts (KWW) relation by using a complex nonlinear least squares fitting procedure given by Bergmann. • The stretching parameter b estimated from the above procedure is found to exhibit MAE. The observed variation in b with Rb mole fraction is explained by taking into considerations the contributions from fast and slow processes, and coupling between different relaxing sites. The manuscript is under preparation. Chapter 5: This chapter illustrates the optical study of RE doped TeO2 based glasses to determine the suitability of a particular RE ion with a given glass matrix. TeO2 based glasses having a general formula (in mol %) 65TeO2 – 5BaF2 – 30ZnF2 (TBZ) were prepared by usual melt quenching technique. RE doping was done at the expense of TeO2. 3 mol % of Eu or Er are added to prepare RE doped glass. Raman, PL, UV-visible absorption studies are carried out on the glass samples. • From the peak shift, intensity variation and FWHM of the Raman spectra of the glass samples it is observed that Eu doped TBZ glass has a greater tendency towards depolymerizing the glass matrix by influencing the conversion of TeO4 units into the formation of TeO3 units. • PL spectra of the glass samples shows emission due to different possible transitions. Position of the peak of the de-convoluted spectra shows the position of the particular Stark component and the FWHM is a measure of the inhomogeneous broadening. • The UV-visible absorption spectra are used to calculate the optical density and fitted to the Mott equation to determine the band edge of the glass samples. It is seen that Eu doped TBZ glass has a lesser band gap than that of Er doped glass. The manuscript is submitted to Bul. Mat. Sci. Appendix : This consists of a collection of details of EDS study carried on the VBL series glasses and some MATLAB codes used to simulate the EPR spectrum for VBL series glasses.

Les plasmas froids sont des gaz partiellement ionisés, très riches d’un point de vue physico-chimique. Cette propriété se retrouve dans des plasmas froids aujourd’hui générés à pression atmosphérique et température ambiante et a été mise à profit depuis une quinzaine d’années environ pour des applications biomédicales (hématologie, dermatologie, cancérologie, odontologie etc…). L’efficacité de ces plasmas froids dans le domaine de la médecine a été prouvée par de nombreuses études. Cependant, les phénomènes biologiques mis en jeu ne sont pas encore bien compris, et il primordial de savoir quels pourraient être les éventuels effets secondaires indésirables de ces milieux ionisés réactifs. Le premier niveau d’interaction des plasmas avec le vivant est celui avec les milieux liquides, qui sont présents en surface des tissus, des cellules in vivo ou en culture. Depuis une décennie, une attention particulière a donc été portée aux interactions des plasmas avec les liquides, pour apporter un niveau de compréhension supplémentaire. La compréhension de ces interactions a constitué l’axe de ce travail. Différents réacteurs à plasmas froids (générés à pression atmosphérique et température ambiante) ont été développés, notamment afin de contrôler les interactions du plasma avec l’air ambiant qui peuvent être problématiques pour les applications visées. La nature du gaz servant à initier le plasma a été modifiée, pour connaître son influence sur la réactivité chimique de la phase gaz. Pour cela, des mesures de spectroscopie d’émission optique (SEO) ont été nécessaires. Par ailleurs, de nouveaux capteurs électrochimiques et des approches méthodologiques ont été développés pour identifier et quantifier les espèces réactives de l’oxygène et de l’azote (RONS) produites dans des milieux liquides physiologiques, exposés à ces gaz ionisés. Les analyses électrochimiques ont été combinées à de la spectroscopie d’absorption UV-visible ainsi qu’à d’autres méthodes de chimie (pH-métrie/conductimétrie). Un des objectifs visés est d’établir une corrélation entre les espèces réactives générées dans la phase gaz et dans la phase liquide. Enfin, des expérimentations nous ont permis d’analyser la production des RONS dans des liquides in situ en temps réel. Les mesures de SEO montrent qu’il existe de nombreuses espèces chimiques excitées au sein des différents plasmas (NO°, HO°, O, N2+ (FNS) etc…). Les analyses de la phase liquide ont révélé la présence d’espèces stables de l’oxygène et de l’azote (H2O2, NO2-, NO3-), directement reliées aux espèces détectées dans les plasmas. De plus, les diverses méthodologies d’analyse chimique mises en place ont permis la détection et la quantification de RONS tels que l’anion peroxynitrite ONOO-. L’ensemble des résultats obtenus devrait permettre d’appréhender de façon plus fine les effets induits par différents plasmas froids dans des milieux liquides physiologiques afin d’établir un lien avec les études menées sur des cellules en culture et sur la peau dans le cadre d’un programme de recherche financé par l’ANR, Agence Nationale de la recherche. / Cold plasmas are partially ionized gases, very rich in a physico-chemical point of view. This property characterizes cold plasmas today generated at atmospheric pressure and ambient temperature and was used since about fifteen years approximately for biomedical applications (haematology, dermatology, cancer research, odontology etc.). The efficiency of these cold plasmas in the field of the medicine was proved by numerous studies. However, the involved biological phenomena are not still well included, and it is essential to know what could be the possible unwanted side effects of these reactive ionized gases. The first level of interaction of plasmas with living matter is the one with the liquid phase, which is present on the tissue surface, in vivo cells or in culture. For a decade, a particular attention was thus worn in the interactions of plasmas with liquids, to bring a level of additional understanding. The understanding of these interactions constituted the axis of this work. Various cold plasmas reactors (generated at atmospheric pressure and ambient temperature) were developed, in order to control the interactions of these plasmas with the ambient air which can be problematic for the aimed applications. The nature of the gas used to initiate the plasma was modified, to know its influence on the chemical reactivity of the gas phase. For that purpose, measurements of optical emissive spectroscopy (OES) were necessary. Besides, new electrochemical sensors and methodological approaches were developed in order to identify and quantify the reactive nitrogen and oxygen (RONS) produced in physiological liquid media, exposed to these ionized gases. The electrochemical analyses were combined UV-visible absorption spectroscopy as well as other methods of chemistry (pH-metry/conductimetry). One of the aimed objectives is to establish a correlation between the reactive species generated in the gas phase and in the liquid phase. Finally, experiments allowed us to analyze the production of RONS in liquids in situ and in real time. OES measurements showed that there are numerous chemical species generated in various plasmas (NO°, HO°, O, N2+ (FNS) etc.). The analyses of the liquid phase revealed the presence of stable oxygen and nitrogen species (H2O2, NO2-, NO3-), directly correlated with the species detected in plasmas. Furthermore, the diverse methodologies of chemical analysis allowed the detection and quantification of RONS such as the peroxynitrite anion ONOO-. The obtained results should allow to arrest in a finer way the effects led by various cold plasmas in physiological liquid media to establish links with the studies led on cultured cells and on skin within the framework of a research program financed by the ANR, National Agency of the Research.