L'importance des effets relativistes dans la chimie a été reconnu depuis les années 1980. Par exemple, sans la relativité (a) l'or aurait la même couleur que l'argent (b) le mercure ne serait pas liquide à la température ambiante et (c) nos voitures ne démarrent pas avec une batterie de plomb. Pour une description théorique de la structure et la réactivité des éléments lourds, la relativité est un ingrédient essentiel. Le hamiltonien pour les calculs moléculaires relativistes à 4 composantes est construit en remplaçant la partie mono-électronique de l'hamiltonien électronique non-relativiste par le hamiltonien de Dirac. La partie bi-électronique est approchée par le terme de r Coulomb comme dans le cas non relativiste, ce qui donnel'hamiltonien de Dirac-Coulomb (DC). Pour réduire le coût de calcul, on peut utiliser des hamiltoniens relativistes à 2 composantes. Parmi eux, l'hamiltonien exact à 2 composantes (X2C) est le plus précise. La corrélation électronique est, cependant, une contribution très importante pour obtenir une description théorique à la fois qualitative et quantitative des spectroscopies moléculaires, réactions, etc. Dans cette thèse, nous avons étudié l'interaction entre la relativité et de la corrélation. à la fois par des développements méthodologiques et par des applications moléculaires. Dans la première partie de la thèse, nous avons calculé les constantes spectroscopiques dimères des gaz rares lourds. La liaison faible de ces dimères ne peut être décrit que par l'inclusion de la corrélation électronique. Les dimères des gaz rares les plus lourds, le radon et l'eka-radon, nécessite de plus un traitement adéquat de la relativité. Nos calculs sont basés sur l'hamiltonien X2Cmmf, à la fois avec des méthodes de corrélation basés sur une fonction d'onde et séparation de porte (srDFT). La deuxième partie de cette thèse concerne la simulation de la spectroscopie des rayons X, où l'on sonde la région du cœur d'une molécule, ou la relativité joue un rôle très important. Nous avons étudié la spectroscopie L-edge de la série isoélectronique: UO22 +, UNO+, et UN2, où le couplage spin-orbite joue un rôle majeur. Au niveau des méthodes, nous avons considéré MP2 à couches ouvertes et la théorie de la fonctionnelle de la densité dépendante de temps (TDDFT). Dans un autre étude, nous avons simulé la spectroscopie K-edge de la série H2X (X = O, S, Se, Te) et XH3 (X = N, P, As) ainsi que les molécules N2 et N2O2. Pour ces systèmes, l'interaction spin-orbite est moins important. Par conséquent, nous avons utilisé un hamiltonien DC sans spin (SF). Certains des systèmes pris en compte dans ce travail sont de caractère multi-référentielles ; nous avons utilisé la methode Coupled Cluster Multi-référentielle de type State Universal et adapté au groupe unitaire (UGA-SUMRCC) comme une méthode de corrélation. Dans la troisième et partie principale de la thèse, l'attention est de nouveau sur la relativité et de la corrélation, mais pour le calcul des propriétés électriques et magnétiques moléculaires. Nous avons développé et mis en œuvre un module pour le calcul des valeurs moyennes au niveau relativiste à 4-composantes coupled cluster monoréferentiel. Les propriétés qui sondent la densité électronique près de noyaux (lourds), telles que la résonance paramagnétique électronique (RPE), les paramètres des gradients de champ électrique et la non-conservation de la parité (NCP) des molécules chirales ,sont parfaitement adaptés pour l'application de cette méthode. Pour l'instant, nous avons étudié que la NCP. Ce module dans le logiciel DIRAC pour les calculs moléculaires relativistes fournit un cadre propice pour la mise en œuvre de méthodes de CC relativistes employant la symétrie de groupes doubles et de permutation de manière très efficace. En perspective, nous ciblons la mise en œuvre de la réponse linéaire CC pour le calcul des énergies d'excitation et propriétés moléculaires de second ordre tels que les paramètres de RMN. / The importance of relativistic effects in chemistry has been recognized since the 1980s. Without relativity (a) gold would have the same colour as silver (b) mercury would not be liquid at room temperature (c) our cars would not start (lead-battery). For a theoretical description of the structure and reactivity of heavy-elements, relativity is considered as an essential ingredient. The Hamiltonian for the 4-component relativistic molecular calculations is constructed by replacing the one-electronic part of the non-relativistic molecular Hamiltonian by the Dirac Hamiltonian. The two-electronic part of the Hamiltonian is approximated by the Coulombic repulsion term as in the non-relativistic case. The resulting Hamiltonian is called the Dirac-Coulomb (DC) Hamiltonian. For chemical applications there exist a class of relativistic Hamiltonians, where one-electronic part of the DC Hamiltonian is transformed to a 2-component one. Among them the eXcact 2-component (X2C) Hamiltonian is the most accurate one. Electron correlation, however, is a very important contribution to achieve a both qualitative and quantitative correct description of molecular spectroscopies, reactions etc. It is, therefore, essential to study the interplay between relativity and correlation. In this thesis, we have studied this interplay both in terms methodological developments and molecular applications. In the first part of the thesis we have studied the spectroscopic constants of the heavy rare gas dimers. The weak bonding of those dimers can only be described by the inclusion of electron correlation. The heavier analogues in the rare gas series i.e, Radon and eka-Radon, in addition require adequate treatment of relativity. Our calculations are based on the eXact 2-Component molecular-mean field (X2Cmmf) Hamiltonian both with wave function methods and range-separated DFT methods. The second part of this thesis simulates X-ray spectroscopy, where one probes the core region of a molecule. In the core region relativity plays a very significant role. Removal and excitation of electrons from that region involve various processes, which are beyond a mean-field description. We have studied L-edge spectroscopy of the isoelectronic series: UO22+, UNO+, and UN2, where spin-orbit coupling plays a major role. For the theory we have considered single reference open-shell MP2 and Time Dependent Density functional Theory (TDDFT). In another work, we have studied K-edge spectroscopy of the H2X (X= O, S, Se, Te) and XH3 (X= N, P, As) series as well as N2, N2O2 molecules. For this study spin-orbit coupling is less important, therefore, we have treated them with the Spin-Free (SF) DC Hamiltonian. Some of the systems considered in this work are Multi-Reference in nature; we have used Unitary Group Adapted (UGA) State Universal Multi-reference Coupled Cluster (UGA-SUMRCC) theory as a correlation method. In the third and major part of the thesis, the thrust is again on relativity and correlation, but for the calculation of molecular electric and magnetic properties. We have developed and implemented a module for the calculation of expectation values at the 4-component Relativistic Single Reference Coupled Cluster level. Properties that probe the electron density near (heavy) nuclei, such as Electron Paramagnetic Resonance (EPR) parameters, electric field gradients and parity non-conservation (PNC) in chiral molecules are ideally suited for the application of this method. However, we have only studied PNC so far. This module in the DIRAC software for relativstic molecular calculations provides a convenient framework for the implementation of relativistic CC methods employing double group and permutation symmetry very efficiently. In the near future we therefore target the implementation of Linear Response CC for the calculation of excitation energies and second-order molecular properties such as NMR parameters.

Uncertainty in rainfall forecasts affects the level of quality and assurance for decisions made to manage water resource-based systems. However, eliminating uncertainty in a complete manner could be difficult, decision-makers thus are challenged to make decisions in the light of uncertainty. This study provides statistical models as an approach to cope with uncertainty, including: a) a statistical method relying on a Gaussian mixture (GM) model to assist in better characterize uncertainty in climate model projections and evaluate their performance in matching observations; b) a stochastic model that incorporates the El Niño–Southern Oscillation (ENSO) cycle to narrow uncertainty in seasonal rainfall forecasts; and c) a statistical approach to determine to what extent drought events forecasted using ENSO information could be utilized in the water resources decision-making process. This study also investigates the relationship between calibration and lead time on the ability to narrow the interannual uncertainty of forecasts and the associated usefulness for decision making. These objectives are demonstrated for the northwest region of Costa Rica as a case study of a developing country in Central America. This region of Costa Rica is under an increasing risk of future water shortages due to climate change, increased demand, and high variability in the bimodal cycle of seasonal rainfall. First, the GM model is shown to be a suitable approach to compare and characterize long-term projections of climate models. The GM representation of seasonal cycles is then employed to construct detailed comparison tests for climate models with respect to observed rainfall data. Three verification metrics demonstrate that an acceptable degree of predictability can be obtained by incorporating ENSO information in reducing error and interannual variability in the forecast of seasonal rainfall. The predictability of multicategory rainfall forecasts in the late portion of the wet season surpasses that in the early portion of the wet season. Later, the value of drought forecast information for coping with uncertainty in making decisions on water management is determined by quantifying the reduction in expected losses relative to a perfect forecast. Both the discrimination ability and the relative economic value of drought-event forecasts are improved by the proposed forecast method, especially after calibration. Positive relative economic value is found only for a range of scenarios of the cost-loss ratio, which indicates that the proposed forecast could be used for specific cases. Otherwise, taking actions (no-actions) is preferred as the cost-loss ratio approaches zero (one). Overall, the approach of incorporating ENSO information into seasonal rainfall forecasts would provide useful value to the decision-making process - in particular at lead times of one year ahead.

This thesis investigates the chemistry of 25 ascidian species collected from Algoa Bay, South Africa with a concerted focus on metal accumulation by these ascidians and the possible interaction of these metals with ascidian metabolites. Chapter 2 details the screening techniques employed to establish the presence of nitrogenous metabolites (1H- 15N HMBC), hyper-accumulated metal ions (ICP-MS) and potential metal ion/ ascidian metabolite complexes (LC-ICP-MS/ESI-MS). Unfortunately, exhaustive attempts to detect intact metal ion/ascidian metabolite complexes through the use of liquid chromatography with parallel inductively coupled plasma mass spectrometry/electrospray mass spectrometry (LC-ICPMS/ ESI-MS) were unsuccessful. However, the LC-ICP-MS/ESI-MS data obtained for the crude organic extracts of six of the Algoa Bay ascidian species, Distaplia skoogi, Aplidium monile, Aplidium sp., Didemnum sp., Leptoclindines sp. and Polycitor sp. enabled identification of a number of ten halogenated metabolites, namely the indoles 2.28-2.30, and the tyramine and tyrosine derivatives (2.31-2.33, 2.41, 2.43, 2.44 and 2.46), within the ascidian extracts. This study confirmed that LC-ICP-MS/ESI-MS is a powerful tool for the dereplication of halogenated metabolites in complex mixtures especially where these compounds are present in very small amounts. This study is also the first report of these compounds (eight of which are known) in African ascidians. Compounds 2.32 and 2.46 have not been reported before from a marine source. Compounds 2.28-2.30 and 2.33 were present in sufficient amounts in the respective ascidian extracts to allow their isolation and structure elucidation using standard spectroscopic techniques Chapter 3 explores the ability of ascidians to accumulate a wide range of metal ions at concentrations which are often orders of magnitude higher than those of the surrounding sea water. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the total ion concentrations of 24 metals in 25 Algoa Bay ascidian species. To the best of our knowledge this is the largest and most extensive investigation of metal concentrations in a group of different ascidians occurring in the same area. Hypotheisizing that the metal ion concentrations for each ascidian specimen screened may represent a unique fingerprint for each specimen principal component analysis (PCA) was used in an attempt to establish whether there were spatial, temporal or phylogenetic relationships associated with the metal concentration fingerprints of the ascidians that formed part of this study. The PCA results showed that there were no statistically significant relationships between ascidian metal ion concentrations and either the collection year or the collection site of the ascidians. However, species from the family Didemnidae provided the clearest statistical evidence supporting a phylogenetic relationship between these ascidians and their hyperaccumulated metal ion profiles. Furthermore, these results suggested that ascidian species are indeed actively concentrating metal ions from the surrounding sea water and are not simply sinks for passively accumulated metal ions. Interestingly, the concentration of vanadium in the set of ascidians studied did not appear to correlate with any of the other metals accumulated by these ascidians suggesting that there is possibly a unique method employed for the accumulation of vanadium by ascidians. Chapter 4 investigated this possibility further after the nucleosides 4.10, 4.11, 4.13, 4.15, 4.17 and 4.40 were isolated from the vanadium accumulating ascidian Aplidium monile. Studies into the interactions between nucleosides and vanadyl are unfortunately rare and usually qualitative in nature with limited information provided about the stability or structures of the complexes formed. The vanadyl accumulating aplousobranch ascidians e.g. Aplidium monile dominated our study of Algoa Bay ascidians therefore providing us with the rationale to investigate the relatively little studied binding ability and stability of vandyl-nucleoside complexes. Potentiometric studies were conducted to determine the stability constants of complexes formed between the oxovanadium ion vanadyl (VO2+) and the commercially available nucleosides 4.10-4.14. The data afforded by this analysis clearly confirmed the complexity of the vanadyl/nucleoside complexation and suggested that guanosine (4.12) formed the most stable complex with oxovanadium ions. We were also able to establish a third protonation constant for the hydroxyl moiety in 4.12 with a logK 8.87 which has not been previously reported. Finally, Chapter 5 revisited the cytoxicity two Algoa Bay ascidians, Clavelina sp. and Atriolum marinense the extracts from which produced promising bioactivity results in previous studies against oesophageal cancer cells. The HP-20 fractionated extracts of Clavelina sp. and Atriolum marinense proved to be similalrly cytotoxic to breast cancer cells. With the exception for the 100% acetone(aq)fractions the NMR data for both species suggested that most active non polar fractions were dominated by what appeared to be structurally unremarkable fatty acid glycerides and as such were not pursued further. Purification of the 100% acetone(aq)fraction of A. marinense resulted in the isolation of a styrene trimer, 5.1, common to both ascidian extracts. The NMR simulation software WIN-DAISY was employed to confirm the structure of 5.1. Attempts to establish if 5.1 was an isolation artefact or a product of marine pollution were inconclusive

Les récepteurs métabotropiques du glutamate (mGluRs) sont des récepteurs couplés aux protéines G (RCPGs) modulant la transmission synaptique au sein du système nerveux central. D'un point de vue structural, ils ont la particularité de posséder un large domaine extracellulaire, le Venus Flytrap (VFT), où se lie leur ligand endogène, le glutamate. Leur domaine transmembranaire à 7 hélices, commun à tous les RCPGs, est connu pour être la cible d'une nouvelle classe de molécules à visée thérapeutique, les modulateurs allostériques. Au contraire, le VFT est le siège du développement de ligands compétitifs du glutamate et peu de choses sont connues quant à l'existence de modulateurs allostériques du VFT. Des études récentes ont mis en évidence une sensibilité des mGluRs aux ions extracellulaires et en particulier au chlore (Cl-), sans que son site de liaison ne soit identifié. Dans ce contexte, ce travail de thèse explore la possibilité d'une modulation allostérique endogène des mGluRs par les ions Cl-, en identifiant leur(s) site(s) de liaison(s) et leur effet sur la dynamique conformationnelle et la fonction des récepteurs. En combinant une approche pharmacologique, biophysique basée sur la technique de FRET, et la modélisation, nous avons tout d'abord confirmé que le Cl- potentialise l'action du glutamate sur tous les mGluRs et qu'il favorise la conformation active des récepteurs en se liant au niveau du VFT. Les mGluRs présentent également une sensibilité différente au Cl-, mGlu4 étant le plus sensible et mGlu2 le moins. Ceci s'explique notamment par le nombre de sites fonctionnels, tous les mGluRs dont mGlu4 possédant 2 sites par monomère à l'exception de mGlu2 qui n'en possède qu'un, en raison d'une mutation « clé » d'une sérine en aspartate dans le lobe 1 du VFT. D'autre part, le récepteur mGlu3 est apparu comme un cas particulier ayant une sensibilité accrue au Cl-, son domaine VFT cumulant la présence et l'orientation adéquate d'acides aminés formant un « verrou » Cl-, qui favorise de manière drastique la conformation active et une activité basale élevée de ce récepteur. Enfin, la modélisation de la variation de la concentration extracellulaire en Cl- lors d'une activité synaptique GABAergique est compatible avec une modulation des mGluRs les plus sensibles. En conclusion, le Cl- est un modulateur allostérique endogène des mGluRs et l'exploitation de ses sites de liaison au sein du VFT pourrait permettre le développement de nouveaux agents thérapeutiques. / Metabotropic glutamate receptors (mGluRs) are G coupled-protein receptors (GPCRs) playing key roles in synaptic transmission in the central nervous system. They display a large extracellular domain, the Venus Flytrap (VFT) where the endogenous ligand, glutamate, binds. Their 7 transmembrane helices spanning domain, common to all GPCRs, is known to be the target of new therapeutic compounds, called allosteric modulators. In contrast, VFT domain is used to develop glutamate competitive ligands and there are only few data about allosteric modulators targeting the VFT. Recent studies have shown mGluRs are sensitive to extracellular ions, particularly to chloride (Cl-), although its binding site has not been elucidated. This thesis work explores the possibility of an endogenous allosteric modulation of mGluRs by Cl-, aiming to delineate its binding site(s) and its effect on receptor conformational dynamics and function. Using pharmacological, FRET based biophysical approaches and modelling, we have first confirmed that Cl- potentiates glutamate action in all mGluRs and that this ion favors agonist induced active conformation by binding to the VFT. mGluRs are also differently sensitive to Cl-, mGlu4 being the most and mGlu2 the least. This difference is notably explained by the number of Cl- functional sites within the VFT, all mGluRs including mGlu4 displaying 2 sites per monomer whereas mGlu2 has only 1 site due to a serine-aspartate “key” mutation in VFT lobe 1. Besides, mGlu3 receptor appears to be a “special case”, as this receptor is highly sensitive to Cl- because its VFT domain is carrying amino acids creating a “Cl- lock”, which dramatically favors active conformation and a high level of basal activity. Finally, modelling of extracellular Cl- concentration variations in a GABAergic synapse is compatible with a modulation of the most sensitive mGluRs. In conclusion, Cl- is an endogenous allosteric modulator of mGluRs and exploiting its binding sites may yield to the development of innovative therapeutic tools.

Wideband, wide-scan antenna arrays are a promising candidate for the future wireless networks and as well as an essential part of experimental radio astrophysics. Understanding the underline physics of the element performance in the array environment is paramount to develop and improve the performance of array systems. The focus of this thesis is to develop novel wideband antenna array technologies and develop new theoretical insights of the fundamental limits of antenna arrays. The developed methodologies have also been extended to include a radio astrophysics application for the global 21cm experiment.   Investigating the fundamental antenna array limits and extracting general performance measures can provide a priori estimates for any application of arrays. In this thesis, a general measure for antenna arrays, the array figure of merit is proposed. This measure couples bandwidth, height from the ground plane and reflection coefficient in a bounded quantity. An extension of the array figure of merit that is able to provide matching, bandwidth and directivity/gain limits is also introduced. The soft Vivaldi array is introduced as a novel wideband, wide-scan angle array technology. Periodic structure loading has been utilized to improve the array's performance and mold the electromagnetic wave behavior to our benefit. The soft condition has been utilized in the same manner as the conventional soft-horn antenna at the Vivaldi element. An integrated matching layer in the form of periodic strip loading is introduced. A single polarized soft Vivaldi array prototype has been developed fabricated and measured. The developed finite array has been loaded with a soft condition in the periphery to mitigate edge effects. The results indicated improved cross-polarization and side-lobe levels. A new class of wideband antenna arrays, the Strongly Coupled Asymmetric Dipole Array (SCADA) was also proposed in this thesis. Exploiting asymmetry in the array element introduces an additional degree of freedom that improves bandwidth and scanning performance. A novel methodology for terminating finite arrays is also proposed. The theory and an experimental antenna array is presented with good agreement between measured and simulated results. An effort to integrate a vertical wide angle matching layer was also addressed and a prototype array with this concept is presented. In the last part of this thesis, a methodology for the detection of the global cosmological 21cm signal from the Epoch of Reionization (EoR) is developed. The main sources of errors in this experiment, the foregrounds and the antenna chromaticity are evaluated. A new algorithmic methodology for extracting the global EoR signal is proposed. The method is based on piecewise polynomial fitting and has successfully been applied and evaluated. An antenna array that is based on the methodologies described in this thesis has been developed and evaluated with the proposed algorithm. / Bredbandiga gruppantenner med stor utstyrningsvinkel är en av de lovande kandidaterna för nästa generations trådlösa kommunikationsnätverk samt en väsentlig del av experimentell radioastrofysik. Att förstå de bakomliggande fysikaliska principerna hos gruppantennens element är avgörande för att kunna utveckla och förbättra prestandan hos ett gruppantennsystem. Denna avhandling är fokuserad på att utveckla nya bredbandstekniker samt nya teoretiska insikter om de grundläggande gränserna för gruppantenner. De här utvecklade metoderna har förutom kommunikationstillämpningar också tillämpats på en radioastrofysik tillämpning i det globala 21cm experimentet. Att undersöka de fundamentala gränserna för gruppantenner och att utröna allmängiltiga mått på deras prestandaegenskaper kan möjliggöra a priori uppskattningar om gruppantenns tillämpbarhet för dess planerade användning. I den här avhandlingen föreslås ett allmänt kvalitetsmått på gruppantenner: gruppantennkvaliten. Detta mått kopplar samman främst bandbredd, reflektionskoefficienten med antennens tjocklek över ett jordplanet. En utvidgning av begreppet gruppantennkvaliten, presenters också i avhandlingen det kopplar samman bandbredd, matchning med antennens direktivitet/förstärkningsfaktor. En Vivaldi-gruppantenn med mjuka ytor introduceras här som en ny sorts bredbandig gruppantenn med stor utstyrningsvinkel. I antennen har en periodisk belastning inkluderats för att förbättra dess egenskaper, och för att forma antennens elektromagnetiska utstrålning till vår fördel. Den mjuka ytan på elementet har används på ett liknande sätt som det välkända korrigerade Vivaldihornets design, och har integrerats direkt i elementets design. Den här utvecklade ändliga gruppantennen har också en mjuk yta på dess yttre delar för att minska kanteffekternas påverkan av antennprestandan. Resultaten indikerade både förbättrad korspolarisations och lägre sidlobsnivåer hos antennen. En ny klass av bredbandiga gruppantenner har utvecklas i denna avhandling, den kallas en Starkt Kopplad Asymmetrisk Dipol-gruppAntennen - SCADA. Genom att utnyttja geometrisk asymmetri i antennelementet introduceras ytterligare en frihetsgrad som möjliggör förbättrad bandbredd och utstyrning. Vidare presenteras här en ny metod för impedansterminering av ändliga gruppantenner. Både SCADA-teorin samt dess verifiering i forma av en experimentell gruppantenn presenteras här. Teori, simulering och experiment visar god överenskommelse, vilket validerar idéerna. En prototyp av ett matchande skikt som stöder stor utstyrbarhet har integrerats med gruppantennprototypen och presenteras i avhandlingen. I den sista delen av avhandling utvecklas också en metod för detektering av den globala kosmologiska 21 cm-signalen från universums rejoniseringsepok - EoR. Huvudkällorna för mätfel i detta experiment utvärderas, de är antennens kromaticiteten och förgrundsstrålningen. En ny algoritmbaserad metod för att extrahera den globala EoR-signalen föreslås. Metoden är baserad på anpassning med multipla polynom och har med framgång tillämpats och utvärderats. En gruppantenn som baseras på de metoder som beskrivs i avhandling har också föreslagits och dess prestanda har utvärderats med den föreslagna metoden. / <p>QC 20171121</p>

Reliability and sensitive information protection are critical aspects of integrated circuits. A novel technique using near-field evanescent wave coupling from two subwavelength gratings (SWGs), with the input laser source delivered through an optical fiber is presented for tamper evidence of electronic components. The first grating of the pair of coupled subwavelength gratings (CSWGs) was milled directly on the output facet of the silica fiber using focused ion beam (FIB) etching. The second grating was patterned using e-beam lithography and etched into a glass substrate using reactive ion etching (RIE). The slightest intrusion attempt would separate the CSWGs and eliminate near-field coupling between the gratings. Tampering, therefore, would become evident. Computer simulations guided the design for optimal operation of the security solution. The physical dimensions of the SWGs, i.e. period and thickness, were optimized, for a 650 nm illuminating wavelength. The optimal dimensions resulted in a 560 nm grating period for the first grating etched in the silica optical fiber and 420 nm for the second grating etched in borosilicate glass. The incident light beam had a half-width at half-maximum (HWHM) of at least 7 µm to allow discernible higher transmission orders, and a HWHM of 28 µm for minimum noise. The minimum number of individual grating lines present on the optical fiber facet was identified as 15 lines. Grating rotation due to the cylindrical geometry of the fiber resulted in a rotation of the far-field pattern, corresponding to the rotation angle of moiré fringes. With the goal of later adding authentication to tamper evidence, the concept of CSWGs signature was also modeled by introducing random and planned variations in the glass grating. The fiber was placed on a stage supported by a nanomanipulator, which permitted three-dimensional displacement while maintaining the fiber tip normal to the surface of the glass substrate. A 650 nm diode laser was fixed to a translation mount that transmitted the light source through the optical fiber, and the output intensity was measured using a silicon photodiode. The evanescent wave coupling output results for the CSWGs were measured and compared to the simulation results.

G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that perform transmembrane signal transduction. Due to their pivotal role in a wide range of essential physiological functions GPCRs represent a high proportion of all drug targets. High resolution X-ray structures of GPCRs are however underrepresented in the Protein Data Bank. This is due to their instability in detergent, low expression levels and the presence of misfolded receptors in many heterologous expression systems. The objective of this project was to engineer the angiotensin II type 1 receptor (AT1R), a human GPCR, to make it suitable for structural studies. It was determined that detergentsolubilised AT1R was thermostable with antagonist bound with an apparent Tm of ~45°C, which was sufficiently stable for purification without further thermostabilisation by rational mutagenesis. Two expression systems were then evaluated for large-scale production of AT1R, namely baculovirus-mediated expression in insect cells and mammalian expression in HEK293 cells. Radioligand binding assays showed that only the mammalian system produced sufficient quantities of active AT1R for structural studies. Expression in the mammalian system was further optimised to approximately 6 mg/L. An AT1R-GFP fusion was created to examine membrane localisation using confocal laser scanning microscopy, to assay expression levels, to select highly expressing monoclonal cell lines using fluorescence activated flow cytometry and to develop a fluorescence size-exclusion chromatographybased assay to examine the suitability of 12 different ligands for co-crystallization. AT1R was also engineered to facilitate crystallisation, including C-terminal truncations to remove predicted disordered regions and bacteriophage T4-lysozyme being added to the third intracellular loop to provide additional points of contact for crystallisation, which increased the apparent Tm by approximately 10°C. All modified versions of AT1R were assessed for expression, stability and monodispersity. Additionally a rapid western blotting based assay was developed for the detection of unfolded membrane proteins, which will have wide applicability in the field.

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated non-covalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism [1]. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD) comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail [2]. Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. The TSH receptor (TSHR) is an especially interesting member of this family as it not only recognizes is cognate ligand, i.e. TSH, but also binds to the non-cognate ligands such as autoantibodies. TSHR autoantibodies come in different flavors; inhibitory antibodies that compete with the hormone for receptor binding and block its action, stimulatory antibodies that activate the receptor in a hormone independent manner and neutral antibodies that bind to the receptor but do not directly influence its functions. The inhibitory autoantibodies cause hypothyroidism and are responsible for Hashimoto’s Thyroiditis, whereas the stimulatory autoantibodies cause Graves’ thyrotoxicosis characterized by hyperthyroid condition [3]. The exact epitopes of these autoantibodies are not well delineated although it has been hypothesized that the blocking type- and the stimulatory type- autoantibodies have predominant epitopes in the TSHR ECD that overlap with hormone binding regions [4]. Insights into the mode of hormone or autoantibody binding to the receptor was primarily derived from the crystal structure of FSHR leucine rich repeat domain (LRRD) bound to single chain analog of FSH, and the crystal structures of TSHR LRRD bound to the stimulatory type human monoclonal antibody M22 [5] and the inhibitory type- monoclonal antibody K1-70 [6]. Both these crystal structures propose LRRDs as the primary ligand binding site which interacts with the hormone through its determinant loop in a hand-clasp fashion [7] while the autoantibodies mimics the hormone binding to a large extent [8] . These structures, while providing detailed understanding of the molecular interactions of the LRRs with the hormone, shed little light on the mechanism by which the signal generated at the LRRD are transduced to the downstream effector regions at the distally situated TMD. Hence, while one understands the ligand binding to a large extent, the activation process is not well understood, one of the central objective of the present study. Ligand-receptor interactions are typically studied by perturbing ligand/receptor structure by mutagenesis or by mapping conformational changes by biophysical or computational approaches. In addition to the above-mentioned approaches, the present work also uses highly specific antibodies against different domains of the receptor as molecular probes due to the ability of antibodies to distinguish between conformations likely to arise during the activation process. Use of antibodies to understand the receptor activation process is especially apt for TSHR due to the presence of physiologically relevant TSHR autoantibodies and their ability to influence hormone binding and receptor activation [9, 10]. Chapter 2 attempts to provide a comparison between the interactions of the hormone and the autoantibodies with TSHR. For this purpose, two assays were developed for identification of TSHR autoantibodies in the sera of patients suffering from autoimmune thyroid diseases (AITD), the first assay is based on the ability of TSHR autoantibodies to compete for radiolabeled hormone (The TSH binding inhibition (TBI), assay) and the second based on the capability of stimulatory antibody to produce cAMP in cells expressing TSHR (TSHR stimulatory immunoglobin (TSI) assay). A stable cell line expressing TSHR capable of recognizing both TSH and TSHR autoantibodies was thus created and used for prospective and retrospective analysis of AITD patients. Based on the TBI and TSI profiles of IgGs, purified from AITD patient's sera, it was recognized that TSHR stimulatory and TSH binding inhibitory effects of these antibodies correlated well, indicating overlap between hormone binding and IgG binding epitopes. It was also recognized that stimulatory IgGs are not affected by negative regulatory mechanism that governs TSH secretion substantiating the persistence of these antibodies in circulation. Kinetics of cAMP production by Graves’ stimulatory IgG was found to be fundamentally distinct, where the autoantibodies displayed pronounce hysteresis during the onset of the activation process when compared to the hormone. This could possibly be explained by the oligoclonality of the autoantibody population, a different mechanism of receptor activation or dissimilarity in autoantibody and hormone epitopes. To gain additional insights into the epitopes of TSHR autoantibodies and the regions that might be critical in the activation process, different overlapping fragments encompassing the entire TSH receptor ECD were cloned, expressed in E.coli as GST fusion proteins and purified: 1] the first three LRRs (TLRR 1-3, amino acid (aa) 21-127), 2] the first six LRRs (TLRR 1-6, aa 21-200), 3] the putative major hormone binding domain (TLRR 4-6, aa 128-200), and 4] the hinge region of TSH receptor along with LRR 7 to 9, (TLRR 7-HinR, aa 201-413). The receptor fragment TLRR 7-HinR was further subdivided into LRR 7-9 (TLRR 7-9, aa 201-161) and the hinge region (TSHR HinR, aa 261-413), expressed as N-terminal His-Tagged protein and purified using IMAC chromatography. Simultaneously, the full-length TSHR ECD was cloned, expressed and purified using the Pichia pastoris expression system. ELISA or immunoblot analysis of autoantibodies with the TSHR exodomain fragments suggested that Graves’ stimulatory antibody epitopes were distributed throughout the ECD with LRR 4-9 being the predominant site of binding. Interestingly, experiments involving neutralization of Graves’ IgG stimulated cAMP response by different receptor fragment indicated that fragments corresponding to the TSHR hinge region were better inhibitors of autoantibody stimulated receptor response than corresponding LRR fragments, suggesting that the hinge region might be an important component of the receptor activation process. This was in contrast to prevalent beliefs that considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance. Mutagenesis in TSHR hinge region and agonistic antibodies against FSHR and LHR hinge regions, reported by the laboratory, recognized the importance of the hinge regions as critical for receptor activation and may not simply be a scaffold [11-13]. Unfortunately, the mechanism by which the hinge region regulates binding or response or both have not been well understood partially due to unavailability of structural information about this region. In addition poor sequence similarity within the GpHR family and within proteins of known structure, make this region difficult to model structurally. In chapter 3, effort is made to model the hinge regions of the three GpHR based on the knowledge driven and Ab initio protocols. An assembled structure comprising of the LRR domain (derived from the known structures of FSHR and TSHR LRR domains) and the modeled hinge region and transmembrane domain presents interesting differences between the three receptors, especially in the manner the hormone bound LRRD is oriented towards the TMD. These models also suggested that the α-subunit interactions in these three receptors are fundamentally different and this was verified by investigating the effects of two α-subunit specific MAbs C10/2A6 on hCG-LHR and hTSH-TSHR interactions. These two α-subunit MAbs had inverse effects on binding of hormone to the receptor. MAb C10 inhibited TSH binding to TSHR but not that of hCG, whereas MAb 2A6 inhibited binding of hCG to LHR but not of hTSH. Investigation into the accessibility of their epitopes in a preformed hormone receptor complex indicated that the α-subunit may become buried or undergo conformational change during the activation process and interaction may be different for LHR and TSHR. Fundamental differences in TSHR and LHR were further investigated in the next chapter (Chapter 4), especially with regards to the ligand independent receptor activation. Polyclonal antibodies were developed against LRR 1-6, TLRR 7-HinR and the TSHR HinR receptor fragments. The LRR 1-6 antibodies were potent inhibitor of receptor binding as well as response, similar to that observed with antibodies against the corresponding regions of LHR. Interestingly, the antibodies against the hinge region of TSHR were unable to inhibit hTSH binding, but were effective inhibitors of cAMP production suggesting that this region may be involved in a later stage of a multi-step activation process. This was also verified by studying the mechanism of inhibition of receptor response and their effect on ligand-receptor association and dissociation kinetics. Hinge region-specific antibodies immunopurified from TLRR 7-HinR antibodies behaved akin to those of the pure hinge region antibodies providing independent validation of the above results. This result was, however, in contrast to those observed with a similar antibody against LHR hinge region. As compared to the TSHR antibody, the LHR antibody inhibited both hormone binding and response. In addition, this antibody could dissociate a preformed hormone-receptor complex which was not observed for TSHR hinge region antibodies. Although unable to dissociate preformed hormone-receptor complex by itself, the TSHR HinR antibodies augmented hormone induced dissociation of the hormone-receptor complex suggesting that this region may be involved in modulation of negative cooperativity associated with TSHR. Molecular dissection of the role of hinge region of TSHR was further carried out by using monoclonal antibodies against LRR 1-3 (MAb 413.1.F7), LRR 7-9 (MAb 311.87), TSHR hinge region (MAb 311.62 and MAb PD1.37). MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of TSH binding. MAb 413.1.F7 did not affect hormone binding or response and was used as the control antibody for different experiments. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region [14] and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. Estimation of apparent affinities of the antibody to the receptor and the cooperativity factor suggests that epitope of MAb 311.87 (LRR 7-9) may act as a pivot involved in the initial events immediate to hormone binding at the LRRs. The anatgonsitic effect of MAB 311.62 on binding and response also suggested that binding of hormone is conformationally selective rather than an induced event. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation. In contrast to the stimulatory nature of Cb-2 antibody such as MAb 311.62, MAb PD1.37, which identified residues aa 366–384 near Cb-3, was found to be inverse agonistic. Unlike other known inverse agonistic MAbs such as CS-17 [15] and 5C9 [16], MAb PD1.37 did not compete for TSH binding to TSHR, although it could inhibit hormone stimulated response. Moreover, unlike CS-17, MAb PD1.37 was able to decrease elevated basal cAMP of hinge region constitutively activated mutations only but not those in the extracellular loops. This is particularly important as interaction of hinge region residues with those of ECLs had been thought to be critical in maintenance of the basal level of receptor activation and are responsible for attenuating the constitutive basal activity of the mutant and wild-type receptors in the absence of the hormone. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain, which returned to the wild-type levels upon reintroduction of the hinge region. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type TSHR indicated that these mutations of the receptor resulted primarily in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Confirmation of the above proposition has been difficult to verify primarily due to a highly transient conformational change in the tripartite interaction of the hinge region/hormone and the ECLs. The current approaches of using antibodies to probe the ECLs are difficult due to the conformational nature of the antigen as well as difficulty in obtaining a soluble protein. In chapter 5, the ligand induced conformational alterations in the hinge regions and inter-helical loops of LHR/FSHR/TSHR were mapped using the exoloop specific antibodies generated against a mini-Transmembrane domain (mini-TMD) protein. This mini-TMD protein, designed to mimic the native exoloop conformations, was created by joining the TSHR exoloops, constrained through the helical tethers and library derived linkers. The antibody against mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the hinge regions, exoloops and TMDs such as those involved precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, dynamic changes in hinge region-exoloop interactions were mapped. The computational analysis suggests that mini-TMD antibodies act by conformationally locking the transmembrane helices by restraining the exoloops and juxta-membrane regions. This computational approach of generating synthetic TMDs bears promise in development of interesting antibodies with therapeutic potential, as well as, explains the role of exoloops during receptor activation. In conclusion (Chapter 6), the study provides a comprehensive outlook on the highly dynamic interaction of ligand and different subdomains of the TSHR (and to a certain extent of LHR and FSHR) and proposes a model of receptor activation where the receptor is in a dynamic equilibrium between the low affinities constrained state and the high affinity unconstrained state and bind to the hormone through the LRR 4-6. Upon binding the βL2 loop of the hormone contact LRR 8-10 that triggers a conformational change in the hinge region driving the α-subunit to contact the ECLs. Upon contact, the ECLs cooperatively causes helix movement in the TMH and ultimately in ICLs causing the inbuilt GTP-exchange function of a GPCR.

Introduction Nucleotide excision repair (NER) is a highly conserved pathway involved in repair of a wide variety of structurally unrelated DNA lesions. One of the well characterized NER systems is from E. coli which involves UvrABC nucleases. NER consists of two related sub-pathways: global genomic repair (GGR), which removes lesions from the overall genome, and transcription coupled repair (TCR), which removes lesions from the transcribed strand of active genes. Bulky DNA lesions such as cyclobutane pyrimidine photodimers (CPD) induced by UV irradiation block RNA polymerase (RNAP) during transcription. In bacteria, a gene product of mfd called transcription repair coupling factor (TRCF) or Mfd is required for TCR. Bacterial Mfd interacts with the stalled RNAP, displaces it from the DNA and recruits NER proteins at the site of damage. Mfd, thus contributes to the faster repair of the transcribed strand compared to the non-transcribed strand for similar kind of lesions. Intracellular pathogens like M. tuberculosis are constantly exposed to a variety of stress conditions inside the host, mainly due to host defense systems and antibiotic treatments. It is therefore, extremely important for bacteria to have DNA damage repair and reversal mechanisms that can efficiently counteract these effects. However, very little is known about DNA repair systems in M. tuberculosis compared to other bacteria. Sequencing of M. tuberculosis genome revealed the presence of NER associated genes including a putative mfd. Additionally, due to the high GC content of genome as well as the DNA damage prone host environment, the transcription in M. tuberculosis may encounter the problems, which are not apparent in other bacteria. Therefore, the gene like mfd may play very important role in physiology of M. tuberculosis. In the present study, we describe the biochemical and functional characterization of Mfd from M. tuberculosis (MtbMfd) and discuss its unusual properties. Biochemical characterization of MtbMfd Genome analysis of M. tuberculosis as well as the sequence alignment studies revealed that MtbMfd is 1234 amino acids long multifunctional protein having various domains specialized for different functions. Cloning of Mtbmfd was carried out by reconstructing the full length gene from three PCR amplified fragments using genomic DNA as a template. Complementation study using Mtbmfd suggested that the gene of interest complements E. coli counterpart and increases survival of UV irradiated cells. To further characterize the function of Mtbmfd, a road block reporter assay was performed, which indicates that the MtbMfd interacts with stalled E. coli RNAP and displaces it from the site of transcription resulting in low reporter gene activity. The MtbMfd protein was expressed and purified by using various chromatographic techniques, and confirmed by mass spectrometry. In addition to full length protein, a number of truncated MtbMfd constructs were generated and purified to homogeneity. Mfd is a motor protein and requires ATP hydrolysis in order to translocate along DNA. The signature motifs of superfamily 2 helicases / ATPases are present at the C-terminal of Mfd along with translocase motif which is highly homologous to motif present in RecG helicase. To analyze the kinetics of ATP hydrolysis of MtbMfd and its truncated proteins, ATPase reactions were carried out using γ32P-ATP as a tracer. Wild-type MtbMfd exhibited ATPase activity, which was stimulated ~1.5 fold in presence of dsDNA. The mutant MtbMfd (D778A), which harbors mutation in one of the key residues of Walker B motif of the ATPase domain showed negligible ATPase activity indicating the importance of residue D778 for ATP hydrolysis. While the C-terminal domain (CTD) comprising amino acids 600 to 1234 showed elevated ATPase activity, the N-terminal domain (NTD) containing the first 500 amino acid residues was able to bind ATP but deficient in hydrolysis. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ~10-fold compared to full-length MtbMfd. The translocase activity of MtbMfd was measured by an oligonucleotide displacement assay and it was found that full length MtbMfd and CTD have a very weak translocase activity whereas, MfdΔC exhibited efficient translocation along DNA in ATP dependent manner. These results provide a direct correlation between translocase and ATPase activity of MtbMfd, and suggest possibly an auto-regulatory function for the extreme C-terminus of MtbMfd. Oligomeric status of MtbMfd was determined using various techniques including gel filtration chromatography and it was found that MtbMfd exists as monomer and hexamer in solution. The monomer showed increased ATPase activity and susceptibility to proteases compared to the hexameric form. MfdΔC, on the other hand, was predominantly monomer in solution implicating importance of the extreme C-terminal region in oligomerization of protein. Taken together, the biochemical evidence suggests that monomeric MtbMfd is an active form and oligomerization provides stability to the protein. One important finding of the present study is the binding of ATP to NTD of MtbMfd. All Mfd NTDs resemble UvrB and possesses the degenerate ATPase motifs. Indeed, on the basis of sequence and structural similarities, it has been suggested that Mfds have evolved from UvrB incorporating an additional translocase activity. UvrB has a cryptic ATPase activity while the NTD of Mfd may have lost the activity as it possesses degenerate Walker motifs. In contrast, NTD of MtbMfd binds ATP but is hydrolysis deficient. A closer comparison of the amino acid sequences in the Walker A motif reveal that conserved K 45 of UvrB has been replaced by R in case of NTD of MtbMfd. It has been shown previously that mutation of K 45 to A, D and R led to a loss of ATPase activity of UvrB. Thus, MtbMfd seems to be a natural mutant of UvrB. Since NTD harbors an intact UvrA interacting domain, when it is expressed it may sequester the cellular pool of UvrA leading to dominant negative phenotype. When UV survival assays were carried out, cells expressing NTD showed hyper-sensitivity to UV light – a typical characteristic of NER deficiency. In addition, in vitro NER assay clearly suggested that NTD sequesters pool of UvrA inside the cell and blocks both GGR and TCR which further affects the mutation frequency of bacterial cells. Influence of MtbMfd on elongation state of RNAP The movement of RNAP along the template during transcription elongation is not uniform and is interrupted due to various factors. To overcome transcription elongation interruptions, a number of proteins viz. Mfd, Gre and Nus act on RNAP and modify its activity. RNAP displacement and transcript release experiments showed that MtbMfd influenced the elongating RNAP by more than one way. MtbMfd displaced stalled RNAP, which was blocked by NTP starvation on T7A1 promoter based template in a concentration and time-dependent manner. RNAP displacement activity of MtbMfd was shown to depend on ATP or dATP hydrolysis. On the other hand nucleotides like ADP, GTP, CTP and ATPγS did not support the RNAP displacement activity. However, in presence of ATPγS, MtbMfd was able to bind stalled complex but unable to displace RNAP suggesting that ATP or dATP hydrolysis is important for MtbMfd function. On the other hand, MtbMfd did not affect initiating RNAP when σ factor was still bound suggesting that upstream DNA is necessary for Mfd function. To assay RNA or transcript release activity of MtbMfd after transcription complex disruption, immobilized transcription complex assay was carried out. Immobilized stalled complex was generated by UTP and CTP starvation on biotinylated T7A1 promoter based template which can be affixed to temporary pellet in presence of streptavidin beads. It was found that MtbMfd released RNA into a supernatant fraction in a concentration-dependent manner suggesting that MtbMfd releases transcript after ternary complex disruption. MtbMfd released transcript in an energy-dependent manner and both ATP and dATP supported the activity, which allows the complete separation of RNA release from RNA synthesis inside the cell. An ATPase mutant of MtbMfd (MfdD778A) failed to release transcript, which further supported that ATP hydrolysis is important for MtbMfd function. Since both Mfds and RNAPs are evolutionary conserved proteins, to analyze the effect of MtbMfd on other bacterial RNAPs, displacement and release assays were carried out. Stalled complexes were generated using EcoRNAP (E. coli), MsRNAP (M. smegmatis) and MtbRNAP (M. tuberculosis) on T7A1 promoter based template. It was observed that MtbMfd was able to displace all the three RNAPs from stalled elongation complex as well as released transcript with varying efficiency. MtbMfd showed optimal displacement and release activity in presence of mycobacterial RNAPs. Transcription elongation complexes adopt various conformations and exist as different isomerized states during elongation. In an active elongation complex the 3'-OH polymerizing end of transcript aligns with an active centre of the RNAP. However, one of the most common and intrinsic properties of RNAP is backtracking or reverse translocation, which leads to misalignment of 3'-OH polymerizing end from an active centre of the polymerase. It is of interest to know if backtracking affects MtbMfd function. It is likely that complexes blocked by lesions inside the cell might tend to backtrack, and different translocational isomers possibly have different sensitivities to MtbMfd action which may illuminate the overall mechanism of MtbMfd. Backtracking of RNAP was induced on +20 and +39 stalled complexes and the effect of MtbMfd was analyzed in presence of NTPs in the reaction. It was found that arrested or backtracked complexes were restored to the forward position by the activity of MtbMfd in presence of NTP resulting into productive elongation. These results suggest that arrested RNAP again resumes transcription if conditions are favorable; otherwise, MtbMfd further assists RNAP to dissociate which leads to release of transcript. Anti-backtracking activity of MtbMfd might have important function in cellular metabolism and it has been speculated that Mfd could play more general role during transcription apart from repair. To explore the role of MtbMfd as a transcription factor and effect of MtbMtb on transcription processes in the mycobacteria, a variety of T7A1 promoter based templates were generated. These templates were derived from genes of M. tuberculosis and E. coli having varying GC content (39-81 %). The rationale behind this experiment is that the high GC content of mycobacteria and the template derived from mycobacterial genes may pose as sequence dependent structural constraints and hence block the RNAP during transcription. By anti-backtracking activity of MtbMfd these paused complexes may get relieved, leading to efficient transcription by RNAP which may lead to the formation of more full length transcript. To analyze the effect of MtbMfd, purified templates of different GC content were incubated with RNAP and MtbMfd to carry out in vitro transcription. Although, in case of multiple rounds of transcription, multiple pauses were observed even in presence of MtbMfd. However, in presence MtbMfd around 1.5 - 2 fold increased full-length transcripts were observed suggesting that MtbMfd assisted RNAP during elongation to overcome sequence dependent pause. To avoid multiple pauses that are likely to occur due to the initiation of multiple round of transcription, and trailing effect of RNAP itself, single round of transcriptions were carried out in presence of heparin. Sequence specific pauses were observed with increasing GC percentage in template suggesting that indeed high GC content contributes to transcription pause. At the same time, MtbMfd in the reaction increased the amount of full length transcript by 1.5 - 2.0 fold probably by pushing paused RNAP forward to resume elongation. Taken together, this study investigates the biochemical properties of MtbMfd and its mechanism of action. In addition, it explores the importance of the coupling of transcription to repair in M. tuberculosis as well as the overall proof reading mechanism of transcription elongation in the GC rich genome of mycobacteria.

This thesis has two parts: In the first part, we study the dispersion characteristics of structural-acoustic waveguides by obtaining closed-form solutions for the coupled wave numbers. Two representative systems are considered for the above study: an infinite two-dimensional rectangular waveguide and an infinite fluid- filled orthotropic circular cylindrical shell. In the second part, these asymptotic expressions are used to study the nonlinear wave propagation in the same two systems. The first part involves obtaining asymptotic expansions for the fluid-structure coupled wave numbers in both the systems. Certain expansions are already available in the literature. Hence, the gaps in the literature are filled. Thus, for cylindrical shells even in vacuo wavenumbers are obtained as part of the objective. Here, singular and regular perturbation methods are used by taking the thickness parameter as the asymptotic parameter. Valid wavenumber expressions are obtained at all the frequencies. A transition in the behavior of the flexural wavenumbers occurs in the neighborhood of the ring frequency. This frequency of transition is identified for the orthotropic shells also. The closed-form expressions for the orthotropic shells are obtained in the limit of slight orthotropy for the circumferential orders n > 0 at all the frequency ranges. Following this, we derive the coupled wavenumber expressions for the two systems for an arbitrary fluid loading. Here, the two-dimensional rectangular waveguide is considered first. This rectangular waveguide has a one-dimensional plate and a rigid surface as its lateral boundaries. The effects due to the structural boundary are studied by analyzing the phase change due to the structure on an incident plane wave. The complications due to the cross-sectional modes are eliminated by ignoring the presence of the other rigid boundary. Dispersion characteristics are predicted at various regions of the dispersion diagram based on the phase change. Moreover, the also identified. Next, the rigid boundary is considered and the coupled dispersion relation for the waveguide is solved for the wavenumber expressions. The coupled wavenumbers are obtained as the coupled rigid-duct, the coupled structural and the coupled pressure-release wavenumbers. Next, based on the above asymptotic analysis on a two-dimensional rectangular waveguide, the asymptotic expansions are obtained for the coupled wavenumbers in isotropic and orthotropic fluid- filled cylindrical shells. The asymptotic expansions of the wavenumbers are obtained without any restriction on the fluid loading. They are compared with the numerical solutions and a good match is obtained. In the second part or the nonlinear section of the thesis, the coupled wavenumber expressions are used to study the propagation of small but a finite amplitude acoustic potential in the above structural-acoustic waveguides. It must be mentioned here that for the rst time in the literature, for a structural-acoustic system having a contained fluid, both the structure and the acoustic fluid are nonlinear. Standard nonlinear equations are used. The focus is restricted to non-planar modes. The study of the cylindrical shell parallels that of the 2-D rectangular waveguide, except in that the former is more practical and complicated due to the curvature. Thus, with regard to both systems, a narrow-band wavepacket of the acoustic potential centered around a frequency is considered. The approximate solution of the acoustic velocity potential is found using the method of multiple scales (MMS) involving both space and time. The calculations are presented up to the third order of the small parameter. It is found that the amplitude modulation is governed by the Nonlinear Schr•odinger equation (NLSE). The nonlinear term in the NLSE is analyzed, since the sign of the nonlinear term in the NLSE plays a role in determining the stability of the amplitude modulation. This sign change is predicted using the coupled wavenumber expressions. Secondly, at specific frequencies, the primary pulse interacts with its higher harmonics, as do two or more primary pulses with their resultant higher harmonic. This happens when the phase speeds of the waves match. The frequencies of such interactions are identified, again using the coupled wavenumber expressions. The novelty of this work lies firstly in considering nonlinear acoustic wave prop-agation in nonlinear structural waveguides. Secondly, in deriving the asymptotic expansions for the coupled wavenumbers for both the two-dimensional rectangular waveguide and the fluid- filled circular cylindrical shell. Then in using the same to study the behavior of the nonlinear term in NLSE. And lastly in identifying the frequencies of nonlinear interactions in the respective waveguides.