La comprensión de los mecanismos moleculares que controlan la resistencia de la planta frente a patógenos biotrofos es un campo de investigación complejo y en expansión donde se impone la identificación de nuevos reguladores. Previamente se había descrito en nuestro laboratorio el gen P69C que codifica una proteasa con homología a subtilisinas y cuya expresión se induce en el transcurso de la interacción planta-patógeno. Con el fin de estudiar nuevos componentes de la planta implicados en la señalización de la respuesta defensiva, se procedió al escrutinio de mutantes de Arabidopsis thaliana que de forma constitutiva y sin la existencia de ningún estímulo externo se encontrara activada la expresión del gen GUS dirigida por el promotor P69C. En la presente memoria de tesis se describe ampliamente la identificación y caracterización del mutante, csb3 (constitutive subtilisin3). Las plantas csb3 poseen elevados niveles de ácido salicílico (SA) y además expresan genes dependientes de la ruta de SA tales como PR-1, PR-2 y GST6. Por otra parte, el mutante csb3 exhibe una elevada resistencia al oomiceto patógeno Hyaloperonospora parasitica de naturaleza biotrofa y a la bacteria patógena también biotrofa Pseudomonas syringae pv.tomato DC3000 (Pst) DC3000. Sin embargo, la resistencia a patógenos necrotrofos tales como Botrytis cinerea y Plectosphaerella cucumerina permanece inalterada en las plantas csb3. Para analizar la participación de los distintos componentes de la ruta de señalización dependiente de SA en la manifestación del fenotipo de resistencia de csb3, se procedió al análisis epistático entre csb3 y pad4, sid2, eds5, nahG, npr1, dth9 y cpr1. Estos estudios indican que la elevada resistencia frente a patógenos biotrofos de las plantas csb3 requiere de todos y cada uno de los componentes de la ruta de señalización dependiente del SA estudiados. El gen CSB3 identificado por clonaje posicional codifica la 1-hidroxi-2-metil-2-butenil 4-difosfato (HDS) sin / Gil Morrió, MJ. (2005). Disección genética del mecanismo de resistencia frente a patógenos biotrofos mediado por el gen CSB3 en Arabidopsis thaliana [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1870

My research work for PhD has focused on: (i) the development and application of new NMR methodologies to solve challenging problems in structural biology and (ii) studying important biological systems to correlate their structural and functional aspects. I have worked on diverse research projects ranging from NMR methodology development to the study of structure and dynamics of protein-based nanotubes. Chapter 1 of my thesis gives brief introduction to bio-molecular NMR spectroscopy and the different biological systems that I have studied. In recent years, several new methods have emerged for rapid NMR data collection. One class of methods is G-matrix Fourier transform (GFT) projection NMR spectroscopy. GFT NMR spectroscopy involves phase sensitive joint sampling of two or more chemical shifts in an indirect dimension of a multidimensional NMR experiment. Chapter 2 describes a new method based on the principle of GFT NMR for increasing further the speed of data collection. In the current implementations of the GFT method, cosine/sine modulation of all chemical shifts involved in the joint sampling are collected and stored as separate FIDs. A post-acquisition data processing step (application of G-matrix) then separates the different inter-modulations of chemical shifts. Thus, joint sampling of K+1 spins results in 2K combination of chemical shifts (also representing 2K projection angles). One limitation of this approach is that even if only a few of the 2K components of the multiplet (or projection angles) is desired, an entire data set containing information for all 2K shift combinations is collected. We have proposed a simple method which releases this restriction and allows one to selectively detect only the desired linear combination of chemical shifts/projection angles out of 2K combinations in a phase sensitive manner. The method involves selecting the appropriate cosine/sine modulations of chemical shifts and forming the desired linear combination by phase cycling of the radiofrequency pulses and receiver. This will benefit applications where only certain linear combination of shifts are desired or/and are sufficient. Further, G-matrix transformation required for forming the linear combination is performed within the pulse sequence. This avoids the need for any post-acquisition data processing. Taken together, this mode of data acquisition will foster new applications in projection NMR spectroscopy for rapid resonance assignment and structure determination. Chapter 3 describes another GFT NMR-based method for rapid estimation of secondary structure in proteins. This involves the detection of specific linear combination of backbone chemical shifts and facilitates a clear separation and estimation of residues in different secondary structures of a given protein. This methodology named as CSSI-PRO (Combination of Shifts for Secondary structure Identification in PROteins), involves detection of specific linear combination of backbone 1Hα and 13C’ chemical shifts in a two dimensional (2D) NMR experiment. Such linear combination of shifts facilitates editing of residue belonging to α-helical/ β-strand regions into distinct spectral regions nearly independent of the amino acid type. This helps in the estimation of overall secondary structure content of the protein. Comparison of the estimated secondary structure content with those obtained from the respective 3D structures and/or the method of Chemical Shift Index (CSI) was carried out for 254 proteins and gives a correlation of more than 90% and an overall RMSD of 6.5%. The method has high sensitivity and data can be acquired in a few minutes. This methodology has several applications such as for high-throughput screening of proteins in structural proteomics and for monitoring conformational changes during protein folding and/or ligand-binding events. Chapter 4 (Part-A and Part-B) describes an area of my research which involves the study of structure and function in the Insulin-like Growth Factor Binding Protein (IGFBP) family. IGFBPs (six in number; IGFBP1-6) belong to the IGF-system, which plays an important role in growth and development of the human body. This system is comprised of the following components: (i) Two peptide hormones, IGF-1 and -2, (ii) type 1 and type 2 IGF receptors, (iii) six IGF-binding proteins (IGFBP; numbered 1-6) and (iv) IGFBP proteases. IGF-1 and -2 are small signalling peptides (~7.5 kDa) that stimulate action by binding to specific cell surface receptors (IGF-1R) evoking subsequent response inside the cell. Six soluble IGF binding proteins, the IGFBPs, which range in 22-31 kDa in size and share overall sequence and structural homology with each other, regulate the activity of the IGFs. IGFBPs bind strongly to IGFs (KD ~ 300-700 pM) to ensure that all the circulating IGF in the blood stream is sequestered and inhibit the action of IGFs by blocking their access to the receptors. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind and activate the cell surface receptors. IGFBPs have been recently implicated in different cancers and HIV/AIDS. However, the nature of their interaction with the ligand: IGF-1 or IGF-2 at a molecular level poorly understood. This is due to the difficulty in over-expressing these proteins in large scale and in soluble amounts which is required for structural studies. We have for the first time developed an efficient method for bacterial expression of full-length human IGFBP-2, a 33 kDa system, in soluble (upto 30 mg/ml) and folded form. Using a single step purification protocol, hIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity as evident from its strong binding to IGF-1 and IGF-2 detected using surface plasmon resonance (SPR). This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization by NMR. Using different NMR methods, we are now in the process of elucidating the 3D structure of this molecule. Chapter 5 (Part-A and Part-B) describes our discovery of nanotubular structures formed by spontaneous self-assembly of a small fragment from the C-terminal domain of hIGFBP-2. The nanotubular structures are several micrometers long and have a uniform outer diameter of ~35 nm. These structures were studied extensively by NMR and other techniques such as TEM, fluorescence and circular dichroism (CD). The water soluble nanotubes form through intermolecular disulphide bonds due to the presence of three cysteines in the polypeptide chain and exhibit enhanced tyrosine fluorescence. Based on different experimental evidences we have proposed a mechanism for the formation of the nanotubes. This was considered as a breakthrough by the journal ChemComm and featured on the cover-page of the journal. An article highlighting the discovery was also published in RSC news. In recent years, a number of novel polypeptide and DNA based nanotubes have been reported. Our study reveals intrinsically fluorescent self-assembling nanotubes made up of disulphide bonds having the following novel properties: (i) their formation/dissociation can be controlled by tuning the redox conditions, (ii) they do not require the support of any additional chemical agent for self-assembly, (iii) they have high stability due to the involvement of covalent interactions, (iv) the monomer is a small polypeptide chain which can be chemically synthesized or produced using simple recombinant methods and (v) they possess high inherent fluorescence and can thus be easily detected against a background of other proteins. In addition, the presence of an RGD motif in this polypeptide fragment offers avenues for novel biomedical applications. The RGD motif is known to be recognized by integrins. The design of such self-assembling polypeptide fragments containing an RGD motif can be utilized to enhance the efficacy of cancer therapeutics. Towards this end, we have investigated the structural basis of formation of these nanotubular structures by NMR spectroscopy and proposed its application for cancer cell imaging.

Vanadium oxides are being used as the thermal sensing layer because of their applications in infrared detectors. They have high temperature coefficient of resistance, favorable electrical resistance and compatibility with the MEMS technology. Of all oxides of vanadium, only vanadium dioxide (VO2)has been highly investigated as it shows first order transition (semiconducting to metal transition-SMT)at 68 oC. First order transition is understood as the sharp change in the electrical resistance. The change in resistivity in this case is of the order of 105 over a temperature change of 0.1 oC at 68 oC in a single crystal. Doping vanadium oxides with elements like Mo and W reduce the transition temperature. This is very important for room temperature electrical and optical detection. Though most of the research groups subscribe to PLD, cost-effective methods with large area deposition are major focus of this research. Hence for synthesizing VO2 in bulk and thin films, Solution Combustion Synthesis (SCS), Ultrasonic Nebulized Spray Pyrolysis of Aqueous Combustion Mixture (UNSPACM) Chemical vapour deposition (CVD)and microwave are explored. Synthesis of doped VO2 films in CVD has not been done extensively to yield optical quality thin films. Chapter I surveys the use of phase transition in oxides system for a variety of practical applications. In particular, Vanadium dioxide (VO2) is chosen as it is found to be very useful for infrared and metamaterials based applications. VO2 is known for its first-order semiconducting to metallic transition (SMT). This chapter attempts to explain the influence of processing, doping, annealing, etc on the SMT characteristics. Important aspects such as the idea of hysteresis in VO2 and similarity to martensitic transformation are discussed. The scope and objectives of the thesis are discussed here. Chapter II explains in detail the materials and methods used to synthesize VO2 both in bulk and in thin lm form and methods used to study their characteristics. Brief description on the principle and the working of the home-built experimental set up needed for this study is elicited. In chapter III, attempts were made to understand the phase stability of VO2 and the evolution of crystal structures during the phase transition. VO2 crystallizes in P21/c space group at room temperatures with lattice parameters a=5.752 Ab=4.526 Ac=5.382 Aα=90 β=122.60 γ=90 . Precise control of synthesis parameters is required in stabilizing pure phase in bulk as well as thin lm form. This study focuses on the novel large scale two step synthesis of VO2 using Solution Combustion Synthesis. This involves synthesis of product utilizing redox reaction between metal nitrate and suitable fuel. Generally the products are nanocrystalline in nature due to self-propagation of the exothermic combustion reaction. First step involved the synthesis of V2O5 by combustion reaction between Vanadyl nitrate and urea. In the second step, the as-synthesized V2O5 has been reduced by a novel reduction technique to form monophasic VO2. The presence of competing phases like M1, M2, M3 and R are investigated by XRD, Raman spectroscopy, DSC, Optical and high temperature X-ray diffraction. Chapter IV deals with the reduction in phase transition temperature by doping the SCS synthesized VO2 with W and Mo. Effect of doping on the transition temperature was studied using differential scanning calorimetry (DSC) in both W and Mo. Electrical characteristics of Mo doped VO2 and Optical characteristics of the W-doped VO2 were also studied using four probe resistivity measurements and UV-VIS Spectroscopy respectively. W addition was found to be more effective in reducing the phase transition temperature. To understand further more on the W addition, X-ray photo-electron spectroscopy measurements were performed. W-addition alters the V4+-V4+ bonding and with W addition it is observed that V was present in V3+state. W was present in W6+ state. The addition of W to VO2 introduces more electrons to the systems and disturbs the V4+-V4+ thus reducing the phase transition temperature of VO2. Chapter V describes the large scale, large area deposition of thin films of VO2 by a cost effective method. A novel technique to deposit vanadium dioxide thin films namely, UNSPACM is developed. This simple two-step process involves synthesis of a V2O5 lm on an LaAlO3(LAO) substrate followed by a controlled reduction to form single phase VO2. The formation of M1 phase (P21/c) is confirmed by Raman spectroscopic studies. A thermally activated metal{insulator transition (MIT) was observed at 61 oC, where the resistivity changes by four orders of magnitude. The infrared spectra also show a dramatic change in reflectance from 13% to over 90% in the wavelength range of 7-15 m. This indicates the suitability of the films for optical switching applications at infrared frequencies. A trilayer metamaterial absorber, composed of a metal structure/dielectric spacer/vanadium dioxide (VO2) ground plane, is shown to switch reversibly between reflective and absorptive states as a function of temperature. The VO2 lm, which changes its conductivity by four orders of magnitude across an insulator{metal transition, enables the switching by forming a resonant absorptive structure at high temperatures while being inactive at low temperatures. The fabricated metamaterial shows a modulation of the reflectivity levels of 58% at a frequency of 22.5 THz and 57% at a frequency of 34.5 THz. Chapter VI explains the W doped VO2 thin films synthesized by UN-SPACM. Morphology of the thin films was found to be consisting of globular and porous nanoparticles having size 20 nm. Transition temperature decreased with the addition of W. 1.8 at. %W doping in VO2 transition temperature has reduced upto 25 oC. It is noted that W-doping in the pro-cess of reducing the transition temperature, alters the local structure and also increases room temperature carrier concentration. The presence of W, as was seen in Chapter IV, altered V4+-V4+ bonds and introduced V3+. W was found to be in W6+ state suggesting W addition increased the carrier concentration. Hall Effect measurements suggested the increased carrier con-centration. The roughness of the synthesized films were very high for them to be of de-vice quality, despite encouraging results obtained by electrical measurements. Hence in order to further improve the smoothness and thereby the optical quality of thin films, Chemical Vapour Deposition (CVD) is employed. Chapter VII outlines the effect of processing parameters and post pro-cessing annealing on the semiconductor-metallic transition of VO2. Here in this chapter, the influence of substrate temperature on the SMT properties of VO2 is explored. At different substrate temperatures, the percentage of phase fraction of V in V3+, 4+ and V5+ differed. Besides, the morphology also varied with substrate temperatures. Similarly it is observed that with annealing the VO2 film deposited on glass substrates, SMT properties enhanced which was attributed to filling out of oxygen vacancies. Si based substrates and non-Si based substrates were used for depositing VO2 thin films by CVD. Their temperature coefficient of resistance and SMT properties were studied in order to understand their potential in bolometer and thermal to optical valve based applications. Chapter VIII involves the study of VO2 thin films for thermal to optical valves. ITO coated glass substrates were used for the purpose. Thin films were deposited by both UNSPACM and CVD. It was observed that the reflectivity pro les of the films synthesized by the above said methods were very different. Hence in the process of understanding the huge difference in the reflectivity pro les, classical harmonic oscillator, Lorentzian model was employed to t the experimental data at room temperature whereas Drude-Lorentzian model was used to t the data at higher temperature (at 100 oC- after transition). With this fitting plasma frequencies of the CVD films were calculated. It was observed that defect chemistry of films synthesized by these methods were different. In order to further improve the smoothness of the films, microwave method was proposed in Chapter IX. The preliminary results showed the presence of uniform spheres and 3 D hierarchical structures of VO2 consisting of nanorods. This was extended to deposit VO2 thin films on ITO. DSC and Infrared reflectance pro le of VO2 nanopowder suggested the phase transition. Chapter X summarizes the work done for the thesis and provides insights to the applications and to the future work. The work reported in this thesis has been carried out by the candidate as part of the Ph.D.program. She hopes that this would constitute a worth-while contribution towards development of VO2 thin film technology and its challenges for reliable infrared device applications.

Helper T cells direct the immunological response to foreign pathogens and cancer. To become activated, helper T cells must recognize unique peptides presented on major histocompatibility complex II (pMHCII) by antigen presenting cells (APCs) with their T cell receptor (TCR). While much is known about helper T cell activation signaling cascades and the subsequent roles of helper T cell subsets, the initiation of helper T cell activation by the TCR and other co-receptors is less well understood. Specifically, the affinity of the TCR for its pMHCII can change helper T cell subset fate, proliferation, and alter the risk for activation induced cell death. High affinity TCRs are attractive targets for immunotherapies, but little is known about how helper T cells respond to high affinity TCRs. Here we describe high affinity TCR activation thresholds for both full length TCRs and chimeric antigen receptor TCRs both with and without the presence of the coreceptor CD4 and propose a mechanism whereby CD4 inhibits T cell activation via Lck sequestration and a CD4-independent method. Dysregulated helper T cells play critical roles in the development and perpetuation of systemic lupus erythematosus (SLE), a systemic autoimmune disease that causes widespread inflammation and organ damage throughout the body. Chronic inflammation in SLE affects the immune response to viruses and the risk of developing cancer. However, in SLE patients, it is unclear if viruses initiate the development of cancer directly or if the effects are non-interacting and concomitant. Here we describe the interactions between SLE, viruses, and cancer risk revealing that viruses and SLE do interact to increase the both the overall cancer risk and the risk for hematological malignancies. Due to vaccine efficacy, vaccine preventable diseases (VPDs) are no longer commonly experienced or understood by the public. Vaccines are a victim of their own success and according to the World Health Organization (WHO), vaccine hesitancy (VH) is one of the top threats to global health. VH is the refusal to accept vaccinations and the reasons for VH vary across time, place, and vaccine. Refuting VH is difficult as directly confronting false assumptions can cause individuals to become more entrenched in their position resulting in confirmation bias. Adults with VH attitudes are often motivated by concerns over personal liberty, harm, independence, and body purity. Here we describe the results of a VPD interview- and education-based intervention geared towards promoting positive vaccine attitudes for young adults and demonstrate that education focused on VPDs is more effective than vaccine safety.

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