Indian Institute of Science Bangalore
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Non-canonical functions of lymphoid-specific proteins AID and RAG1: Understanding mechanism, regulation, and implications in genomic instability
No full textRecombination-activating genes (RAGs) and Activation-induced cytidine deaminase (AID) are lymphoid cell-specific proteins that play crucial roles in the development of adaptive immunity. Diversification of the immune system is initiated by a carefully orchestrated DNA rearrangement process, V(D)J recombination, in the Immunoglobulins (Ig) and T-cell receptor (TCR) genes during lymphocyte development. The lymphoid-specific recombinases, RAG1 and RAG2, mediates the process of V(D)J recombination to generate antigen receptor diversity. All B lymphocytes that successfully complete V(D)J recombination express IgM on surface and migrate to the secondary lymphoid organs, where they are activated after encountering antigens. Activated B cells further undergo other genetic alterations, namely somatic hypermutation (SHM), class-switch recombination (CSR) and gene conversion (GC), carried out by a B cell-specific enzyme: Activation-induced cytidine deaminase to further expand the immunoglobulin repertoire.
On the other hand, the development, differentiation, and maturation of T lymphocytes are known to be dependent on transcription factors like GATA-3, c-MYB, members of the E2A/HEB family, BCL11B etc. Among these, BCL11B is a key transcription factor known to play a role in the survival and differentiation of T cells during early stages of T cell development in both mice and humans. Deletion of Bcl11b confers natural-killer cell-like properties to T cells, resulting in increased proliferation and survival of immature T lymphocytes. Several studies have reported that BCL11B is very frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL) patients. These mutations which are significantly more frequent in exon 4 of BCL11B disrupt the structure of the zinc finger domains and prevent the binding of the transcription factor to DNA, blocking the transition from Double negative 2-Double negative 3 (DN2-DN3) stage of T cell development. However, the mechanism behind the generation of such mutation hotspots in BCL11B is yet to be deciphered.
In the first part of the study, we have investigated the molecular mechanism of fragility of BCL11B in T-ALL. The high frequency of C to T or G to A conversion at the AID-hotspot motifs (DGWY/WRCH/WRC) in the deregulated gene prompted us to investigate the role of the enzyme, AID behind the BCL11B gene mutagenesis. Although AID primarily involves in somatic hypermutation and class-switch recombination of the Ig genes in activated B cells, its aberrant expression is reported across B cell lymphomas/leukemias and T cell lymphomas, suggesting that AID could play a role in tumorigenesis. Furthermore, AID transgenic mice spontaneously develop T cell lymphomas and micro-adenomas in the lung and harbour frequent point mutations in the TCR and c-MYC genes. In the present study, we show that AID is aberrantly expressed in T-ALL patients and cell lines. Endogenous expression of AID generates a signature mutation pattern specifically in BCL11B fragile region in T-ALL cells. To establish the role of AID in the mutagenesis of BCL11B, we generated AID-knockout (KO) in one of the T-ALL cell lines, MOLT4 using CRISPR-Cas9 technology. Cloning and DNA sequencing of the clones confirmed mutations in AID exon 2 at the target sequence in the knockout cells. Loss of AID expression was confirmed using immunoblotting. Chromatin immunoprecipitation studies revealed the specific binding of AID to BCL11B fragile region in exon 4 which was significantly decreased in the AID-KO cells. Interestingly, when both the wild type (WT) and AID-KO cells were grown in culture for a long period of time to assess the generation of de novo mutations in BCL11B fragile region, we observed a reduced mutation load in the AID knockouts compared to WT cells. Further, overexpression of AID in the knockout cells led to a two-fold increase in AID-induced hypermutation (C to T and G to A conversion), indicating that AID play a significant role in mutating BCL11B exon 4. When BCL11B fragile region was further analysed by isolating genomic DNA from lymphocytes of healthy individuals, we did not observe AID-induced mutations in this region.
The action of AID requires single-stranded region in the genome, generated by transcription bubbles or stem-loop structures which are natural targets of this enzyme. Bioinformatic analysis revealed the presence of G4 sequence motifs, inverted repeats and mirror repeat sequences in the exon 4 of BCL11B of human and mice. Fragile region of exon 4 had majority of the non-B DNA motifs and hence was used for further studies. Sodium bisulfite modification assay revealed the single-strandedness of BCL11B fragile region at the chromosomal DNA level, indicating the propensity of these sequences to fold into non-B DNA structures. Using various biochemical and biophysical assays like Electrophoretic mobility shift assay (EMSA), Circular Dichroism (CD), DMS protection assay, P1 nuclease assay etc. we determined the formation of non-B DNA structures like parallel intramolecular G-quadruplex structure in the template strand, triplex DNA structure in the non-template strand and hairpin DNA in both the strands. Using overexpressed and purified GST-AID, we show that AID
preferentially binds to such non-B DNA over linear ssDNA. Taken together, our results suggest that binding of AID to the single-stranded region due to the formation of non-B DNA in BCL11B exon 4 and subsequent deamination of unpaired cytosines results in a U:G mismatch. When repaired erroneously, deleterious point mutations/deletion/insertion are generated in the coding region of BCL11B gene, resulting in loss of its function, contributing towards leukemiagenesis.
The aberrant expression of AID in T cell leukemia and the generation of thymic lymphomas in AID-transgenic mice prompted us to determine the endogenous expression of AID across the developmental stages of mouse and human thymocytes. Normalized count matrix was obtained from the publicly available NCBI GEO dataset (GSE139242) for the human T cell stages. RNA-seq dataset of 42 T cell samples from early T cell progenitors to terminally differentiated T helper cell subsets of the mouse were analyzed. The result revealed a significant expression of Aid in the double negative stages of mouse T cells and a moderate Aid expression in CD4 and CD8 population of human T cells. Interestingly, Bcl11b is highly expressed and transcriptionally active in the early T cell developmental stages, specifically the double-negative and double-positive stages. To determine the expression of Aid in the T lymphocytes of both young and adult mice, we sorted CD3+ T cells from mice thymus, wherein we observed a low but consistent expression of Aid in T cells of both the age groups. Sequencing of the transcript confirmed its identity. We further demonstrated that AID could bind to Bcl11b exon 4 in vivo, owing to the single-strandedness of this region in the genome of mouse thymocytes. Interestingly, nucleotide alterations in Bcl11b exon 4 were observed only in adult mice (7-8 months old) compared to young mice (2-3 weeks old). Whether the observed mutations are indeed generated due to Aid activity in this region warrants further investigation.
In the second part of the study, we have explored the regulation of RAG1 activity by microRNAs (miRNAs), in addition to its regulation by transcription factors, enhancers etc. The discovery that RAGs can act on non-B DNA structures and cryptic RSS, that are abundant in the genome, significantly increases the spectrum of illegitimate recombinase activity by RAGs which can lead to the generation of genomic instability and cancer. Therefore, cells must possess stringent regulatory mechanisms for RAG expression to ensure genomic stability. Restriction of RAG expression to early stages of lymphocyte development, allowing unequal access to transcription factors/coactivators, controlling RAG expression through chromatin remodelling are some of the mechanisms operating in lymphocytes to prevent the occurrence of chromosomal translocation and development of B cell leukemias and lymphomas.
microRNAs represent an additional layer of gene regulation that affects different arms of the living system, including the mammalian immune system. They are a class of small, non-
coding RNAs, around 20-25 nucleotides in length, that fine-tune gene expression by either degradation of target mRNAs or inhibiting their protein expression following its binding to the 3’UTR region of its target gene. Although miRNAs regulating B cell development, maturation etc. are well explored, regulation of RAG1 expression and function in lymphoid cells by microRNAs have only been recently reported.
Using in silico, ex vivo and in vivo studies, recently we identified miR-29c as a novel and direct regulator of RAG1 which modulates V(D)J recombination during B cell development. Interestingly, unlike miR-29c, knockout of miR-29a, a member of the miR-29 family, contributed to poor survival, impaired immunity, thymic involution etc. in mice. Since miR-29a shared the exact same seed sequence as that of miR-29c, we investigated the regulation of RAG1 by miR-29a and its impact on the immunity of mice. While overexpression of miR-29a in pre-B cell line, Nalm6 led to reduced expression of RAG1, inhibition of miR-29a using miRNA-specific inhibitor resulted in its enhanced expression in B cells. A significant downregulation of RAG1 expression at the protein level was also observed in miR-29a stable overexpression Nalm6 cells. On the other hand, enrichment of 3'-UTR of RAG1 within the cells led to enhanced expression of RAG1. The generation of mature miR-29a transcripts inside the cell following transfection with pre-miR-29a construct, pUR2-8, revealed the endogenous processing of the pre-miRNAs by the RISC (RNA-induced silencing) complex. Therefore, to determine its association with the Argonaute protein, we performed native RNA immunoprecipitation from bone marrow cells of mice and Ago HITS-CLIP (GEO: GSE137071) wherein we observed a strong interaction of Ago2 protein with miR-29a and RAG1 3’ UTR.
Consistent with this, we observed generation of mature miR-29a transcripts in mice after intramuscular injection with the pre-miR-29a construct. This further led to a decrease in RAG1 protein expression in bone marrow cells of mice highlighting the significance of this regulation in the physiological context. However, there was no major alteration in T and B cell population, or other blood parameters of mice. Expression of GFP gene in bone marrow cells of mice post intramuscular delivery of pmaxGFP plasmid reiterates the delivery of plasmid DNA to bone marrow cells via the intramuscular route.
Analysis of miR-29a and RAG1 levels across developmental stages of B cells in mice revealed a strong negative correlation between them. The early pre-B cells exhibited high RAG1 and low miR-29a expression, whereas robust miR-29a and low RAG1 level was observed in mature B cells. Interestingly, several reports suggest an impaired immune response in miR-29a knockout mice which die as early as 7 months after birth. The early death of these animals could be possibly attributed to increased DNA breaks and apoptosis caused due to unchecked expression of RAG1. Ubiquitous expression of Rag genes in mice is known to cause severe lymphopenia, growth retardation and a severe block in both B and T cell
lymphopoiesis, a phenotype reminiscent of those reported for mice deficient in DNA double-strand break repair.
Furthermore, we determined the level of miR-29a and RAG1 in lymphoid cell lines where we observed a negative correlation between them. Analysis of RNA-seq datasets from T-ALL and B-CLL patients also revealed a negative correlation between miR-29a and RAG1, underlying the possibility of using it as a biomarker and therapeutic target in cancer. Our results highlight the significance of miR-29a mediated regulation of RAG1 activity during B cell development in mice and its possible impact on immunoglobulin diversity, immune response, oncogenesis, and its potential use in cancer therapeutics. Further, this suggests potential role of miR-29a mediated regulation of RAG1 in maintaining the genomic stability in lymphoid cells.
Thus, the first part of the study reveals that in addition to its physiological role, illegitimate action of AID outside the Ig loci could lead to mutations and deregulation of a key T cell transcription factor, BCL11B, essential for the survival and proliferation of the T cells. We show that AID is primarily responsible for generating point mutations in BCL11B exon 4, which is important for its binding to DNA and subsequent functional activity in T cells. Interestingly, we also find a consistent AID expression in the early stages of mice T cell development, which is in the same window where Bcl11b is expressed. From our study, it appears that AID binding to BCL11B exon 4 could be the initiating point of genomic fragility in this region, which warrants further investigation. In the second part of the thesis, we report the post-transcriptional regulation of RAG1 by a microRNA, miR-29a in B cells of mice and humans and its implications on different immunological parameters when pre-miR-29a is overexpressed in mice. Our study is consistent with observed defects in the survival of B and T cells in miR-29a knockout mice (van Nieuwenhuijze et al., 2017). Further, an inverse correlation of miR-29a with RAG1 in leukemia patients suggests the potential use of this microRNA in cancer therapeutics. Taken together, our study suggests that tight regulation of RAG1 and AID expression is crucial to prevent frequent DNA breaks, deleterious mutations, and genomic rearrangements mediated by these lymphoid-specific proteins inside the cell
Short Circuit and Open Circuit Natural Frequencies of 3-Φ Transformers: Derived Analytical Expressions and its Applications
No full textFrequency Response Analysis (FRA) method is perhaps the most sensitive tool that can detect even the slightest of winding/core movements. High sensitivity, non-invasiveness, non-destructiveness, and on-site capability are some salient features − making it an ideal monitoring and detection tool. The existence of Standards (IEEE, IEC, and CIGRE) is ample testimony of its global acceptance and superior detection capabilities. The detection principle is based on observing a deviation between two measured FRAs which implies a possible fault. Naturally, the next logical step is to analyze these deviations to determine the type of fault, estimate the extent of damage and its severity, and as a bonus, predict its location, if possible. However, even after three decades, arriving at these inferences is still at the research level. Even though there is a consensus among all the standards on FRA test/measurement procedures, best-suited terminal connections, cable layout, grounding practices, etc., they remain largely silent regarding interpretation and diagnostics.
A detailed analysis of literature compiled in Chapter 1 reveals that lack of a mathematical foundation might be one reason for the present plight of FRA. So, developing a generic mathematical-based approach for interpretation and location of incipient mechanical winding damages in actual 3-Φ transformer windings using measured FRA, is imperative. Development of such a generic method necessitates derivation of closedform expressions, which can directly link measured FRA quantities to the electrical parameters of the winding. For assessing damage severity, the challenge is to identify a quantity which is not only extractable from measured FRA, but also be sensitive, monotonic, and traceable to the fault. Driven by this philosophy, this thesis aims to address the following -
1. Propose a unified and general approach to derive closed-form analytical expressions (for each multiphase winding) to link the measured open and short circuit natural frequencies to electrical parameters of the winding and valid for any condition of the neutral
2. Define a quantity calculable from the measured FRA’s peak/trough frequencies which is physically related to mechanical damage in the winding and capable of yielding some physical insight about damage
3. Develop novel methods using the derived analytical expressions to identify an incipient, discrete and localized axial and/or radial displacement in any multiphase winding and applicable for any condition of the neutral
In the second chapter, a generic and unified analytical method is developed (applicable to any 1-Φ or 3-Φ winding) starting from the basic mutually coupled lossless ladder network model to derive equations which relate the harmonic sum of squares of short circuit natural frequencies (SCNF) and open circuit natural frequencies (OCNF) to the elemental winding inductances and capacitances. Complete derivation details are discussed, and all the derived formulae were cross-verified by extensive numerical circuit simulations.
Each of these derived expressions has a strikingly similar structure and possesses a unique property, viz., the contribution of series capacitances and ground capacitances are decoupled. This important property paves the way for estimating a physical quantity directly responsible for the winding resonances, viz., the effective air-core inductance (Leff). This estimation requires multiple FRA measurements. Chapter 3 presents complete details of the concept, its derivation, measurements, and experimental results for all 1-Φ and 3-Φ windings.
Loss of clamping pressure in a winding is not directly identifiable by any means other than an FRA measurement. But, this damage cannot be judged by merely comparing two FRAs. So, a clamping pressure measurement experiment was carried out on a single isolated winding to ascertain the sensitivity and monotonicity afforded by the quantity, Leff, to a change in clamping pressure. Driven by the promising results, author proceeds to build a method based on Leff to find the location of a discrete and localized axial displacement (AD) in any 3-Φ winding configuration. Details of this method, experimental results, and measurement steps are presented in Chapter 4.
Proceeding further, Chapter 5 discusses concept of a new method, measurement steps and experimental results to identify presence of a Radial Displacement (RD) in a 3-Φ star winding with neutral-open, as well as, in a delta connected winding. Driven by success, the concept was extended to identify the simultaneous occurrence of a discrete and localized AD and RD in one phase of a 3-Φ star winding, with neutral-open. Preliminary experimental results proved the method could successfully identify faulted phases that contained AD and RD.
All experiments reported in the thesis were carried out on transformer windings rated at 33 kV, 3.5 MVA. The results are encouraging, and the author believes that true potential of the proposed methods can be judged when implemented on actual transformers.
In summary, this thesis presents, perhaps for the first time, a mathematical basis for identifying and diagnosing axial and radial displacements in 1-Φ and 3-Φ windings using the peak/trough frequency data from the measured FRA. The author believes this is a small step forward in advancing FRA as a diagnostic tool.IIS
Coalescence of Polymeric Droplets
No full textCoalescence is an energy minimization phenomenon in which two equilibrium droplets undergo
a transition to attain a final equilibrium state, i.e., a coalesced state. Coalescence begins
with a point contact between the two drops followed by a liquid bridge of size comparable to
the diameter of the droplets. This phenomenon is more complex for macromolecular fluids
like polymeric solutions than its counterpart Newtonian fluids due to molecular relaxations
and chain entanglements. Under experimental conditions, coalescence can be achieved in
three different configurations: sessile-pendant, sessile-sessile and pendant-pendant. This study
demonstrates the coalescence dynamics of polymeric droplets in sessile-pendant and sessilesessile configurations. To probe this phenomenon in various configurations, we quantify the
growth of liquid neck.
The dynamics of the sessile drop coalescing with the pendant drop is governed by the growth
of neck radius R with time t. In this configuration, we unveil the existence of three regimes
based on concentration ration c/c
∗
, namely, inertio-elastic (IE) c/c
∗ < ce/c
∗
, viscoelastic (VE)
ce/c
∗ < c/c
∗ < 20 and elasticity dominated (ED) regimes c/c
∗ > 20. Our results suggest that
the neck radius growth follows a power-law behaviour R ∼ t
b
. The coefficient b is constant in
IE, VE and with a monotonic decrease in ED. Based on the variation of b in ED, we propose a
new measurement technique named Rheocoalescence, which can possibly predict the relaxation
times of the fluids in elasticity dominated regimes.
The constant value of b in IE and VE regimes is found to be 0.37 and is distinct from the
value of 1, 0.5 in viscous and inertial regime respectively of Newtonian fluids. Further, we reveal
the existence of universality in the neck radius evolution following a scale of R ∼ t
0.36. This
universal behaviour is probed across various polymers like polyacrylamide (PAM), polyethylene oxide (PEO), Polyvinyl alcohol (PVA) and polyethylene glycol (PEG) of different molecular
weights using high-speed imaging. Our findings are substantiated by a theoretical model using
the linear Phan-Thein-Tanner (PTT) constitutive equation.
In comparison, coalescence in sessile-sessile configuration is relatively more complex due
to the contact line motion and energy interaction between the solid and liquid interface. In such
a configuration, coalescence can be triggered by volume filling (VFM) or droplet spreading
method (DSM). Coalescence of sessile polymeric fluid drops on a partially wettable substrate via
DSM exhibits a transition from inertio-elastic (IE) to viscoelastic (VE) regime at concentration
ratio c/c
∗ ∼ 1. Our findings unveil that the temporal evolution of the bridge height follows
a power-law behaviour t
b
, such that the coefficient b continuously decreases from 2/3 in the
inertial regime (c/c
∗ < 1) to an asymptotic value of 1/2 in the viscoelastic regime (c/c
∗ > 1).
To account for fluid elasticity and characteristic timescale in the viscoelastic regime, a modified
thin film equation under lubrication approximation has been proposed using the linear PhanThien-Tanner constitutive equation. The temporal evolution of the droplet has been evaluated
by solving the modified one-dimensional thin-film equation using a marching explicit scheme.
The initial droplet shapes are obtained by resorting to energy minimization. A good agreement
between numerical and experimental results is obtained.
The coalescence of two droplets on a solid substrate via the volume filling method (VFM)
has a contrasting behaviour compared to DSM. Similar to DSM, we identify four different
regimes, namely, inertial dominated (ID), inertio-elastic (IE), viscoelastic (VE) and elasticity
dominated (ED) regimes on the basis of c/c*. Our results reveal that the temporal evolution of
bridge height for VFM follows a power law behaviour, such that b remains constant at 2/3 in
ID, IE, VE, followed by a monotonic decrease in ED. Our study unveils that the coalescence
dynamics of polymeric drops are not universal and, in fact, are contingent on the method by
which the coalescence is triggered. Additionally, we demonstrate the spatial features of the
bridge at different time instants by similarity analysis. We also theoretically obtain a universal bridge profile by employing the similarity parameter in a modified thin film lubrication equation
for polymeric fluids
Room Temperature Phosphorescence and Circularly Polarized Luminescence Characteristics of Phosphine Oxides and Phosphoramides
No full textRoom temperature phosphorescence (RTP) materials have attracted much attention in recent years owing to their potential applications in organic light-emitting diodes (OLEDs), security writing, time-gated bio-imaging, sensing, etc.1 The spin-forbidden phosphorescence phenomenon is rarely observed for organic molecules as the spin-orbit coupling (SOC) facilitated spin-flipping Sn↔Tn process is weaker in these compounds than their inorganic counterparts. Nonetheless, a significant number of organic RTP materials have been developed by breaking the symmetry of Sn and Tn excited states by way of incorporating heteroatoms having at least one lone pair of electrons or empty p orbital (B, N, O, S, P, Se, etc.) (El-Sayed’s rule).2 Despite these developments, the fundamental understanding of the relationship between photoluminescence (PL) lifetime, RTP quantum yield, and specificity of the orbital configurations of the excited state is still elusive in literature. Thus, this thesis aims to unravel the RTP characteristics of fluorophores, mainly phosphorus-containing compounds such as arylphosphine oxides and phosphoramides.
The geometric and electronic features of the tricoordinate phosphorus atom in Ar3P favour n → π* transition and promote the spin-forbidden S1 → Tn intersystem crossing to populate triplet excited states. However, how different the P-centered lone pair electrons (LPE) in Ar3P compared to O-centered LPE in Ar3P=O in the production of triplet excitons through n(O) → π* transitions is not known in the literature. To understand the difference between Ar3P and Ar3P=O based n → π* transitions in the production of triplet excitons, we set to investigate the RTP characteristics of a series of simple triarylphosphines (3·1, 3·2, 3·3, and 3·4) and corresponding triarylphosphine oxides (3·5, 3·6, 3·7, and 3·8). By combining theoretical and experimental investigations, we have established that n → π* transitions in Ar3P=O stabilize the triplet state better than Ar3P; consequently, the former shows ultralong/persistent room temperature phosphorescence (ULRTP/pRTP) with a lifetime exceeding 100 ms. This study also established that intermolecular interactions are essential for stabilizing the triplet states. Unfortunately, the PL quantum yield of Ar3P=O is poor, limiting its practical applications.3 To widen the scope of these investigations, we aimed to study PL characteristics of Ar2P=O(H) 4·1, 4·2, and 4·3. We envisioned that replacing one of the aryl moieties in Ar3P=O with a hydrogen atom would make more room for intermolecular interactions and consequently improve the PLQY by stabilizing the triplet state better. The Ar2P=O(H) showed pRTP with improved PL quantum yield than Ar3P=O. Furthermore, we established that the n(O)→σ*(P-C) transitions in Ar2P=O(H) stabilize the triplet state better than n(P)→σ*(P-C) in Ar3P.4
To understand the effect of resonance interaction between N-centered LPE and P=X (X= O, S, Se) bond on the RTP characteristics of phosphoramides, we developed a series of donor-acceptor (D-A) systems comprising phenothiazine donor and P=X acceptor (5·1-5·3, 6·1-6·6).5-6 The donor-acceptor electronic interactions in these phosphoramides are judiciously fine-tuned by varying the number of phenothiazine units, and the Lewis acidity of the phosphorus center is controlled by the nature of heteroatoms (O, S, and Se) and the number of phenyl moieties (C6H5) attached to it. All compounds exhibit phosphorescence in the solid state under ambient conditions with an afterglow lifetime in the millisecond range (~ 8 – 40 ms). Through this study, we established that the steric crowding and the number of C6H5 moieties around the P=X unit play a crucial role in controlling the electronic coupling between the donor and acceptor moieties in phosphoramides containing non-planar donor moieties.5-6 Furthermore, for the first time, we demonstrated the chiroptical and magnetic chiroptical properties of phosphoramides. For the first time, we demonstrated the utility of cyclotriphosphazene as an optically innocent dendritic core for developing novel chiroptical materials.7 We have also developed a few donor-acceptor (D-A) architecture-based multifunctional organic luminophores for differential imaging of hypoxia/normoxia cancer cells.8 All these intriguing results are discussed in detail in this thesis
2D Piezotronics: Performance to Functionality
No full textIn the pursuit of interactive electronic devices, there is a need for smart materials which can serve multiple functionalities. 2D (two-dimensional) layered materials have gained attention in semiconductor technology because of their versatile electrical and optical properties. Furthermore, some materials exhibit piezoelectricity at 2D scale and can withstand enormous strain. These properties make them suitable as smart materials involving electromechanical signals. In the literature, materials which are semiconducting and piezoelectric are termed piezotronic (piezo+electronic) materials. Theoretical studies have indicated many materials as piezoelectric in 2D form. However, experimental tools to investigate the extent of piezoelectric coupling in 2D materials are limited, and their relevance for piezotronics has not been studied in detail. This dissertation presents some key aspects of 2D Piezotronics for improved performance and to achieve additional functionalities with heterojunctions. The work constitutes proposing a technique to estimate piezoelectric coupling coefficients, choice of flexible substrates for piezotronics, methods to reduce the charge screening effects, measurement strategies to extract the actual piezoelectric output from the bending measurements, and the study of heterojunctions for rectifying behaviour. In this work, Molybdenum disulfide (MoS2) is used as active piezoelectric material.
In the initial part of the work, I propose a technique to estimate in-plane piezoelectric coupling quantitatively for 2D materials. The method involves a novel approach for in-plane field excitation in lateral Piezo force microscopy (PFM). Contact resonance gain of the tip-sample system is leveraged to measure the piezoelectric coupling coefficients in a few pm/V to sub pm/V range. However, I have shown that operating PFM at contact resonance can cause pseudo piezoelectric signals. Therefore, a detailed methodology for signal calibration and electrostatic background subtraction is developed in this work. The technique is verified by estimating the in-plane piezoelectric coupling coefficients (d11) for freely suspended MoS2 of one to five atomic layers. The technique presented is useful in estimating the piezoelectric coupling strengths in emerging 2D materials.
Piezotronic devices are made on flexible substrates for practical applications. Fabrication on flexible substrates often poses great difficulties in handling them, depositing inorganic materials, and carrying out lithography processes. I propose the commercially available nano flex film as a prospective substrate for piezotronics. Carrying out fabrication on these substrates is as seamless as that on rigid substrates. Substrates such as PET, Nano flex and TPU can be used for low-temperature (200 deg C). However, they tend to twist when heated, making the fabrication difficult. I have proposed a gel-based bonding for the Kapton substrates wherein the debonding process is automatic. The method is helpful for the fabrication of 2D material devices on Kapton. Besides selecting the substrates, suitable base layers and passivation techniques are studied to reduce the charge screening effects and thus improve the performance of piezotronic devices. It is verified that open circuit voltages and strain gauge factors obtained for the current monolayer MoS2 device on SiO2 are three folds higher than those presented in the literature.
A simple measurement setup which does not require probe needles or wire bonding is developed for the bending strain measurements. The open circuit voltage and short circuit current signals obtained from a single 2D material device are very small. The noise signals that originate from various triboelectric and electrostatic sources of the measurement setup can be of similar magnitude. Consequently, the electrical outputs from these devices during bending measurements are often misinterpreted. Thus, it is essential to analyse various noise sources in bending measurements. I then discuss ways to reduce the background noise and identify the valid piezoelectric output.
Finally, I have studied some homogeneous and heterogeneous junctions of MoS2 to achieve good rectifying junction behaviour, which can add extra functionalities for piezotronics. The rectification ratio values as high as 5000 could be achieved at 1 V bias. Besides the rectifying ratio, I have observed that the heterojunctions of MoS2 and MoTe2 have superior piezoelectric behaviour compared to other 2D material junctions reported so far with open circuit voltages as high as ~1 V and peak power density of ~200 mW/m2 at 0.44% bending strain. Formation of the p-n and Schottky junction hybrid in MoS2-MoTe2 heterojunction could achieve high rectification ratios and open circuit voltages and is fascinating for further study
Isolation, structure elucidation and increasing anticancer efficacy of an anti-cancer secondary metabolite from a marine-derived endophytic fungus, Aspergillus species
No full textCancer is the leading cause of mortality globally, contributing to ~10 million fatalities in 2020, or roughly one in every six deaths, according to global cancer statistics 2020. Around 50% of all small molecules approved for treatment of cancer between the 1940s and the end of 2014 are comprised of either naturally occurring substances or compounds that were synthesized using those substances. The majority of bioactive chemicals are obtained from terrestrial microorganisms, and while the terrestrial environment is abundant in bioactive producers, the finding of new metabolites is withering. Marine microorganisms are a possible sustainable source of new physiologically bioactive chemicals since the biodiversity of the oceans makes up 50% of the world's total biodiversity. The endophytic fungal community in the marine ecosystem has been demonstrated to be a relevant source of novel and pharmacologically active secondary metabolites. This study aimed to identify potential lead compounds for therapeutic development by evaluating natural compounds produced by endophytic fungi of marine algae, with an emphasis on chemotherapeutic leads. The seaweed S. muticum, a brown alga was collected from the Kovalam Coast, Thiruvananthapuram, Kerala, India. To accomplish this, five fungal species associated sargassum muticum were isolated, grown, and their extracts evaluated for anticancer potential. Two fungal species out of five were chosen as they exhibit the best cytotoxicity on HeLa (based on IC50). The two fungi, Aspergillus sp. and Talaromyces purpureogenus, were grown in liquid media, extracted with ethyl acetate, and their ethyl acetate extracts were tested for their potential to induce cell death on the HeLa cell line. Aspergillus sp, which showed the best cytotoxicity was used for purifying specific compounds responsible for cytotoxicity. Aspergillus sp, ethyl acetate extract as an organic solvent was used for the bioactivity-guided fractionations that resulted in the identification of an anticancer compound. The compound was successfully isolated in significant proportions using silica gel column chromatography, followed by crystallization of the Aspergillus sp ethyl acetate extract. Single crystal XRD, 1D NMR (1H, 13C, DEPT), FT-IR and HRMS analysis revealed that the structure of the isolated molecule is kojic acid (5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). The cytotoxic potential of the kojic acid was studied in vitro against A-431 (human skin carcinoma). Kojic acid-induced cell death on the A431 cell line in a concentration-dependent and Reactive oxygen species (ROS)-independent manner. To enhance the efficacy of kojic acid as a potent anti-cancer compound and solve its limitations. Carbon dots (CDs) are photoluminescent nanostructures with numerous applications. Carbon dots derived from kojic acid were synthesized hydrothermal method and examined using XPS, TEM, and FT-IR. The optical properties were analysed by spectrophotometer and spectrofluorometer. The CDs were tested for cytotoxicity on HeLa, A549, A375, and MDA MB 231 cancer cell lines. The CDs showed best cytotoxicity on MDA MB 231 cell line. Cell death induced by CDs in MDA MB 231 cells is independent of ROS, results in increased mitochondrial membrane depolarization, and arrests cell cycle progression at the sub G1 phase. This opens new avenues for investigating their potential use as anticancer drugs in the treatment of cancer development.DB
Extended Reality based Digital Twin of Arc Welding
No full textArc welding is an important permanent joining process widely used across multiple industries such as transportation, infrastructure construction and equipment manufacturing. However, the inherent high-energy physics and toxic fumes generated during the process poses serious health risks for the human operator. In addition, there is an acute shortage of welding professionals across the world leading to significant disruptions in industry. This risk is partly reduced from the use of automation and welding robots which provide stability and accuracy in movement. However, welding robots need to be taught by humans and this requires significant programming efforts and on-site presence of the skilled operator. This thesis proposes a solution to these problems by leveraging the complementary strengths of Digital Twin (DT) and Extended Reality (XR). The advancements in connected and immersive technologies have led to new reality in design and manufacturing. The recent pandemic and hybrid work culture have pushed companies to adopt new ways of digital transformation and global collaboration. This work starts with the development of Virtual (VR) and Mixed Reality (MR) interfaces for facilitating remote welding scenarios. The differences in VR and MR were investigated with an ISO 9241 pointing and selection task and a weld path definition task with actual robot movement. Comprehensive user studies with an array of quantitative measures such as task completion time, qualitative scores such as NASA TLX and SUS, and physiological parameters such as EEG and ocular parameters revealed crucial insights about users’ cognitive workload and preferences. Trajectory analysis with curve similarity measures uncovered important considerations for path planning scenarios in XR environments. To improve the realism of the VR environment in the next phase, the influence of multi-modal elements such as vibrotactile feedback and spatial audio cues was studied with the raycast based pointing and selection task. Results from user studies were used to derive design guidelines for implementation and selection of frequency and duration of these elements. In the final phase, a multi-modal VR based digital twin was developed by leveraging the obtained results. A novel multi-modal collision-based path definition method for welding was developed and evaluated with various mapping functions. A novel and intuitive robot simulation method was also developed to plan and simulate various weld paths for reachability and safety prior to actual execution by the robot. The developed framework was then integrated with a robotic welding station at an industrial plant and was found to be successful in producing welds of acceptable quality. The system was built using commercially available devices and tools and is suitable for industrial deployment at various stages of engineering development and operations. Thus, this user centered immersive digital twin facilitates the creation of a high-performance Human Robot Team (HRT) which utilizes the intelligence and skill of the human operator and the stability and accuracy of the robot
Investigating the role of miR-198 in oral squamous cell carcinoma pathogenesis
No full textEvidence supports the critical role of microRNA (miRNA) dysregulation in several diseases including cancers. The aberrant expression of numerous tumor suppressor and oncogenic miRNAs is a well-established driving factor in tumor progression. Though several mechanisms are reported to cause downregulation of tumor suppressor miRNAs, epigenetic silencing seems to play a major role. Pharmacological drugs are mostly employed for the reactivation and identification of epigenetically silenced tumor suppressor miRNAs. To this end in our laboratory, the miRNA microarray analysis of miRNA enriched RNA samples from OSCC SCC131 cells treated separately with DMSO (vehicle control) and 5-Azacytidine (a DNMT inhibitor) had previously revealed the upregulation and downregulation of 50 and 28 miRNAs respectively. In the present study, we have elucidated the role of miR-198 which is one of the miRNAs found to be upregulated following the 5-Azacytidine treatment. We confirmed the upregulation of miR-198 induced by 5-Azacytidine treatment using qRT-PCR and established its tumor suppressive function by checking its cell proliferating ability using trypan blue dye exclusion assay in OSCC cells from SCC084 and SCC131 cell lines. Since miRNAs bind to their target genes to bring about changes in gene expression, we wanted to identify a novel target gene for miR-198. Using bioinformatics and molecular approaches, we showed that miR-198 regulates the expression of the oncogene TOPORS at both the transcript and protein levels in OSCC cells in a dose-dependent manner. Using the dual-luciferase reporter assay, we also established the direct interaction of miR-198 with the 3’UTR of TOPORS in a sequence-specific manner. We hypothesised that miR-198 downregulation might be the cause of increased expression of TOPORS in OSCC and miR-198 might play a critical role in tumorigenesis. Thus, we analysed the expression of miR-198 and TOPORS in six different cancer cell lines and 39 matched normal oral tissue and OSCC patient samples and established the biological relevance of this interaction. We also showed that miR-198 suppressed the cell proliferation and anchorage-independent growth, and enhanced apoptosis of OSCC cells, in part, by targeting the 3’UTR of TOPORS. To elucidate the downstream effectors of miR-198 and TOPORS interaction, we analysed the p53/p21 signaling, and our results showed that miR-198 enhances this signaling pathway, in part, through the miR-198-TOPORS-p53-p21 axis. We also investigated the expression of TP53 (p53) and CDKN1A (p21) transcripts in our OSCC patient cohort and showed the clinical relevance of the p53-p21 pathway in these patients.
To uncover the mechanism for upregulation of miR-198 following 5-Azacytidine, we identified an independent promoter for the MIR198 gene using the dual-luciferase reporter assay. We then analysed the methylation status of the MIR198 promoter in DMSO and 5-Azacytidine treated SCC131 cells, using bisulphite sequencing PCR. The results showed that the promoter methylation of MIR198 is responsible for its downregulation/silencing in SCC131 cells. We also showed that the promoter methylation of MIR198 causes differential miR-198 expression in MCF7 and MDA-MB-231 cells (breast cancer) and speculate the involvement of the same mechanism in other cancers. We also showed that the differential methylation pattern of the MIR198 promoter is inversely correlated with p21 levels in MCF7 and MDA-MB-231 cells, indicating the relevance of miR-198-TOPORS-p53-p21 axis in these cell lines. Taken together, our study suggests that miR-198 is epigenetically silenced by hypermethylation of the MIR198 promoter in OSCC, and exerts its tumor suppressive effect, in part, by regulating TOPORS, and the interaction between miR-198 and TOPORS is biologically relevant
Dynamical systems biology approach to identify mediators of the Epithelial-Hybrid-Mesenchymal spectrum
No full textCancer metastasis – the spread of cancer cells from one organ to another – remains the major cause of cancer-related deaths. A hallmark of metastasizing cells is their ability to adapt quickly and reversibly to their dynamic microenvironment. This ability to switch among different cell-states is called as phenotypic plasticity. A well-studied example of phenotypic plasticity in carcinomas (cancers originating in epithelial tissues) is Epithelial-Mesenchymal Transition (EMT) and its reverse Mesenchymal-Epithelial Transition (MET). EMT is characterized by cancer cells losing their cell-cell adhesion and gaining migration and invasion traits, enabling cancer cell dissemination. During MET, the disseminating cells, upon reaching distant organs, regain the epithelial traits, facilitating metastatic colonization. Initially, EMT and MET were considered as binary processes, but recent studies have discovered that cancer cells can acquire one or more hybrid epithelial/mesenchymal (E/M) phenotypes that can be highly aggressive and are associated with worse patient outcomes. While the molecular drivers of EMT have been extensively investigated, the molecular factors that can stabilize hybrid E/M phenotypes or drive MET are ill understood.
In my work, I have used dynamical systems approach to identify two transcription factors that can stabilize hybrid E/M phenotype(s) – NFATc and SLUG – and two transcription factors that can drive MET – ELF3 and KLF4. Our computational model predictions are validated by extensive transcriptomic data analysis at both bulk and single-cell analysis levels. Further, modeling results collected over an ensemble of kinetic parameters – using a tool called RACIPE (RAndom CIrcuit PErturbation) – suggest that the role of these players in stabilizing hybrid E/M phenotype or driving MET emerges from underlying network topology rather than specific parameter values.
First, I incorporated experimentally reported interactions of NFATc with key molecules influencing EMT dynamics, such as E-cadherin, SNAIL and ZEB, in a mechanism-based model. Bifurcation analysis reveals that NFATc can prevent the progression towards a full EMT and expand the parameter region for the existence of hybrid E/M phenotype. Further, RACIPE analysis demonstrated the role of NFATc in augmenting the co-existence of epithelial, hybrid E/M and mesenchymal phenotypes. Knockdown of NFATc in H1975 cells (lung cancer cells exhibiting a stable hybrid E/M state) drove them towards a complete EMT, thus validating the role of NFATc as a stabilizer of hybrid E/M state. Next, via dynamical modeling and transcriptomic data analysis, I examined the role of EMT-inducing transcription factor SLUG in mediating EMT/MET. I found that SLUG, unlike its family member SNAIL, drove a weak
EMT and stabilized cells in hybrid E/M state. Overexpression of SLUG led to an enrichment of hybrid E/M state, highlighting its role in maintaining this phenotype.
Second, I investigated the role of KLF4 and ELF3 through expanding abovementioned regulatory networks governing EMT/MET. Mechanism-based modeling suggested that both KLF4 and ELF3 can delay the onset of EMT, and their overexpression can drive MET, with ELF3 being a relatively more potent inducer. In both cell lines and primary tumors, KLF4 and ELF3 correlate negatively with EMT-inducing factors, and their expression is inhibited during EMT. Thus, both ELF3 and KLF4 are associated with an epithelial phenotype and are putative drivers of MET.
Finally, I observed that while high levels of NFATc, SLUG, KLF4 and ELF3 associated with worse patient survival in some solid tumors, the trends were tissue specific. These observations reveal the complex association of EMT/MET with patient survival. Overall, my research showcases how a dynamical systems biology approach can help identify potent regulators implicated in phenotypic plasticity, thus suggesting putative therapeutic targets to be considered for curtailing metastasis
Effect of High Pressure Torsion Processing on Precipitate Containing Alloys
No full textSevere plastic deformation (SPD) processes yield excellent grain refinement in metallic materials by application of very large shear strains while retaining the initial sample dimensions. Its effects on simultaneously enhancing the strength and ductility in pure metals and single-phase alloys has been studied in great details since the past three decades. The focus of the recent studies has been on the metallurgical alchemy achieved during SPD processing that yields good structure – property correlations. Most of the studies investigating the compositional modulation achieved during SPD have been performed in binary alloys using the high-pressure torsion (HPT) processing technique. However, detailed investigations on the effect of large strains on multicomponent alloy systems, containing precipitate phases, have rarely been performed till date. Hence, a systematic study on the microstructure evolution with dissolution of precipitate phases and their reprecipitation during severe plastic deformation processing is highly desirable.
The work carried out in the present thesis focuses on the effect of severe plastic strains applied by HPT technique on the state of precipitates in two ductile cubic alloy systems, namely the niobium alloy (BCC) system and the aluminium alloy (FCC) system. The first part of the thesis focusses on the niobium alloys, pertaining to the composition of the dissolved and reprecipitated carbide phases and the corresponding change in its strength. The results obtained from multiscale characterization techniques have been used to further assess the additivity of strengthening mechanisms in niobium alloys. The plasticity of the HPT deformed niobium alloys has also been investigated and correlated with the microstructures to determine the optimum applied strain. The second part of the thesis aimed at increasing the plasticity and irradiation damage tolerance of Nb – 1Zr alloy, which is popularly used in the nuclear industry, by increasing the sites for absorption of the irradiation induced defects by HPT processing. In the last part of the thesis, the effect of Sc addition in the strengthening of AA-2195 Al alloy deformed by HPT was examined. The microstructural features and metallurgical alchemy yielded by large plastic strains were investigated at the nanoscale to understand the post HPT mechanical response. The role of excess vacancies, dislocations and non-equilibrium grain boundaries were investigated by theoretical calculations and corroborated experimentally by atom probe tomography to understand the contributors to compositional modulations of various alloying additions