Open Access Repository of Indian Theses

Vyas, Brinda K.

Undetermined

Studies on Structure-Property Correlations in Mixed Valent Manganites and Functional Oxides

Pradhan, Atul

viii 
 
Synopsis of the PhD Thesis Entitled 
The Influence of Glycerol and Specific Genes 
on the Asymmetric Cell Division in Mycobacteria 
Submitted by Atul Pradhan (S.R. No. 03-04-00-10-11-14-1-11678) 
Background Information: Mycobacteria maintain heterogeneity in cell-length, morphology, and 
metabolic differences in in vitro cultures, infected mice and guinea pigs, and tuberculosis patients. 
We had reported that ~20-30% of the septating mid-log phase cells of Mycobacterium smegmatis, 
Mycobacterium tuberculosis, and Mycobacterium xenopi undergo highly deviated asymmetric 
constriction during division (ACD) to generate short-sized cells (SCs) and normal/long-sized cells 
(NCs) as sister-daughter cells having differential susceptibility to oxidative/nitrite/antibiotic stress. 
The remaining 70-80% of the septating cells undergo more-or-less symmetric constriction during 
division (SCD) generating sister-daughter cells of more-or-less comparable length, as shown by 
us and other groups. We found that cell-length heterogeneity was achieved in mycobacteria by 
generating differently sized sister-daughter cells through ACD of NCs and SCD of SCs and NCs, 
as one of the mechanisms. Subsequently, we discovered Ap6A as the molecule, which gets secreted 
into the growth medium, that induces ACD in M. smegmatis and M. tuberculosis in a 
paracrine/autocrine manner and the levels of which in change with growth phase. Ap4A, which 
was introduced into the medium, was also found to induce ACD. 
In Escherichia coli and Bacillus subtilis, cell-size heterogeneity is affected by the availability 
of glucose in the culture medium for which UDP-glucose (UDPG) serves as the intracellular proxy 
for nutrient availability and its concentration decides the cell size during the division. In the present 
study on M. smegmatis, despite the absence of the orthologues of UgtP of B. subtilis and OpgH of 
E. coli that are involved in UDPG metabolism, we examined the influence of glycerol on the 
proportions of cells undergoing division by ACD and SCD. Further, we identified the genes that 
influence the synthesis and degradation of Ap4A, which is another ApnA, probably the precursor 
to Ap6A, also found to be involved in inducing ACD. 
Layout of the Thesis: The Chapter 1, which forms the Introduction to the thesis, gives an 
extensive literature survey on different areas of research in the bacterial physiology that are linked 
to the present study. These areas of research include bacterial cell division, different mode of cell 
division in mycobacteria, cell division related proteins in bacteria, relation between cell division 
and nutrient status, different carbon sources used by bacteria for survival, estimation of free 
ix 
 
glycerol in the medium, genes and its biochemical activity, related to diadenosine polyphosphates 
synthesis and phosphorylation in bacteria. 
The Chapter 2 forms the Material and Methods used in the present study. Here a detailed 
description of the methods used to study growth phase of bacteria, cell division and bioassay used 
to score for the proportion of cells undergoing symmetric and asymmetric constriction during 
division, assay for free glycerol concentration in the medium, Ap6A exposure to M. smegmatis 
(Msm) and its effect on ACD/SCD, RNA extraction and qPCR analysis, Protein overexpression of 
MSMEG_2932 and its biochemical activity using different substrate followed by identification 
and quantitation of different products with the help of HPLC. 
The Chapter 3 forms the first data chapter that presents the results on the effects of growth 
phase of Msm and free glycerol concentration in the medium on the proportions of cells dividing 
by SCD and ACD. Here we have shown as to how the changes in the glycerol (main carbon source) 
concentration with the growth phase affect the proportion of cells dividing by SCD and ACD in 
the population. The population shows change in the proportion of SCD and ACD. The proportion 
could be maintained by maintaining free glycerol concentration in the medium. We had also tested 
the effect of spent medium from different growth phases on the ACD:SCD proportions. 
The Chapter 4 forms the data chapter that presents the levels of expression of 
MSMEG_2932, MSMEG_2933, and MSMEG_2936, which were found to influence ACD. Hence 
the proportions of cells dividing by ACD:SCD, in a growth phase dependent manner, were 
determined using the respective gene knockout mutants. The ACD:SCD proportion change with 
growth phase and glycerol concentration in the medium was more or less reversed or unaffected 
in the KO strains. Quantitative PCR was performed for the expression levels of the three genes at 
0.8 OD and in the Ap6A-exposed cells. 
This data chapter also presents the results of MSMEG_2932 protein overexpression, 
purification and study of activity as an Ap4A hydrolase/phosphorylase and as a Ap4A synthase at 
a variety of conditions involving different pH, buffers, temperatures, and divalent ions. Thus, the 
present study for the first time has shown the influence of glycerol on ACD:SCD proportions in 
the dividing population of Msm cells. The present study also reports the in vitro synthesis of Ap4A 
by MSMEG_2932, using ATP as the substrate. 
The thesis is concluded with a section on general discussion, salient findings from the work, 
followed by an extensive bibliography. 


English

The Influence of Glycerol and Specific Genes on the Asymmetric Cell Division in Mycobacteria

IKHLAS, SHOEB

TOXICITY ASSESSMENT OF BISPHENOL-B, A POTENTIAL ENDOCRINE DISRUPTING CHEMICAL

P., SAKTHI KUMARAN

ENRICHMENT OF MILLET FOODS FOR DIETARY BIOACTIVE MOLECULES 
THROUGH FERMENTATION 
 
ABSTRACT 
 
Ferulic acid known for its antioxidant, antidiabetic, anticancer and antihypertensive properties 
is a hydroxycinnamic acid derivative ubiquitously present in the plant kingdom. Due to its 
health beneficial effects, food industries are improving their processed products for enrichment 
of ferulic acid. In this regard, importance of the enzyme feruloyl esterases has attracted the 
attention of food processing industries owing to its ability to release bound ferulic acid making 
it available in free form. The therapeutic properties of ferulic acid have also instigated research 
towards exploration of unique feruloyl esterases for use in the development, and preparation of 
functional foods.  
The thesis describes isolation and characterization of Feruloyl esterase producing probiotic 
lactic acid bacteria, Lactobacillus fermentum CFR5, from pearl millet porridge (kambu koozh). 
The zymogram assays were useful for rapid screening for the enzyme producing bacteria. The 
properties of the enzyme and its ability to release bound ferulic acid, specifically the activity 
retention in presence of metal ions, were suited for applications in millet processing. The 
enzyme activity increased free phenols in Indian traditional millet fermented food and thereby 
its antioxidant properties. Further, the fermented food retained aminoacid content and 
significantly reduced anti-nutritional phytic acid, trypsin inhibitors and tannins. Feeding the 
formulated millet porridge to diabetic rats reduced free radical formation with concomitant 
increase in antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidases. 
Thus the results apart from establishing the scientific basis of nutritional benefits in the 
traditionally fermented millet food, describes a cleaner technology for its preparation.  
 


ENRICHMENT OF MILLET FOODS FOR DIETARY BIOACTIVE MOLECULES THROUGH FERMENTATION

O.V., Manila

 
Electrochemically Engineered Tungsten Disulfide and Phosphorene Quantum Dots for Electrocatalytic 
Applications 
 
The discovery of Graphene paved the way to a brand-new scientific realm: Two-dimensional (2D) 
materials. Explicit research on graphene ignited the research on other 2D materials and we have now 
a wide spectrum of 2D materials of unique structural and electronic properties. One major reason for 
the search of other 2D materials is its intrinsic zero band gap which restricts them for optoelectronic 
applications. Consequently, 2D materials beyond graphene such as Transition Metal Dichalcogenides 
(TMDs, e.g. MoS2, WS2, MoSe2 and WSe2) and Phosphorene have emerged to tackle the bottlenecks 
associated with graphene. Band gap opening in 2D materials can be more realized by nano structuring 
TMDs and phosphorene to their quasi-0D quantum dots (QDs). Such a transformation presents 
constructive impacts as it endows enhanced quantum confinement effect and surface to volume ratio, 
ease of heteroatom doping and functionalization enabling them to be utilized for various applications 
such as electrocatalysis, bioimaging and optoelectronics. Though MoS2 QDs are explicitly studied as 
‘flagship’ TMD QDs, limited abundance of Mo and its high industrial consumption necessitates the 
search of alternative TMDQDs. Therefore, WS2 QDs which resemble the lattice structure of MoS2 is a 
good alternative for MoS2 QDs. To date, the research progress in synthesizing WS2 QDs is mainly 
centred on liquid phase exfoliation (LPE). LPE has a major disadvantage of using high-boiling point 
solvent which presents destructive impact while separation. Similarly, phosphorene, single or few-
layer of black phosphorus, was rediscovered in 2014 as a potential 2D material due to its remarkable 
performance in field effect transistors. Later, phosphorene QDs (PQDs) were first synthesized by LPE 
in 2015. These methods have their own limitations in terms of size tunability and choice of solvents. 
Therefore, synthesizing QDs from TMDs and Phosphorene is still in infancy and necessitates new 
synthetic approaches and studies on their properties.  
Electrochemical synthesis of QDs from their bulk 2D bulk is a promising approach as it allows exact 
control of electrochemical behaviour at the electrode- VIII electrolyte interface and presents 
constructive impact by disrupting the van der Waals interaction between the layers. ‘Customized QDs’ 
can be obtained by tuning various factors such as electric field gradient and current across the 
electrode-electrolyte interface, nature of electrolyte, area of the electrode and anions/ cations 
offered by the supporting electrolyte. Factors such as duration of the experiment, temperature, 
dielectric constant and conductivity of the solvent also endow high degree of flexibility for 
electrochemical transformation. To be precise, tailor-made QDs (layer specific and size-tuned) for 
specific applications can be obtained by appropriate combination of applied potential, electrolyte and 
duration in an electrochemical synthesis. These are often a single step synthesis at room temperature 
which makes them industrially viable for obtaining QDs in bulk with large area electrodes. Towards 
this direction, this thesis contains six chapters, out of which four chapters discuss the electrochemical 
approach to synthesize WS2 and Phosphorene QDs from their bulk counterpart, their physicochemical 
properties and electrocatalytic applications.  
Chapter 1 provides an overview of various aspects of 2D materials and their QDs. A thorough literature 
survey has been carried out to identify the present scenario of the existing approaches for WS2 & 
Phosphorene QDs synthesis and their limitations. A brief section of the chapter is dedicated to discuss 
some of the important properties and applications of TMDQDs and PQDs. Further, the 
characterization techniques adopted to study the physicochemical properties of the 
electrosynthesized QDs are also discussed.  
Electrochemical exfoliation of 2D WS2 has not been explored aiming to understand the electric field 
induced morphological changes underwent by WS2.Towards this direction, Chapter 2 presents the 
electrochemical synthesis of WS2 QDs from bulk WS2 and their enhanced electrocatalytic activity 
towards hydrogen evolution reaction than their bulk 2D form.  
Deployment of the electrosynthesized QDs towards fabrication of various QDs based devices, 
necessitates development of methods to organize such QDs and a thorough understanding of their 
stability when assembled onto a substrate. Accordingly, Chapter 3 deals with the self-assembly of the 
electrosynthesized WS2 QDs onto a polycrystalline gold electrode and investigation of their 
adsorption kinetics. Changes in the open circuit voltage of the electrode were used as a key to monitor 
the adsorption. Further, thermodynamic parameters such as free energy of adsorption, enthalpy and 
entropy of adsorption are deduced using Langmuir model.  
The next two chapters are centered on the electrochemical exfoliation of black phosphorus to yield 
blue luminescent PQDs. Chapter 4 provides an understanding on the role of structural distortion and 
oxygen content in maintaining the structural integrity of electrosynthesized PQDs. The experimental 
findings are also complemented by the DFT calculations carried out on PQDs of various oxygen 
content.  
Heteroatom doped QDs are anticipated to have altered electronic structure which can exhibit 
intriguing physicochemical properties. Accordingly, Chapter 5 deals with the electrochemical 
preparation of nitrogen doped PQDs using nitrogen containing electrolyte such as acetonitrile with 
Tetraethylammonium tetrafluoroborate as supporting electrolyte. The products have about 3.8 at.% 
of nitrogen with nitrogen being present in two different chemical environments. Further the possibility 
to tune the electrochemical doping of nitrogen is demonstrated with appropriate combination of 
electrolyte and supporting electrolyte. The high quantum yield of the pristine and doped PQDs (83 
and 88 %) could be utilized for light emitting devices.  
The conclusions from the present work along with outlook are presented in Chapter 6.  


Electrochemically Engineered Tungsten Disulfide and Phosphorene Quantum Dots for Electrocatalytic Applications

SURANA, KARAN

v 
 
 
SYNOPSIS 
Over the centuries quite a few sustainable energy sources have been discovered and 
put to efficient use, such as, solar, wind, water, geothermal, tidal, and biomass. Solar 
energy being available for a considerable period of the day makes it a strong 
candidate for the present and future power generating resource. The solar panels can 
be installed anywhere with an open space and the shining sun, such as, rooftops, 
deserted lands, electric poles and so on. Solar cells have been around for over half a 
century now and have evolved from crystalline silicon solar cells (first generation), to 
semiconductor thin film based cells (second generation) to the present dye solar cells 
(third generation). 
QDSC came into picture for replacing the expensive dyes used in DSSC with low cost 
and more efficient QD (Plass et al., 2002). The third generation of solar cells dealing 
with quantum dots has attracted wide research interest owing to the excellent optical, 
electronic and size-tunable properties of QD (Zhu et al., 2010; Raj et al., 2013; 
Chuang et al., 2014). The engineering or modifications of the solar cell electrodes, 
precisely the working electrode is vital as it shall facilitate efficient transfer of charges 
with minimal loss of the excited charges due to recombination or otherwise (Yu et al., 
2011; Zhu et al., 2014; Man et al., 2015).  
This thesis embodies research on synthesis of cadmium chalcogenide quantum dots 
and their use as photoelectrodes. The band-alignment with conventional TiO2 and 
other synthesized QD, consequent tuning of the working electrode has been the main 
focus, in order to achieve better solar cell characteristics. 
The Thesis deals with the following chapters: 
Chapter I start with basic introduction of the renewable energy resources available to 
mankind and why solar energy has a huge contribution in it. Further, a brief review is 
given on the various types of third generation solar cells while explaining their basic 
working principle. The importance is laid down on quantum dots which are promising 
candidates for replacing the expensive dye while increasing the efficiency due to 
vi 
 
multiple exciton generation property. Further the interest was narrowed down to the 
working electrode engineering of the cell. 
Chapter II gives an insight on the working principle of major characterization tools 
and measurement techniques used in the study viz., UV-Vis Spectroscopy, 
Photoluminescence (PL) spectroscopy, X-Ray Diffraction (XRD), Transmission 
Electron Microscopy (TEM), I-V Characteristics, and Electrochemical Impedance 
Spectroscopy (EIS). Also the entire process for device fabrication and cell 
measurements has been detailed. 
In chapter III, details about the synthesis of QD of CdSe via wet chemical method at 
temperatures of 40, 50, 60 and 70 °C has been presented. A prominent red shift in the 
absorption peaks in UV-Vis Spectroscopy and emission peaks in PL spectroscopy 
have been obtained along with a lowering in energy-gap with the rise in temperature. 
The QD were mixed in different ratios and coated directly onto the working electrode, 
leading to a high Voc of 1.45 V with an efficiency of 0.69% and FF of 74%. The 
obtained Voc value is a reflection upon the optimum band alignment on the working 
electrode of the cell. 
In chapter IV, studies on core-shell CdSe-CdS QD prepared at 50 °C via wet chemical 
method has been presented. The core-shell QD was prepared both with and without 
the capping agent; the one without capping agent showed red shift in UV-Vis 
absorption pointing towards the existence of bigger sized particles. The capped QD 
had an average particle size of 4 nm as confirmed by TEM. XRD confirmed the core-
shell QD to be of cubic structure having face-centered lattice. The core-shell QD was 
coated directly onto the working electrode which resulted in a Voc of 1.27 V with an 
efficiency of 0.43%. The cell parameters were tested for a period of 60 days and were 
found to be stable. 
In the final experimental chapter, i.e., chapter V, studies on CdSe and CdS QDs 
prepared with mixed solvent of water and methanol has been presented. In addition, 
the synthesis of rGO and its use as electrode has been studied.  The combination of 
solvents has been shown to result in excellent luminescence under UV light. 
Crystalline natured rGO was prepared by microwave method and applied on the 
working electrode prior to TiO2. CdS and CdSe QDs were deposited later along with a 
ZnS passivation layer resulting in an efficiency of 1.81% with Jsc of 4.50 mA/cm
2
 and 
vii 
 
Voc of 0.92 V. The obtained results point towards the effective engineering of the 
working electrode and a significant possibility in further enhancement of the cell 
structure for achieving increased solar cell efficiency. 
Chapter VI reflects upon the conclusion of the obtained results while exploring the 
future scope of the reported work. The prepared QDSCs were prepared effectively 
with entire emphasis on the working electrode engineering which resulted in cells 
with good band-alignment, improved charge transfer and consequent enhancement in 
efficiency compared the equivalent DSSCs. 
References cited in the synopsis are listed at the end.


ELECTRODE ENGINEERING IN QUANTUM DOT SOLAR CELLS FOR EFFICIENT CHARGE TRANSPORT

Roy, Pratik

Software Reliability Assessment Under Soft Computing Environment 
 
Software reliability measures the chance of failure-free operation of software for a specified 
period of time in a specified environment. In this thesis, several new types of software 
reliability models have been developed considering more practical situations that arise during 
testing and we derive novel soft computing techniques to predict reliability of software more 
justifiably. 
 
Initially, the following statistical parametric software reliability growth models (SRGMs) are 
presented with some new practical development assumptions.  
 
 
 Original Jelinski and Moranda model is modified assuming imperfect debugging 
process in fault removal activity during testing.  
 SRGM based on non-homogeneous Poisson process (NHPP) imperfect debugging with 
error generation is presented. We modify the fault content function by taking into 
account the increased skill of testing team.  
 NHPP based s-shaped SRGM is presented with imperfect debugging and we present 
fault content and fault detection rate functions by considering the increasing efficiency 
of testing team. 
Besides the following robust soft computing-based nonparametric software reliability models 
are presented.  
 Multi-layer feedforward artificial neural network (ANN) based logistic growth curve 
model is presented for software reliability prediction. We present a neuro-genetic 
approach by optimizing the weights of the network using proposed genetic algorithm 
(GA).  
 We develop feedforward and recurrent neural network based dynamic weighted 
combination models to improve software reliability prediction accuracy. Traditional 
SRGMs are combined based on dynamically evaluated weights determined by learning 
algorithm of the networks. We present a real-coded GA based learning algorithm to 
train the ANNs. 
 We also present an efficient neighborhood based adaptive particle swarm optimization 
(PSO) algorithm to train the proposed dynamic weighted combination model for 
improvement of software reliability prediction accuracy.  
 ANN based software reliability model is developed considering the effect of fault 
generation with different levels of fault complexity. We present a neighborhood based 
fuzzy PSO algorithm for learning of the proposed network. 
Each of these proposed models are validated using real software failure data sets. Software 
reliability prediction capability of these models are much superior than established models 
and application of soft computing methodologies enables us to develop models with great 
prediction accuracy than traditional parametric models. Presented models can be cordially 
accepted by testing engineers to assess software reliability precisely during testing phase of 
software development. 


Software Reliability Assessment Under Soft Computing Environment

S., Preethi

IV 
 
ABSTRACT 
 
Improving the ionic conductivity of Samarium doped cerium oxide electrolyte at low 
temperature is essential to enhance the performance of solid oxide fuel cell (SOFC). 
Lower grain boundary resistance and higher densification of the electrolyte at low 
temperature is of paramount importance for achieving good performance. This thesis 
reports different strategies to engineer the grain boundary for enhanced oxygen ionic 
conductivity. Enhancing the oxygen vacancy concentration and lowering the space charge 
potential at the grain boundary enables faster ionic migration. The addition of both 
sintering aid and scavenger together influences the grain and grain boundary conductivity 
to a great extent.  Reactive sintering of the co-dopants during sintering can provide liquid 
phase to densify and enhance ionic conduction. Modification of ionic conduction by 
doping alkaline earth metals (Mg2+, Ca2+, Sr2+ and Ba2+) of different ionic radii results in 
high grain boundary conductivity by enhancing the oxygen vacancy concentration and 
lowered space charge potential. Increasing oxygen vacancy beyond optimum level is 
deleterious ionic conduction due to the association of oxygen vacancy defects. Increase 
in configurational entropy in the lattice by doping different dopants supress the oxygen 
vacancy ordering. Synergistic effect of sintering aid and scavenger enhances the low 
temperature densification and configurational entropy which in turn increase the oxide 
ion conduction. An overall optimization of dopants concentration, ionic radii and 
sintering temperature significantly improved the efficiency of grain boundary scavenging 
for the realization of potential solid oxide fuel cell. 
 
 
 
 


Grain Boundary Engineering of Ceria Based Electrolytes for Solid Oxide Fuel Cells

V., GAYATHRI

Recombinant Expression of Heat Shock Proteins (HSPs) from Artemia franciscana and Exploration of their In Vitro Chaperone Activity

Dasari, Ramakrishna

Molecular mechanisms underlying the role of 
Tumor protein D52 (TPD52) in the 
Prostate cancer progression 
A Thesis 
Submitted for the Degree of 
DOCTOR OF PHILOSOPHY
In 
BIOLOGICAL SCIENCES 
To the 
Academy of Scientific & Innovative Research 
By 
DASARI CHANDRASHEKHAR 
(AcSIR Enrollment No: 10BB13J18062) 
Under the supervision of 
Dr. Ramesh Ummanni 
DEPARTMENT OF APPLIED BIOLOGY 
CSIR-INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY 
HYDERABAD – 500007, TELANGANA, INDIA. 
MARCH-2019 
Summary of the work done 
Summary of work done 
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Page: v 
Title of the work: 
Molecular mechanisms underlying the role of Tumor protein D52 (TPD52) in the Prostate 
cancer progression. 
Introduction: 
Prostate cancer (PCa) is the most leading cause of deaths worldwide after Lung and 
Colorectal cancers. One in nine men is diagnosed for PCa after the age of 60 in the western 
world. PCa arises due to inheritance, food habits, culture, environmental causes, and age. 
When cancer cells morph to become sophisticated enough to move beyond the prostate, 
they learn evasive maneuvers. Evolution of drug-resistant mechanisms and recurrence of 
tumors vitiate current therapeutic approaches. These conditions necessitate the increased 
understanding of oncogenes responsible for PCa and its progression. Oncogenes arise due 
to the accumulation of mutations due to genetic and conditional factors which are essential 
to study in order to provide advanced therapeutic solutions. Tumor Protein D52 (TPD52) 
is among the oncogenes which frequently overexpressed due to gene amplification in 
Breast cancer, PCa and Colon cancers. In vitro studies on TPD52 evidenced that it 
intervenes in Lipid droplet formation, Vesicle trafficking, Calcium-dependent signaling, 
Cell cycle regulation, Docetaxel drug resistance, Migration, Invasion, and Proliferation. 
These versatile mechanisms of TPD52 make it as an essential gene of study, in cancer 
progression. Especially in PCa, TPD52 was reported to be overexpressed by Robert Bright 
et al. in 2004 and Ummanni et al. in 2008. There are various reports suggesting the 
oncogenic functions of TPD52 in PCa, but the detailed mechanisms responsible for them 
remain elusive.  
     To untangle the TPD52 association with oncogenic pathways, we have carried out 
research through which we found its role in alteration of the proteins which are members 
of the NF-κB pathway. We also found the transactivation of NF-κB and STAT3 by TPD52 
overexpression. In addition to this, we have performed a proteomic-based study of TPD52-
interactome which evidenced us TPD52’s novel function in regulating the oxidative stress 
during cancer progression. In order to perform experiments to define TPD52’s functions in 
vitro, we used two PCa cell lines, i.e., androgen-dependent LNCaP cells and androgen 
insensitive PC-3 cells in as the major tools in our experiments. 
Summary of work done 
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Page: vi 
Objectives of the study: 
1. Studying the altered mechanisms of prostate cancer cells due to aberrant TPD52
expression during prostate cancer progression.
2. Identification of novel interaction partners for TPD52 (Interactome) through a
proteomic approach.
Methodologies and Results: 
1. Studying the altered mechanisms of prostate cancer cells due to aberrant TPD52
expression during prostate cancer progression.
To find the novel roles of TPD52, in cancer progression we have carried out in silico 
analysis using online bioinformatics tools like Cytoscape, Genemania, String, and others. 
In silico analysis of TPD52 and TPD52’s interacting partners, structural and functional 
interactions showed more extensive network through its family members. One of the 
subnetworks contains NF-κB pathway proteins, p65, and Ik-Bα which are associated with 
cancer progression by defining the tumor microenvironment through generation pro-
inflammatory markers. Based on this analysis, we measured the p65 NF-κB promoter 
reporter assay by luciferase activity. Interestingly, TPD52 overexpression was able to 
induce p65 promoter, but the downregulation fails to do so. While the altered expression 
of TPD52 exogenously, showed aberrations in proliferation and cell cycle regulation. 
TPD52 upregulation increased the rate of cell proliferation in LNCaP cells increased while 
the downregulation suppressed the cells proliferation. Overexpression of TPD52 
augmented the LNCaP cell growth might be through increasing cyclinD1, Bcl2, XIAP 
protein levels; whereas the downregulation of it leads to decreased cell proliferation could 
be by the induction of pro-apoptotic factors Bax, p27Kip1 and cleavage of PARP. We 
pursued the study to find whether these oncogenic functions are mediated by the NF- κB 
pathway using western blotting, ELISA and other molecular techniques. Whereas, the 
TNFα-induced p65NF-κB activation was also be nullified by downregulation of TPD52. It 
proved that TPD52 could become a crucial factor for NF-κB activation in PCa. Further 
study on TPD52 reveals that it can regulate the expression of matrix metalloproteinases, 
cytokine secretion, and cell adherent proteins thereby contributing to tumor progression. 
We found that the transactivation of STAT3 was mediate by NF-κB in LNCaP cells by 
Summary of work done 
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Page: vii 
using the Stattic and parthenolide drugs which inhibits the activation STAT3, NF- κB 
respectively.     
2. Identification of novel interaction partners for TPD52 (Interactome) through a
proteomic approach.
TPD52 involved in versatile functions has been reported in vitro.  It might regulate different 
pathways by forming homodimers and/or heterodimers owing to having its water soluble 
and flexible structural motifs.  In order to study its novel functions, we destined to study its 
interacting partners by pull-down assays coupled with the proteomic approach. TPD52 
binding proteins were pulled down by the recombinant TPD52 tagged with GST. 
Immobilized proteins were subjected to 2- Dimensional gel electrophoresis followed by 
identification of differentially bound proteins by using LC-MS/MS coupled with in silico 
analysis and peptide search in MASCOT peptide database. Identified proteins contain 
already reported interacting partners along with novel interacting partners. Upon 
bioinformatics analysis of Protein interaction networks using identified proteins, as input, 
we found a variety of protein-protein interaction networks depicting different signaling 
pathways. One of the proteins is Peroxiredoxin 1 which is a novel interacting protein, 
connecting TPD52 with Redox signaling mechanism which may assist us to decipher 
TPD52 role in counteracting the increased oxidative stress, during enhanced migration and 
proliferative processes.  
TPD52 interaction with PRDX1 was validated using in vitro pull-down assays followed by 
functional characterization. In cell-free peroxidase assays using pyrogallol, we found that 
TPD52 presence could increase the peroxidase activity of PRDX1. The retroviral-mediated 
stable expression of TPD52 in PCa cells, provided evidence that the TPD52 enhances the 
dimerization of PRDX1, thereby effecting the detoxification of peroxides. The conditional 
expression of TPD52 using doxycycline-dependent Tet-On system reciprocated our 
assumption about dimerization of PRDX1.  Apart from this, we found, the interaction was 
dependent on oxidative stress where we found increased binding of PRDX1 with NTAP-
TPD52. In addition, the accumulation of peroxides increased due to the combined 
downregulation in the cells evidencing that TPD52 interaction with PRDX1 is essential to 
counteract oxidative damage in cancer cells. In our effort to map the region of TPD52 
binding with PRDX1, we found that the C-terminal region of TPD52 plays crucial role 
interest.  In addition, we found the positive correlation of their combined expression with 
Summary of work done 
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Page: viii 
poor patient survival in clinical samples data obtained from The Cancer Genome Atlas 
(TCGA) which makes our study important by deciphering the novel interaction of TPD52 
with PRDX1. 
Main Document Chapter-4: Objective-2 
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Page: 121 
cell growth was supported by inhibition of cell cycle due to elevated p27Kip1, a cell cycle 
inhibitor and decreased antiapoptotic signal Bcl-2 expression. This observation 
hypothesizes that PRDX1 and TPD52 complex play a role in the survival of PCa cells 
evading apoptosis. 
 Frequent aberrant expressions of TPD52 and PRDX1 is reported in multiple cancers [46, 
48, 104, 128, 134, 152, 162]. To understand the expression pattern of TPD52 and PRDX1, 
analysis of gene expression data on PCa from TCGA database showed a positive correlation 
between TPD52 and PRDX1 expression. Further, the higher expression of TPD52/PRDX1 
affected the overall survival of high-grade PCa patients. Consequently, elevated TPD52 
and PRDX1 in PCa patients may interact with each other and this interaction is colligated 
with the aggressiveness of PCa thereby poor patient survival. 
4.6 Conclusion: 
In conclusion, this study has reported novel candidate partner PRDX1 for TPD52 
overexpressed in prostate cancer by GST pulldown assay coupled with 2DE and in vitro 
validation. Through the functional characterization of the interaction, we identified that the 
TPD52 ability to promote peroxidase activity of PRDX1. Functionally, TPD52 induced 
increased peroxidase activity of PRDX1 might be indulging in mitigating the cellular 
damage due to higher oxidative reservoirs in cancer cells (survival) or in maintaining the 
higher oxidative capacity within cancer cells (aggressiveness). In our attempt to find the 
PRDX1 interacting domain with the TPD52, we found the C-terminal region was crucial 
in the binding of these two proteins. In addition to this, the migratory potential was 
increased in PCa cells with combined overexpression, which infers us that the interaction 
might play an important role in developing the aggressiveness of PCa cells.  Our 
understanding of clinical data obtained from The Cancer Genome Atlas (TCGA) through 
web tools, supported the positive correlation of co-expression (Pearson correlation 
coefficient value=0.35) and the importance of the interaction during the development of 
aggressive phenotypes. Be of the significance of TPD52 and PRDX1 interaction in in vitro, 
and it is noteworthy to demonstrate the druggability of the interaction to design and develop 
chemical inhibitors disrupting the interaction. This study of our imparts and supports the 
previous literature in the understanding of TPD52 functions in prostate cancer. 


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