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Investigating The Role Of Platelet Proteins In The Regulation Of Atherosclerosis In Coronary Artery Disease
Full text linkThe role of inflammation in all stages of atherosclerotic process is well-known and soluble
TREM like transcript 1 (sTLT1), a platelet protein, is reported to be linked to chronic
inflammation. Yet, no information is available about the involvement of sTLT1 in
atherosclerotic cardiovascular disease. Initially, our results indicated plasma level of sTLT1
was significantly (p<0.05) elevated in clinical (2342±184pg/ml) and subclinical cases
(1773±118pg/ml) than healthy controls (461±57pg/ml). Additionally, statistical analyses
indicated that sTLT1 was associated with Coronary Artery Disease (CAD). Ex vivo studies
on macrophages indicated that sTLT1 binds to Fcɣ RI to activate MAP kinase signalling
cascade to activate NF-kB which promotes secretion of pro-inflammatory cytokine TNF-α
from macrophage cells. Atherosclerotic apoE-/- mice also showed high levels of sTLT1 and
TNF-α in virtually occluded aortic stage indicating the contribution of sTLT1 in
inflammation. As, sTLT1 is a platelet secreted protein with a significant role in CAD, we
explored the plasma secretome of Coronary Artery Disease subgroups (STEMI, NSTEMI,
UA) which may identify new candidate proteins responsible for the development of CAD.
It resulted into the identification of several unique proteins in each subgroup. Employing a
case-control design, more than 2500 annotated proteins were identified using Orbitrap mass
spectrometer in both STEMI and healthy control subjects; whereas in NSTEMI and UA the
numbers were a little less (>800). Quantitative proteomics study on STEMI patients revealed
that 26 proteins were decreased and 38 proteins were increased significantly in STEMI
compared to healthy control. AZGP1, ABCA5, Calicin, PGLYRP2, HAVCR2, C17ORF57
appeared to be relevant to STEMI whereas soluble Galectin-3 seemed relevant to NSTEMI
after cross-validation in human samples. Mechanistic significance in foam cells indicated the
imbalance of RCT through the interaction of AZGP1 with CD36. Additionally, in silico
studies of soluble Galectin-3 with Dectin-1 showed the activation of Dectin-1 mediated
signalling which led to the secretion of pro-inflammatory cytokines. In summary, this study
revealed a unique relationship of some novel proteins apparently responsible for impaired
RCT and chronic inflammation leading to atherothrombosis and myocardial infarction in
CAD
Exploring the role of mechanical cues in T cell function
Full text linkT lymphocytes are constantly subjected to mechanical cues from their microenvironment. The
past few decades have seen a significant development in scientific research concerning the
crucial role of biophysical forces in governing various T lymphocyte-mediated processes.
Mounting research have confirmed that T lymphocyte activation is critically dependent on its
capacity to sense and respond to mechanical forces which are generated during its interaction
with cellular partners and with its microenvironment. Despite extensive research on the role
of mechanical forces on T lymphocyte biology, no dedicated T lymphocyte-intrinsic
mechanosensory module was identified until recently. In the following thesis, I have
described the role of Piezo1 mechanosensors in T lymphocyte function, particularly in the
contexts of T lymphocyte activation and migration. Upon interaction with cognate antigen
presenting cells (APCs), T lymphocytes experience significant mechanical force which
activates Piezo1 channels. Activated Piezo1 allows influx of extracellular calcium which
triggers calpain-dependent polymerisation and remodelling of the actin cytoskeletal scaffold.
This event is crucial for formation and stabilisation of the T cell-APC immunological synapse,
thereby facilitating optimal T lymphocyte activation. Moreover, Piezo1-mediated
mechanotransduction also plays a critical role in T lymphocyte chemotactic migration.
Downregulation of Piezo1 resulted in dramatically impaired motility of T lymphocytes in
response to stimulus. Thus, we have identified a previously unknown pathway of Piezo1-
mediated mechanoregulation of T lymphocyte function
Understanding The Intra And Inter-Cellular Interaction Complexities And Flexibilities Using Systems And Sequence Analysis Approach
Full text linkThe present thesis work has been undertaken to gain an understanding of intra-cellular
or inter-cellular interactions between bio-molecular entities utilizing either a systems
analysis based perspective or different sequence analysis approaches. During this study
different principles likely to be prevalent among intra-cellular and inter-cellular
interactions have been studied with the help of computational approaches. Broadly, the
complexities in intra-cellular interactions have been studied by determining the effect
of perturbations such as over-expression or down-regulation of a key regulator on the
intra-cellular interaction network architecture or its components. In particular, network
analysis of regulatory network proteins in association with the intra-cellular proteinprotein
interaction network, led to a key observation that topologically important
effector proteins in the regulatory network could be important signaling proteins.
Identification of such important effector proteins essential for the regulatory network
integrity of a key regulator may be performed by network analysis. It is likely that
alterations in these important effector proteins may lead to disruptions in cellular
physiology and as such in this manner probable disease associated entities can be
determined. Alternately, the flexibility among protein-protein interactions has been
studied by analyzing homologous sequence families of interacting proteins with the
help of information theory based measures like mutual information and Bhattacharyya
co-efficient. Since interacting proteins may co-evolve, co-variation may allow the
preservation of a functional interaction between co-evolving proteins and interdependent
residue pair alterations may occur as a result of evolutionary pressure.
Analysis of molecular co-evolution in inter-cellular protein interaction complexes
determined that co-evolutionary pairings may be present among interface and noninterface
residue pairs and such positions are likely to be crucial for a functional
interaction between these sets of proteins. Therefore, utilising information contained in
biological sequences, co-evolutionary pairings involving structurally or functionally
crucial residue positions in disease associated inter-cellular protein-protein interaction
complexes were predicted. Thus, different computational approaches have been utilised
to study a particular hypothesis in a disease scenario in order to delineate certain
themes prevalent in intra-cellular or inter-cellular interactions among bio-molecular
entities while predicting disease associated entities or studying interaction patterns
among them
Etiopathological Study of Type 2 Diabetes Patient from India
No full textType 2 Diabetes (T2DM) is an endocrine disorder characterized by high blood glucose level
arising from combination of insufficient insulin secretion from pancreatic β cell and insulin
resistance in peripheral tissues. High dipeptidyl peptidase 4 (DPP4) activity attenuates
insulin secretion from pancreatic cells by inactivating gut derived incretin hormones and
interestingly incretin based therapy is one of the central treatment tools available for
clinicians today.
DPP4 is a type II transmembrane protein and it is released from the membrane through
unknown mechanisms. Plasma DPP4 activity is often increased in metabolic diseases and
associated with hyperglycaemia. We show that plasma DPP4 activity is significantly high in
Indian T2DM patients. However, mechanism of DPP4 shedding in T2DM is largely unknown.
This thesis shows an unknown cellular source and mechanism behind the enhance plasma
DPP4 in T2DM patients. It uncovered that kallikrein-related peptidase 5 (KLK5) is the
enzyme responsible for cleaving DPP4 from the surface of circulating CD4+ Th17 cells and
shedding them into the plasma of T2DM patients. Similar cleavage and shedding activities
were not seen in controls. Thus our study could be a potential approach for developing novel
therapeutic strategies for hyperglycaemia.
Increased DPP4 activity was shown to be independently associated with diabetes as well as
obesity. As obesity is among the most important risk factors for the pathogenesis of T2DM,
the relative contribution of obesity in T2DM with respect to DPP4 activity remains unclear.
Using two different cohorts of non-diabetic and T2DM patients, this thesis shows that DPP4
activity is preferentially related with glycaemic control rather than obesity in T2DM patients.
Hence, the research work underlying this thesis aims to examine the scope of
therapeutic opportunities of DPP4 inhibitor in precision medicine
Designing Platforms and Peptides for Monitoring Microtubule Function and Neuroprotection
No full textEnhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation
No full textLocalized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes,
including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase–promoter open-complex structure showed four bases of the non-template strand, the master base �11A, �7, �6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the �11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several
mutant s70s. This may be interpreted as the protein playing an important role in enhancing the flipping
rate. These results suggest that flipping of �11A, and perhaps other flipped bases observed in the opencomplex,
is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation
