http://hdl.handle.net/123456789/1709 Dissertation submitted by MP -2018 batch as part of their course. Sun, 04 Jun 2023 15:58:58 GMT 2023-06-04T15:58:58Z http://hdl.handle.net/123456789/1783 Title: Understanding the Role of the Hippo-YAP Signaling Pathway in the Process of Retina Regeneration in Zebrafish Authors: Joshi, Pallavi; Ramachandran, Rajesh Abstract: Sight is probably one of the most important senses that a number of animals rely on in their daily lives. While the exact anatomy of the eye varies among animals, most vertebrates possess retina as the inner most layer that helps them with vision. Needless to say, any injury or disease to the retina can cause severe aberrations in vision or even the loss of it. Hence, the body has an intrinsic regenerative capacity to help in such crises. While mammals like humans have an extremely limited potential for regeneration, naturally regenerative organisms like the zebrafish possess excellent reparative mechanisms that can result in full reconstitution of all retinal layers and hence lead to complete restoration of vision. This process of retina regeneration in zebrafish is mainly characterized by the acquisition of a stem cell-like state by the Muller Glial cells which allows them to generate proliferating progenitor populations that can then differentiate into the different cell types. This process requires the interplay of multiple signaling cascades like the TGF-β, Delta-Notch, JAK-Stat and Wnt signaling pathway. However, one such pathway that is not completely understood in this context is the Hippo-YAP signaling pathway. Though recent reports in mice have shown the Hippo pathway to be associated with the quiescence exit of Muller Glial cells and MG cell reprogramming, little is known about its role in a naturally regenerative organism like the zebrafish. In this study, we try to elucidate the role of the Hippo-YAP signaling pathway by looking at the effects of inhibition of the YAP-TEAD interaction on the proliferative response of the Muller Glial cell-derived Progenitor cells (MGPCs) and on the expression levels of various Regeneration Associated Genes (RAGs) like ascl1a, hdac1, oct4, sox2 and tgfbi in the three phases of retina regeneration, namely, the dedifferentiation phase, the proliferation phase and the redifferentiation phase. Furthermore, this study also looks into the effect of this inhibition on the cell fate by quantifying the redifferentiated cell types like the Muller Glial cells, Amacrine cells and the Bipolar cells. In addition, the study has also looked at how the response to injury is affected when the Hippo-YAP pathway is constitutively kept in the ON state in the homeostatic conditions. Together these experiments show that the Hippo pathway plays an important role in the process of zebrafish retina regeneration and has different functions and underlying mechanisms depending on the phase of retina regeneration. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1783 2021-07-28T00:00:00Z http://hdl.handle.net/123456789/1782 Title: Role of Mutually eXclusive Exons (MXEs) in the functional shift of isoforms: A case study of human Pyruvate Kinase M (PKM) Authors: Sawant, Shabduli Arjun Pandit; Pandit, Shashi B. Abstract: The process of alternative splicing (AS) is known to account for a major source of human proteome diversity. Among various known types of AS events, Mutually eXclusive Exon (MXE) splicing results in isoforms having one (or more) out of two (or more) exons mutually eliminated in a coordinated manner. MXE events are known to generate highly diverse function protein variants from the same gene. These splicing events can lead to proteins with similar length and scaffold but highly specific functions. It has been of great interest to understand how change(s) in region(s) of a protein can significantly alter the function of protein. In order to gain insights into structural changes in isoforms generated from MXEs events, we considered human Pyruvate Kinase M (PKM) as a model system because tertiary structures are known for both isoforms (PKM1 and PKM2). It is known that PKM1 is a constitute enzyme and PKM2 shows allostery on binding various effectors. In the present study, we have systematically analyzed the origin of allosteric behavior in monomeric structures by extensive analyses of structural features of both PKM1 and PKM2. Our analyses showed that differences in the inherent dynamics of loop, which is a region encoded by the mutually exclusive exons, in PKM1 and PKM2 could affect oligomerization as well as affect allosteric transitions. The analyses of allosteric paths suggest that FBP-mediated allostery is greatly enhanced in PKM2 whereas in PKM1 the path has low significance. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1782 2021-07-28T00:00:00Z http://hdl.handle.net/123456789/1781 Title: Mechanistic basis of Wound healing in adult Drosophila melanogaster Authors: Bhattacharyya, Saswata; Mandal, Lolitika Abstract: A wound triggers a number of local and systemic responses whose sole aim is closing the wound and eliminating the pathogens entered through the wound in order to maintain homeostasis. Drosophila melanogaster has been used extensively to study the wound healing process. In different stages of Drosophila, wounding experiments have shown to evoke a number of molecular players near the wound such as ROS (hydrogen peroxide) in embryo, larval and adult stages, JNK and Hippo in adult stages, VEGF (pvr/pvf) and insulin signals at larval stage. Hydrogen peroxide is known to initiate most of the processes, i.e; recruitment of hemocytes to the wounded area, activating the recruited hemocytes. Additionally, ROS is also known to activate JNK responses which inturn helps in the re-epithelization process. Hippo, insulin and pvr/pvf signals are important for proper wound closure and the re-epithelization process. A wound should also have systemic responses (depends upon size and extent of the wound). In murine models it has been seen that bone marrow participates actively in the wound healing process (which is a systemic response). The hematopoietic hubs located in the dorsal side of the adult Drosophila seems to be a simple version of the vertebrate bone marrow. Like bone marrow these hubs have been seen to participate actively when the body faces an immune challenge. In this thesis I show that the hematopoietic hubs also respond to sterile wounds. Since such changes should be signalled by the body and since the changes seen are in response to the wound, I tried to identify the signals evoked near the wound. JNK and Hippo were seen to be evoked at 9 and 8 hrs post wounding respectively, thus reconfirming the known data. Since the hemocytes are seen to leave the hubs at 3- 4 hrs post wounding we can rule out the possibility of JNK and Hippo of being the signals responsible for the phenotype seen. Further experiments are required to elucidate the signals responsible for the hemocyte migration from the hematopoietic hubs post wounding. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1781 2021-07-28T00:00:00Z http://hdl.handle.net/123456789/1780 Title: Study of the Self-assembly of a Nonionic Surfactant at Aqueous-Liquid Crystal Interfaces Authors: Dutta, Sukanya; Pal, S.K Abstract: Nematic liquid crystals (LCs) are anisotropic materials, whose long orientational order and high fluidity help to direct molecular assembly and amplify interfacial events into easily observable optical signals. Owing to the environment-sensitive fast response of LCs, they have been substantially studied to understand different complex biomolecular events at the interface. The self-assembly of surfactants, polymers, and other amphiphilic molecules at the nematic LC-aqueous interface, as well as the subsequent optical and orientational transitions in LC, have been extensively studied over the past few decades. In our project, we are trying to investigate various interfacial phenomena at aqueous-LC interfaces, using the polarised optical microscope (POM), that triggers orientational ordering transition of liquid crystal in the presence of the self-assembly of a nonionic surfactant tetra (ethylene glycol) mono-n-dodecanoate (Surf-LTE) and its cleaved fragments to understand the underlying mechanisms of such interactions. Keeping the goal of sustainable chemistry in mind, we are working with an eco-friendly bio-degradable nonionic surfactant system (Surf-LTE) that was synthesized in our lab. We have determined the critical micelle concentration (CMC) of Surf-LTE using the fluorescence probe method (DPH assay). Next, we have performed a set of polarized optical microscopy (POM) experiments and observed that Surf-LTE could spontaneously assemble at aqueous-LC interfaces to induce homeotropic orientations of the LC mesogens. The stability of such anchoring transition was also checked by varying the pH and salt concentration of the aqueous medium. In presence of a targeted stimulus (enzymes such as lipase) cleavage of the ester bond triggered a surface-driven ordering transition to a planar alignment of LC mesogens leading to a macroscopic bright optical signal. The optical response of precleaved moieties and cleaved fragments at the aqueous-LC interfaces was also found to be similar. The results of our study so far exhibit a straightforward and broad approach to the rational design of nonionic surfactant systems that can be used to program stimuli responsiveness into nematic LC-aqueous interfaces. The results provide an easy readout of interfacial events over time without the use of complex instruments and it is also label-free. The results of this study can have further applications in drug delivery thus enhancing its potential in the advancement of therapeutics. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1780 2021-07-28T00:00:00Z