http://hdl.handle.net/123456789/1311 Thesis submitted by MP -2012 batch as part of their course Sun, 14 May 2023 20:10:01 GMT 2023-05-14T20:10:01Z http://hdl.handle.net/123456789/1699 Title: Exploring the implication of cholesterol in regulating pore-formation mechanism of Vibrio cholerae cytolysin, a -barrel pore-forming toxin Authors: Kathuria, Reema; Chattopadhyay, K. Abstract: Vibrio cholerae cytolysin, a -barrel pore-forming toxin constitutes an important virulence factor of Vibrio cholerae, contributing towards its pathogenesis. The mode of action of Vibrio cholerae cytolysin (VCC) involve three distinct steps as initially the toxin monomers need to interact and bind to the target host membrane, the membrane-associated seven monomeric subunits then interact with each other to generate the oligomeric assemblies and finally concomitant insertion of pre-stem loop from monomers takes place, leading to generation of a functional pore. In order to efficiently form the functional trans-membrane pores, VCC binds to the host membrane by exploiting the presence of membrane cholesterol which is exclusively present in the eukaryotic cells. However, there is no detailed evidence of how cholesterol regulates the pore-forming activity of VCC. Thus, in this study, we have examined the critical role of cholesterol in cellular membranes for functional pore-formation by VCC. For this, an array of experiments using artificial membrane system and biomembranes were employed. The liposome membranes were constituted having controlled cholesterol concentration, ranging from 0 to 50% in these liposomes. In this study, we have observed that the presence of optimal level of membrane cholesterol governs the efficient interaction of toxin to the target membranes that affects the subsequent oligomeric species formation and further generation of functional pores. The membrane cholesterol regulates initial binding of VCC to liposomes, leading to abrogation of functional pore-formation in artificial membranes having limited cholesterol or completely devoid of cholesterol. For understanding the physiological relevance of cholesterol in context of eukaryotic cells, human erythrocytes were treated with methyl -cyclodextrin to sequester cholesterol from erythrocyte membranes that clearly indicated the compromised activity of VCC in membranes devoid of cholesterol. In erythrocytes membranes where cholesterol is being dislodged by employing methyl -cyclodextrin, the formation of functional channel by VCC is affected. Further in the same direction, we explored the segregation and binding of VCC to cholesterol-enriched membrane microdomains, known as lipid rafts. VCC appears to sequester in these cholesterol-rich detergent-resistant membranes (DRMs) fractions. Surprisingly, even the biochemical evidence indicates that the variants of VCC having mutation in specific regions that are responsible for defining different steps of pore-formation associate with lipid rafts in the same way as wild-type VCC. Hence, the results from this study suggest the obligatory presence of cholesterol in target cellular membranes for functional pore- formation and the partitioning of VCC to liquid-ordered membrane domains takes place during pore- formation event. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1699 2021-07-28T00:00:00Z http://hdl.handle.net/123456789/1698 Title: A conserved and essential motif in the pre-mRNA splicing factor Snu66 and SRC1 alternative splicing factors in S. cerevisiae Authors: Choudhuri, Poulami; Mishra, Shravan Kumar Abstract: Eukaryotic gene expression requires the removal of non-coding introns and splicing of the coding exons, which is executed by the dynamic ribonucleoprotein (RNP) complex, spliceosome. In addition to cis-acting RNA elements, spliceosomes have evolved to require trans-acting factors. These factors help in assembly and activation of the spliceosome, leading to constitutive and regulated RNA splicing. In my thesis work I report function of the conserved tri-snRNP factor Snu66/SART1 in RNA splicing. A novel and highly conserved motif at the C-terminus of Snu66, termed Snu66-CM, is essential for its splicing function. Biochemical and splicing assays in the budding yeast Saccharomyces cerevisiae show that Snu66-CM is critical for splicing of precursor mRNAs with non- canonical 5’ splice sites (5’ss). We have extended our study to intron-richer fission yeast Schozosaccharomyces pombe and found that not only Snu66 but also Snu66-CM is essential for cell viability. We isolated a conditional mutant of Snu66-CM which was temperature sensitive and showed defects in constitutive RNA splicing. Importantly, splicing of pre-mRNAs with non-canonical splice signals was severely affected in this mutant, suggesting an additional role of Snu66 in regulated RNA splicing. We next identified that S. cerevisiae Snu66 interacts with Sap1, a AAA-ATPase which binds to covalent conjugates of the ubiquitin-related protein modifier SUMO (AAA-ATPase family proteins are reported to be involved in diverse functions like protein degradation, DNA replication & repair, microtubule motors, etc.). We studied function and regulation of Sap1. From the single gene, two protein isoforms of Sap1 are expressed from alternatively transcribed mRNAs in a carbon source- dependent manner. Interestingly, Sap1 bound Snu66 through its CM. While Sap1-Snu66 interaction indicated a role of this complex in RNA splicing, we could not find any splicing role for the complex. Instead, we discovered an unexpected and intriguing role of the complex in homologous recombination. The lack of Sap1 made yeast cells prone to homologous recombination involving DNA circles, and this activity appears to be mediated by Sap1 interaction with Snu66-CM. Thus, the splicing factor Snu66 is critical not only for constitutive and regulated RNA splicing; the protein also regulates homologous recombination by associating with the AAA-ATPase Sap1. S. cerevisiae SRC1 intron has two non-canonical 5’ splice sites (referred to as alternative and constitutive 5’ss). Spliceosomes uses both 5’ss to promote SRC1 alternative splicing. The usage of the alternative 5’ss requires non-covalent interactions of the ubiquitin-like protein Hub1 with splicing factors Snu66 and Prp5. However, additional proteins are likely to participate in SRC1 alternative splicing. Here we show that this process requires proteins of the spliceosomal core, including thePrp8-101 surface, and subunits of the retention and splicing (RES) complex. Hub1’s proximity to the spliceosomal core proteins is needed for SRC1 alternative splicing, whereas the RES complex appears to function by a different mechanism. The collective list of SRC1 alternative splicing factors indicates a general mechanism; spliceosomes slow down at SRC1 5’ss thereby promoting splicing using both sites. Thus, since SRC1 alternative splicing via the competing 5’ss needs a set of diverse spliceosomal proteins and regulators, other forms of alternative splicing might similarly require concerted action of distinct molecules. Wed, 28 Jul 2021 00:00:00 GMT http://hdl.handle.net/123456789/1698 2021-07-28T00:00:00Z http://hdl.handle.net/123456789/1421 Title: Dynamics on Spatially Extended Systems Authors: Moitra, P.; Sinha, Sudeshna Abstract: We present the analysis and characterization of emergent behaviour, arising from the interplay of local dynamics and the form and structure of coupling, in complex dynamical systems motivated by biological phenomena. First, the dynamics of a non-fatal infection spreading across a population is modeled, and the emergence of persistent infection in a closed region is explored. In this system, the disease progression of an individual is given by the SIRS model, with an individual becoming infected on contact with another infected individual. The persistence of contagion is qualitatively and quantitatively investigated, under increasing heterogeneity in the partitioning of the population into different disease compartments, as well as increasing heterogeneity in the phases of the disease among individuals within a compartment. It is observed that the system’s asymptotic behaviour is attracted to two distinct states - an absorbent state devoid of infection and a persistent state with regular spatiotemporal oscillations - depending on the initial composition of the individual disease phases in the population, with initial heterogeneity aiding the emergence of long-term persistent infection. Next we focus on the role of synchronization in the persistence of infection in such a closed region. The following key result is observed: higher degree of synchronization in the individual states, both globally in the population and locally in the neighbourhoods, hinders persistence of infection. Importantly, it is demonstrated that early short-time asynchrony appears to be a consistent precursor to future persistence of infection, and can potentially provide valuable early warnings for sustained contagion in a population patch. In a following study, it is established that populations where the initial distribution of the disease cycle is strongly compartmentalized leads to persistent infection in the complete region. Even after transience, the patterns of disease spreading in the two communities may be completely dissimilar, even though both communities settle down to the same average infected sub-population size, displaying periodic waveforms with different amplitudes, but similar frequencies. In a final study we investigate a collection of populations modeled by the prototypical chaotic Ricker map. Such chaotic maps are widely utilized to model the population growth of species with non-overlapping generations. The feedback received by each population patch is modeled to be influenced by the local mean field of its neighbourhood. The dynamics and distribution of the local populations, as well as the total biomass, in the coupled system described above, is examined. The significant observation is the following: When the range of coupling is sufficiently large, namely when enough neighbouring populations influence the growth rate of a population, the system yields remarkably large biomass values that are very far from the mean. These extreme events are relatively rare and uncorrelated in time. It is also found that at any point in time, exceedingly large population densities emerge in a few patches, analogous to an extreme event in space. These results indicate a new mechanism in coupled chaotic systems that naturally yield extreme events in both time and space. Wed, 01 May 2019 00:00:00 GMT http://hdl.handle.net/123456789/1421 2019-05-01T00:00:00Z http://hdl.handle.net/123456789/1420 Title: Utilizing Noise to implement Logical operations in Bistable Systems Authors: V, Manaoj Aravind; Sinha, Sudeshna Abstract: The central theme of this thesis is the study of bistable dynamical systems in the presence of noise and the subsequent use of this understanding to implement highly reliable logic gates that work in the presence of noise. Specifically, we identify the various regimes of behaviours that arise when bistable systems interact with noise. We use this understanding to construct logic gates and identify the parameter ranges where reliable logic operation is achieved. First, we explore the behaviour of two coupled bistable systems that are subject to noises from two independent uncorrelated noise sources. We answer the question, “When do the random hopping events induced by noise, become synchronous in coupled bistable systems?”. Next, we use the above understanding to implement Logical Stochastic Resonance (LSR) in coupled bistable systems. We show that, two coupled bistable sub-systems each individually driven by an input signal, yield outputs that can be mapped to specific 2-input logic gate operations in a robust manner, in an optimal window of noise strength. The collective response of the system due to coupling, in the presence of the noise floor leads to reliable logic operations. We term this phenomena coupling induced Logical Stochastic Resonance (cLSR). The clear advantage of such a system being employed in the generation of reliable logic is that the two inputs can be fed to the two bistable subsystems in completely different noise environments. Lastly, we demonstrate the implementation of a noise-aided logic gate using both LSR and cLSR in a second order autonomous memristive circuit. Memristors are resistors with memory which have been considered promising candidates to implement neuromorphic computing architectures. Here, we construct, simulate and measure the reliability of operation of both LSR and cLSR based logic gates in a memristive circuit. We demonstrate that in an optimal window of noise the memristive circuit produces a very reliable logic output. We also show that due to the inherent symmetry in this system, complementary logic outputs are obtained in parallel from the response of the second state variable. Thus, in this thesis we explore and quantify the behaviour of nonlinear bistable systems interacting with noise and use the understanding to physically implement robust noise-aided logic gates using both simple piece-wise linear elements and memristors which will further the future forms of computing Wed, 01 Jan 2020 00:00:00 GMT http://hdl.handle.net/123456789/1420 2020-01-01T00:00:00Z