Dissection of nucleosome positioning and spacing mechanisms in Saccharomyces cerevisiae

Eukaryotic genomes are packaged as chromatin that contains an ever-repeating succession of nucleosomes like beads-on-a-string. Nucleosomes, together with linker histones, promote packaging of genome into higher-order chromatin structure. Nucleosomes regulate access to the underlying DNA as well as all nuclear processes, including transcription, replication, and double-strand break repair. Defects in these processes lead to genome instability and diseases. Therefore, it is essential to understand the fundamental mechanisms which regulate nucleosome organization. Nucleosomes attain a stereotypical organization near major regulatory sites in the genome. At these regions, a nucleosome-free region is followed by well-positioned nucleosomes which are phased to a known genomic point, such as transcription start sites in S. cerevisiae and at CCCTC-binding sites in higher organisms. The average distance between these nucleosomes, called nucleosome repeat length (NRL), is surprisingly constant and lead to a regular nucleosome array. How this primary structure of chromatin is established is not completely clear. Several nuclear factors, like transcription, ATP-dependent nucleosome remodelers, barrier factors and DNA sequence are known to contribute to this process. Many of these factors act in a redundant manner in the cell. Therefore, it is difficult to understand the mechanism behind regular arrays and the role of individual factor towards it. In this thesis, we employed the baker’s yeast (S. cerevisiae) to dissect the mechanism of regular array formation. We made use of a yeast strain lacking bona fide spacing remodelers of the ISWI- and CHD- families to cleanly dissect the role of cellular factors and function of regular arrays. In chapter 2.1, we show that the RNA Pol II-dependent transcription destroys regular nucleosome arrays and overrides effects of the spacing remodelers. By inhibiting transcription in cells, we identify residual spacing activity and assign it to the INO80 nucleosome remodeler. Several orthogonal approaches establish INO80 as a bona fide spacing remodeler in vivo. We dissected the spacing mechanism of INO80 and show that the Arp8 module determines NRL by INO80, while the Nhp10 module is dispensable. We determine the interplay of histone amounts and remodelers to show that the spacing remodelers critically depend on high histone density to establish the WT-like nucleosome array. ISWI and Chd1 remodelers possess “clamping” activity to establish regular arrays, but this activity is rather weak in vivo. Finally, we find that the DNA sequence co-determines NRL in most part of the genome and spacing remodelers override DNA sequence-influenced extremely short- or long- NRLs in the genome. We show that the regular arrays established by the spacing remodelers protect the genome from genotoxic stress such as DNA damage and ectopic recombination, and regulate chromatin accessibility in the gene body. We propose a four-step model for the establishment of regular nucleosome arrays and suggest that it has evolved to regulate as well as to protect the genome. In chapter 2.2 and 2.3, we investigated the regulatory mechanism of the ISW1 and ISW2 spacing remodelers in vivo. We find that ISW2 requires the N-terminus of its accessory subunit Itc1 to position and space nucleosomes. Unlike ISW1 and Chd1, ISW2 resolves dinucleosomes in the gene body. We present a genome-wide cell-type specific nucleosome architecture analysis and dissect the role of ISW2 in this process. Lastly, we investigated the role of individual domains and subunits in the ISW1 remodeler towards nucleosome positioning and spacing. We show that ISW1 depends on the N-terminus and NegC motif within its ATPase subunit for nucleosome positioning. Surprisingly, the AutoN motif is not required for ISW1 spacing mechanism. Overall, this study establishes a unifying model of the biogenesis of regular nucleosome arrays and provides a basis for future investigation of the interplay of transcription and spacing remodelers towards establishing the primary structure of chromatin

Role of syndecan-1 in tumor cell proliferation and epithelial-mesenchymal plasticity

Syndecan-1 (SDC1) is a heparan sulfate proteoglycan (HSPG) intercalated in the cell membrane but also translocated to the cell nucleus in a regulated manner. SDC1 is involved in several malignancy-associated processes such as proliferation and migration. Altered SDC1 expression can induce changes along the epithelial-mesenchymal axis and it may influence the prognosis of cancer. Transforming growth factor-ß (TGF-ß) plays a pivotal role in many cellular functions, including epithelial-mesenchymal transition (EMT). In the early stages of tumorigenesis TGF-ß inhibits cell growth and induces cell apoptosis, while in the later stages it promotes tumor growth. The overall aim of this thesis was to study the role of SDC1 in mesenchymal tumors and functions related to the presence or absence of SDC1 in the nucleus. Understanding the role of cell surface and nuclear SDC1 and its interactions could be of importance for the understanding of tumor growth, proliferation, differentiation, and migration in these tumors. In paper I we used fibrosarcoma cell sub-lines to study the functions of SDC1, especially the molecular targets and signaling pathways regulated by its nuclear translocation. The TGF-ß pathway was activated by nuclear SDC1, and three genes were altered with the deletion of nuclear localization signal: EGR-1, NEK11 and DOCK8. The study shows the importance of the localization of SDC1 for its effect on tumor cells. The aim of paper II was to further study the role of nuclear SDC1 through characterizing its nuclear interactome, using a mesothelioma cell line. SDC1 was immunoprecipitated to identify co-precipitating interacting proteins. The results indicate a previously unknown role for SDC1 in RNA biogenesis. In Paper III we investigated if SDC1 plays a role in regulating TGF-ß-induced EMT. The knockdown of SDC1 in a carcinoma cell line resulted in decreased expression of E-cadherin, and increased expression of N-cadherin. In fibrosarcoma cells, with its low basic SDC1 levels, overexpression of SDC1 was sufficient to repress N-cadherin and vimentin. The results indicate that SDC1 regulates epithelial-mesenchymal plasticity in tumor cells. Together, these studies provide new insights into the role of SDC1 in tumors and the functional importance of the transport of SDC1 to the nucleus, as well as the connection between SDC1 and TGF-ß

Biometric Identification using Phonocardiogram

Phonocardiogram (PCG) signals as a biometric is a new and novel method for user identification. Use of PCG signals for user recognition is a highly reliable method because heart sounds are produced by internal organs and cannot be forged easily as compared to other recognition systems such as fingerprint, iris, DNA etc. PCG signals have been recorded using an electronic stethoscope. Database of heart sound is made using the electronic stethoscope. In the beginning, heart sounds for different classes is observed in time as well as frequency for their uniqueness for each class. The first step performed is to extract features from the recorded heart signals. We have implemented LFBC algorithm as a feature extraction algorithm to get the cepstral component of heart sound. The next objective is to classify these feature vectors to recognize a person. A classification algorithm is first trained using a training sequence for each user to generate unique features for each user. During the testing period, the classifier uses the stored training attributes for each user and uses them to match or identify the testing sequence. We have used LBG-VQ and GMM for the classification of user classes. Both the algorithms are iterative, robust and well established methods for user identification. We have implemented the normalization at two places; first, before feature extraction; then just after the feature extraction in case of GMM classifier which is not proposed in earlier literature

Phase separation kinetics of block copolymer melts confined under moving parallel walls: a DPD study

We use dissipative particle dynamics (DPD) simulations to study the effect of shear on domain morphology and kinetics of microphase separating critical diblock copolymer (BCP) bulk melts. The melt is confined within two parallel solid walls at the top and bottom of the simulation box. The shear is induced by allowing the walls to move in a direction with a specific velocity. We explore the following cases: (i) walls are fixed, (ii) only the top wall moves, (iii) both walls move in the same direction, and (iv) both walls move in opposite directions. After the temperature quench, we monitor the effect of shear on evolution morphology, the scaling behavior of the system, and the characteristic length scale and growth. The characteristic length scale follows typical power-law behavior at early times and saturates at late times when both walls are fixed. The length scale changes significantly with shear caused by wall velocities. The usual lamellar morphology, which is not achieved for case 1 within the considered simulation time steps, is noticed much earlier for the nonzero wall velocity cases. Specifically, it is seen much before in case 4 than in the other cases. We find that the shear viscosity decreases (shear-thinning) with wall velocity (shear rate) for all the cases at a given coarsening time. Overall, we report the influence rule of shear rates on microphase separation kinetics of BCP melts. This study can provide a scheme to anticipate and design anisotropic microstructures under the application of externally controlled wall shear that may further guide in producing the various composite materials with superior mechanical and physical properties.Comment: 44 pages, 16 figures, articl

Growth, yield and nutrient uptake of hybrid rice as influenced by nutrient management modules and its impact on economic of the treatments

Field experiment was conducted at Instructional Farm of Narendra Deva University of Agriculture and Technology, Kumarganj, Faizabad to develop nutrient management modules for efficient cultivation of hybrid rice. Results showed that growth and yield characters viz. plant height, number of tilers and panicles per hills were high-est under nutrient management module of 100% NPK + 5 t press mud (T3). Significantly higher grain and straw yield was recorded under treatment T3 over all the treatment except treatment T1, T2, T4, T5 and T6 which were recorded on par. Uptake of NPKS and Zn was significantly improved under treatments having organic manure along with inorganic levels (100%, 75% and 50% NPK) of fertilizer over alone levels of inorganic fertilizer. NPKS and Zn uptake was higher in treatment module T3 followed by treatment T2 (100% NPK + 10 t FYM ha-1). The net return Rs. 27373.70 ha-1 and 26087.0 ha-1 and benefit: cost ratio (1.53 and 1.45) was maximum in treatment T3 closely followed by T2. Findings of this study warranted that treatment module T3 resulted higher growth and yield of rice crop. Net return was also higher in this treatment

Tegument based in-silico Drug Targeting of Herpes Simplex Virus-1

Alpha Trans Inducing Factor (a-TIF) is a herpes simplex virus type 1 (HSV-1) virion tegument protein present in the tegument layer between the capsid and the envelope, in association with cellular proteins and trans-activated viral activities. a-TIF stimulates the transcription of HSV-1 Immediate Early genes during lytic virus replication. The presence or absence of functional a-TIF protein in vivo is always associated with viral activities. Its role behind latent HSV-1 infection is ambiguous. No drug is designed for HSV till now based on this protein and thus its conformational details can be very important for drug designing purpose. Methods. Docking studies on this protein becomes logical. Four different ligand molecules viz. Adenosine-3'-5'-Diphosphate, P1-(5'-Adenosyl) P4-(5'-(2'-Deoxy-Thymidyl)) Tet-raphosphate, 9-Hydroxymethylguanine, 9-(4-Hydroxybutyl)-N2-Phenylguanine were screened for the study. Results. P1-(5'-Adenosyl) P4-(5'-(2'-Deoxy-Thymidyl) Tetraphophate was found to be the best ligand as it showed the lowest docking energy of - 9.238 kcal/mol. Conclusions. The best ligand was found to bind different sites of the a-TIF protein and hence can be utilized to combat HSV-1 successfully, with a synergistic effect of multiplicity of drug molecules on the target protei

A Systematic Survey of Classification Algorithms for Cancer Detection

Cancer is a fatal disease induced by the occurrence of a count of inherited issues and also a count of pathological changes. Malignant cells are dangerous abnormal areas that could develop in any part of the human body, posing a life-threatening threat. To establish what treatment options are available, cancer, also referred as a tumor, should be detected early and precisely. The classification of images for cancer diagnosis is a complex mechanism that is influenced by a diverse of parameters. In recent years, artificial vision frameworks have focused attention on the classification of images as a key problem. Most people currently rely on hand-made features to demonstrate an image in a specific manner. Learning classifiers such as random forest and decision tree were used to determine a final judgment. When there are a vast number of images to consider, the difficulty occurs. Hence, in this paper, weanalyze, review, categorize, and discuss current breakthroughs in cancer detection utilizing machine learning techniques for image recognition and classification. We have reviewed the machine learning approaches like logistic regression (LR), Naïve Bayes (NB), K-nearest neighbors (KNN), decision tree (DT), and Support Vector Machines (SVM)

A Review on Cloud Data Security Challenges and existing Countermeasures in Cloud Computing

Cloud computing (CC) is among the most rapidly evolving computer technologies. That is the required accessibility of network assets, mainly information storage with processing authority without the requirement for particular and direct user administration. CC is a collection of public and private data centers that provide a single platform for clients throughout the Internet. The growing volume of personal and sensitive information acquired through supervisory authorities demands the usage of the cloud not just for information storage and for data processing at cloud assets. Nevertheless, due to safety issues raised by recent data leaks, it is recommended that unprotected sensitive data not be sent to public clouds. This document provides a detailed appraisal of the research regarding data protection and privacy problems, data encrypting, and data obfuscation, including remedies for cloud data storage. The most up-to-date technologies and approaches for cloud data security are examined. This research also examines several current strategies for addressing cloud security concerns. The performance of each approach is then compared based on its characteristics, benefits, and shortcomings. Finally, go at a few active cloud storage data security study fields