Developing Molecular Tools for Applications in Metabolic Engineering and ProteinPurification

lahiry, ashwin The Ohio State University ProQuest Dissertations & 2017 해외박사(DDOD)

Biotechnology is an important field of science that utilizes the principles of biology and engineering to build applications in multi-disciplinary research areas. This dissertation discusses the development of molecular tools in two such research areas: 1) industrial biotechnology and 2) medical biotechnology.Chapters 2 and 3 of this dissertation focus on the development of a multi-targeting small regulatory RNA (sRNA) as a potentially useful tool for metabolic engineering applications in industrial biotechnology. sRNAs can govern gene expression by base-pairing with the translation initiation regions (TIRs) of mRNAs. These sRNAs can be engineered to target the TIRs of non-cognate mRNAs by altering their antisense sequences. Due to their modular, tunable and portable nature, they possess unique regulatory qualities for optimizing metabolic pathways. In Chapter 2, an Escherichia coli-based genetic system was created for the development of dual-acting sRNA variants. These variants were built on the DsrA scaffold, a well-characterized multi-acting E. coli sRNA and assayed for the regulation of two separate TIRs fused to independent reporter genes. Expression of the sRNA and each reporter mRNA was independently controlled, and regulatory effects were quantified in vivo using a microtiter plate assay. Using this system, semi-rationally designed DsrA variants were screened in E. coli for their ability to simultaneously regulate two key TIRs from the Clostridium acetobutylicum n-butanol synthesis pathway. In Chapter 3, this system was utilized to successfully design and test the functionality of DsrA variants in two industrially relevant microorganisms, Bacillus subtilis and Clostridium acetobutylicum. This study demonstrates the value of the genetic system and the sRNA scaffold for designing DsrA variants that specifically target desired mRNAs, and thus provides a platform for retargeting and characterizing multi-acting sRNAs for metabolic engineering applications.Chapter 4 discusses the development of a self-cleaving affinity tag for the purification of recombinant proteins and has applications in medical biotechnology. Process development of new recombinant biotherapeutic proteins involves complex optimization and scale up based on the characteristics of each protein. There is no simple, low-cost platform that can be utilized to purify these diverse proteins. Thus, it is desirable to create an affinity tag-based platform for purification of any recombinant protein, but with the requirement that the purified protein be tagless and traceless. Consequently, our lab has developed a pH-controllable self-cleaving tag based on the Npu DnaE intein from Nostoc punctiforme for affinity-based purification of recombinant proteins. Previous work done with self-cleaving inteins has shown that the target protein residues (extein residues) at the cleavage junction can strongly affect cleavage kinetics of the tag. Therefore, in Chapter 4, the extein dependence of this tag is characterized using model (eGFP) and biotherapeutic (streptokinase (SK) and granulocyte colony stimulating factor (GCSF)) proteins. Through these studies, N-terminal extein residues that result in accelerated or diminished cleavage kinetics were identified. An eGFP model system was also established to predict the effect of the primary extein sequence on the cleavage kinetics of this tag. Finally, the information from this study was utilized to improve cleavage kinetics of the tag with GCSF.

Some Problems of Asymptotic Quantum Statistical Inference

Lahiry, Samriddha ProQuest Dissertations & Theses Cornell University 2022 해외박사(DDOD)

Recent breakthroughs in quantum technology, suchas quantum computing, communication, and metrology have given rise to questions related to quantum measurements which can be formulated in the language of mathematical statistics. Since quantum mechanics is fundamentally non-commutative in nature, statistical inference for these problems also involves dealing with such non-commutative structures. Moreover, inference in quantum statistics based on the laws of quantum probability deviates from inference in classical statistics, and the results often turn out to be different in a non-trivial way.In classical statistics a fundamental paradigm is approximating complicated experiments (families of laws, or models) by simpler ones. In particular, one establishes asymptotic equivalence between i.i.d. models indexed by a local parameter and a Gaussian shift model (with the shift given by the same local parameter). This approximation is called local asymptotic normality (LAN) and allows one to construct an estimator from a procedure in the Gaussian model with similar risk bounds. Local asymptotic equivalence can also been established between quantum i.i.d. models and quantum Gaussian models. In this thesis, we explore quantum statistical inference through the lens of local asymptotic equivalence and establish quantum counterparts of several results in classical statistics.

Knowledge spillovers: The role of geography, technology and intra-firm linkages in the global semiconductor manufacturing industry

Lahiri, Nandini University of Michigan 2003 해외박사(DDOD)

Geographic collocation of firms facilitates flow of knowledge spillovers between industry peers. Yet, why firms vary in their ability to use existing geographic locations to acquire external knowledge is not well understood. In an attempt to examine the process, this dissertation relaxes two simplifying assumptions of prior work. First, I allow for the possibility that firms may be multi-unit which introduces complexity to the process. The complexity arises from the simultaneous existence of multi-unit firms in different geographic and different technology domains. Multiplicity of units brings not only opportunities but also challenges in the absorption of knowledge spillovers. Multi-unit firms are able to overcome the challenges of geographic dispersion only when integrative mechanisms, or intra-firm linkages, that allow knowledge sharing across locations, exist within the firm. Second, since the flow of knowledge spillovers is caused by the interaction of firms, hence there is a need to model both the role of the recipient (focal) and source (spillor) firms. Characteristics of the spillor determine where the focal firm is most likely to acquire knowledge spillovers. Finally, characteristics of the dyad of the focal and the spillor firms also contribute to the process. I test the hypotheses in the context of the global semiconductor manufacturing industry. The sample consists of 100 firms over a period spanning 1990–1997. I find strong support for my hypotheses to indicate that geographic concentration of units enhances firm's ability to acquire knowledge spillovers. However, for firms that are geographically dispersed, intra-firm linkages are crucial in the process of absorption. Similarly, intra-firm linkages of the spillor enhance the distributiveness of proprietary knowledge to the focal firm. Additionally, the impact of intra-firm linkages of the focal firm is significantly greater than the impact of intra-firm linkages of the spillor firm, in the flow of spillovers in firm dyads. Thus, firms are benefited overall by such intra-firm linkages. This research enhances our understanding of the role of firm boundaries on flows of knowledge spillovers. The sharing of distributed knowledge within the firm has implications for firms' ability to leverage, not just local but also geographically distant resources for maximum advantage.

Three essays on the interaction of international trade and environmental outcomes and policies

Lahiri, Bidisha The University of North Carolina at Chapel Hill 2007 해외박사(DDOD)

Economic literature on international trade identifies the sources of comparative advantage like endowments and technology that drive international trade and result in gains from trade. Comparative advantage is however also affected by environmental standards. Stricter environmental standards are commonly believed to erode an existing comparative advantage of developing countries and hence result in lower gains from trade. Based on this popular belief, developing economies might legislate weak environmental standards or fail to enforce existing standards in the hope of encouraging "dirty" industries. My essays take a more sophisticated look at the trade and environment relation and find that the relation between welfare gains from trade and environmental quality is not one of simple trade-off of one against the other as popularly believed.

Spectroscopic Studies of Photo Reagents for Precision Chemistry

Lahiri, Jurick Michigan State University ProQuest Dissertations & 2022 해외박사(DDOD)

Precision chemistry offers temporal and spatial control of reaction and requires active photo reagents. In this dissertation, the excited state dynamics of photo reagents is discussed. This is part of a collaboration that included synthesis, theory, and spectroscopic characterization with the goal of developing more active super photobases. The rotational dynamics study revealed significantly slower rotational diffusion of FR0-HSB+* than FR0-SB*. The microscopic solvent interactions play a crucial role in the excited state proton transfer. The spatial resolution can be improved from one-photon excitation (OPE) by utilizing two-photon excitation (TPE). The spectroscopy of FR0-SB following TPE revealed higher reactivity in comparison to OPE. The quantum mechanical aspects of two-photon excitation were examined to demonstrate that the dipolar pathway plays an important role in these transitions even though it is far from resonance. Finally, the efforts to stabilize the higher excited states of cyanine dyes, with the goal of harnessing the energy of photons to achieve greater reactivity, have been described. Using this as inspiration, the S2 spectroscopy of a similar cyanine dye has been used to monitor the binding of the dye with human serum albumin protein.

Modulation of the aromatic-aromatic stacked organizations of phenylene ethynylene macrocycles and oligomers through structure modification

Lahiri, Shreyasi University of Illinois at Urbana-Champaign 2002 해외박사(DDOD)

In the field of biopolymer mimetics, supramolecular assembly has emerged as an important tool for constructing tertiary structure. Our interest in supramolecular chemistry is focused on studying the intermolecular assembly and intramolecular organization of phenylene ethynylene macrocycles and oligomers. The focus of this thesis will be on the effect of subtle manipulations of the structure of phenylene ethynylene macrocycles and oligomers on both their solution conformation and solid-state properties. In Chapter 2, the effect of modulating the side chain linking group and the solvent environment on the aromatic-aromatic stacking propensities of phenylene ethynylene macrocycles and oligomers is discussed. It was observed that electron withdrawing functionality, such as an ester, promoted aromatic-aromatic stacking, whereas electron donating functionalities, such as phenyl ether and benzyl ether groups, hindered this stacking. A solvophobic effect on the stacking propensities of the macrocycles and oligomers was also observed, such that aromatic-aromatic interactions were promoted in polar solvents such as acetonitrile relative to non-polar solvents such as chloroform. In Chapter 3, the relationship between helical cavity packing density and oligomer folding propensity is discussed. It was determined that modifying the packing density of phenylene ethynylene oligomers has a significant effect on their solution and solid-state organizations. The results imply that there is an optimal packing density within the cavity of the helices of phenylene ethynylene oligomers. In summary, it has been observed that subtle modifications of phenylene ethynylene architectures can influence their aromatic-aromatic stacking propensities, and that the nature of these architectures can be modulated by the design of appropriate hetero-sequences.

Unbounded system verification using decision procedure and predicate abstraction

Lahiri, Shuvendu K Carnegie Mellon University 2004 해외박사(DDOD)

Modeling and analysis of systems with large, infinite or parameterized state spaces has received much attention in the last decade. These systems include microprocessors with unbounded buffers and memories; parameterized cache-coherence and communication protocols with unbounded channels; and distributed algorithms for mutual exclusion. Most previous works have either used general purpose theorem provers with considerable manual guidance or techniques specific to a particular class of systems. In this work, we express unbounded systems in the Logic of Counter arithmetic with Lambda expressions and Uninterpreted functions (CLU), a quantifier-free fragment of first-order logic. We illustrate the strengths and limitations of the logic with respect to the expressiveness in modeling systems and efficient tools for reasoning in this logic. We exploit efficient Boolean translation of CLU formulas as a basis for constructing a decision procedure for CLU that exploits recent advances in Boolean Satisfiability (SAT) solving. We use a case study of complex out-of-order microprocessors to illustrate the effective use of decision procedures in reducing manual guidance in proving the verification conditions. To automate the construction of inductive invariants, we use predicate abstraction. Since previous predicate abstraction based tools require a large number of theorem prover calls, they often failed to scale to large problems. We provide a symbolic approach for performing predicate abstraction that alleviates the exponentially large number of decision procedure calls. We leverage recent advances in Boolean methods to perform the task efficiently. We illustrate the effectiveness of the approach for distributed systems and benchmarks from the verification of device drivers in Microsoft Windows. For verifying unbounded systems, it is crucial to construct quantified invariants. In this work, we describe indexed predicate abstraction , a variant of predicate abstraction where free variables are allowed inside the predicates. We formalize the extension and provide a weakest precondition transformer based mechanism for discovering indexed predicates automatically. This has facilitated automatic safety verification of a number of interesting systems including directory based cache coherence protocols and mutual exclusion algorithms.

Structural Origin and Environmental Mediation of Dispersive Optical Activity in the Vapor and Solution Phases

Lahiri, Priyanka Yale University 2013 해외박사(DDOD)

Chiral molecules are ubiquitous in nature, with examples ranging from the building blocks of life such as amino acids and DNA to larger macromolecular systems. The interactions of chiral molecules with polarized light, known as optical activity, have been studied for over two centuries largely in the condensed phases, however analogous studies of the properties of isolated chiral molecules are comparatively limited. This dissertation presents a synergistic experimental and computational investigation of dispersive optical activity (i.e., circular birefringence or CB) in diverse chiral species, with particular emphasis directed towards elucidating the structural provenance of intrinsic chiroptical response and demonstrating the effect of solvation on isolated-molecule behavior. The wavelength-resolved specific rotation of several cyclic molecules has been explored under complementary solvated and vapor-phase conditions, where the latter studies have exploited the ultrasensitive technique of cavity ring-down polarimetry (CRDP) to measure specific rotation parameters at excitation wavelengths of 355 and 633 nm. Linear response calculations at analogous density-functional and coupled-cluster levels of theory have been enlisted to predict the chiroptical response of the isolated species and unravel the structural causes of observed behavior while an implicit polarizable continuum model (PCM) has been utilized to explore the influence of non-specific solute-solvent interactions on intrinsic optical activity. Targeted species include the isomeric terpenes, (S)-(+)-3-carene and ( S)-(+)-2 carene, wherein the transposition of a double bond within the bicyclic carbon skeleton imbues the latter with pronounced flexibility and an attendant enhancement of chiroptical response. Other molecules of interest include the bicyclic ketone (IR, 4R)-norbornenone, where the spatial arrangement of distal alkene and carbonyl moieties gives rise to unusually large optical rotation parameters and marked solvent effects, as well as the model cycloketones (R)-3-methylcyclopentanone and (R)-3-methylcyclohexanone, which exhibit distinct chiroptical properties stemming from differences in their ring morphology, and a conformationally flexible cyclic ether, (R)-(--)-glycidyl methyl ether. Comparison of the solvated and isolated-molecule response for these varied species has revealed the pronounced effect of solvation on the magnitude and sign of intrinsic optical activity, highlighting the importance of the solvent-free measurements as appropriate benchmarks for assessing the reliability of ab initio predictions of dispersive optical activity. The dependence of optical activity on bulk properties of the solvent environment such as dielectric constant, refractive index and polarizability has also been explored in an attempt to bridge the gap between isolated and solvated chiroptical properties.

Consumed by commitment: Suicide protest in the contentious politics of South Asia

Lahiri, Simanti The University of Wisconsin - Madison 2008 해외박사(DDOD)

This dissertation addresses two important questions: Why do groups use suicide as a protest tactic? When does suicide protest succeed and when does it fail? I conduct a comparative analysis of four movements in India and Sri Lanka that use different types of suicide protest in order to investigate these questions. I argue that groups choose suicide protest because, unlike other forms of political violence, it can generate positive affective ties---sympathy and pride---between movement and audience, while simultaneously aspiring for a specific political outcome from the state. Movements also actively direct emotional appeals towards the state through suicide protest. Movements hope to incite the twin emotional responses of fear and shame from the state. The relative success or failure of suicide protest is important to analyze. I contend that four factors shape the efficacy of suicide protest: the level of tactical depth within the movement, the intensity of the protest act, the ability of the movement to make emotional appeals to the state and members of the movement, and the strength of competitive political forces present in the state. These four factors work together to help or hinder the ability of the tactic to produce policy shifts in favor of the movement. I examine four different movements in South Asia. They are: the movement for the linguistic state of Andhra Pradesh (India), the Anti-Narmada Dam movement and its use of fasting (India), self-immolation by the anti-reservation movement (India), and finally the Liberation Tamil Tigers (LTTE) use of suicide bombing in Sri Lanka. I contend that the extent to which these four factors work together will increase the likelihood of movement success, which I define as long term policy shifts in favor of movement goals. Of the four cases, only one, the movement for the linguistic state of Andhra, results in movement success. Both the NBA and the LTTE exhibit short-term success, while the anti-reservation movement is defined as a failure.

On-chip communication: System-level architectures and design methodologies

Lahiri, Kanishka University of California, San Diego 2003 해외박사(DDOD)

The rapidly growing number and variety of components integrated onto a single System-on-Chip is resulting in an unprecedented increase in the volume and diversity of on-chip communication traffic. However, as technology scales into the deep submicron era, chip-level communication structures are challenged by large propagation delays, signal integrity problems, and increased power consumption. Consequently, the design of the on-chip communication architecture is expected to play a significant role in determining several system-wide metrics, such as overall system performance, power consumption, and battery life. However, state-of-the-art system design methodologies, tools, and architectures are not sufficiently geared towards handling the increasing role of on-chip communication. This thesis introduces the on-chip communication architecture as a primary concern in system-level design. It addresses challenges in communication architecture design from the standpoint of methodologies and architectures for high-performance and battery-efficient System-on-Chips. The specific contributions of this thesis include: (i) system-level performance and power analysis techniques to drive the design of the communication architecture, (ii) techniques for customizing the communication architecture to characteristics of the communication traffic generated by the application, (iii) techniques for run-time adaptation of on-chip communication protocols, and (iv) a communication-based, system-level power management methodology for the design of battery-efficient systems. Experiments conducted on variety of System-on-Chip designs demonstrate that incorporation of the developed techniques into the system design process enables significant improvements in design metrics such as performance and/or battery life. In addition, the techniques described in this thesis will go a long way in facilitating research and development of design methodologies for systems based on the emerging “network-on-chip” paradigm.